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Data Processing Handbook for Complex Biological Data Sources
Data Processing
Handbook for
Complex Biological
Data Sources
Edited by
Gauri Misra
Amity Institute of Biotechnology, Amity University, Noida, India
Academic Press is an imprint of Elsevier
125 London Wall, London EC2Y 5AS, United Kingdom
525 B Street, Suite 1650, San Diego, CA 92101, United States
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The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

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This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be
noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes
in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information,
methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own
safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury
and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any
methods, products, instructions, or ideas contained in the material herein.

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ISBN: 978-0-12-816548-5

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Typeset by MPS Limited, Chennai, India

 Dedication

Dedicated to
The lotus feet of Shri Radha Krishna
and
Goverdhannathji
 List of contributors
Usha Agrawal
CRIB Lab, ICMR-National Institute of
Pathology, New Delhi, India
Mohd Tashfeen Ashraf
School of Biotechnology, Gautam
Buddha University, Noida, India
Marcio Chaim Bajgelman
Brazilian Biosciences Laboratory, National
Center for Research in Energy and
Materials, Campinas, Brazil
Ankur Baliyan
NISSAN Analysis and Research Center,
Yokosuka, Japan
Bhaswati Banerjee
School of Biotechnology, Gautam
Buddha University, Noida, India
Nar Singh Chauhan
Department of Biochemistry, Maharshi
Dayanand University, Rohtak, India
Sudhir K. Gupta
Faculty of Chemical Sciences,
Department of Chemistry, Harcourt
Butler Technical University (Formerly
HBTI), Kanpur, India
Hideto Imai
NISSAN Analysis and Research Center,
Yokosuka, Japan
Gagandeep Jhingan
Valerian Chem Private Limited, New
Delhi, India
Mahima Khandelwal
School of Chemical Engineering,
University of Ulsan, South Korea
Amit Kumar
ICMR-AIIMS Computational Genomics
Centre, Convergence Block, All India
Institute of Medical Sciences, New Delhi,
India
Atul Kumar
Amity Institute of Engineering &
Technology, Amity University, Greater
Noida, India
Vinit Kumar
Amity Institute of Molecular Medicine
and Stem Cell Research, Amity
University, Noida, India
Gauri Misra
Amity Institute of Biotechnology, Amity
University, Noida, India
Dibyabhaba Pradhan
ICMR-AIIMS Computational Genomics
Centre, Convergence Block, All India
Institute of Medical Sciences, New Delhi,
India
Jyotika Rajawat
Molecular & Human Genetics
Laboratory, Department of Zoology,
University of Lucknow, Lucknow, India
Reshma Rani
Amity Institute of Biotechnology, Amity
University, Noida, India
Harpreet Singh
ICMR-AIIMS Computational
Genomics Centre, Convergence Block,
All India Institute of Medical Sciences,
New Delhi, India
Vijay Kumar Srivastava
Amity Institute of Biotechnology, Amity
University Rajasthan, Jaipur, India
Leonardo Vazquez
Key Laboratory of Pathogenic
Microbiology and Immunology, Institute
of Microbiology, Chinese Academy of
Sciences, Beijing, China
Rupali Yadav
Dr. Reddy’s Institute of Life Sciences,
University of Hyderabad Campus,
Hyderabad, India
ix
 Foreword

The recent explosion of data in the biological sciences presents both clear opportunities and con-
siderable challenges. Much of these data arise from high-throughput sequencing and associated
computations. However, once a protein sequence is determined, its three-dimensional structure
and interactions must be characterized, in a process that experimentally is much less high-
throughput. The present book melds these two approaches in an extensive description of structural
and genomic techniques. This is the third book by Editor Gauri Misra published by an interna-
tional publisher. Her earlier works have been well accepted in scientific community and we hope
she continues her good work enriching technical scientific literature resources.
Some of the work in the book treats topics related to the generation of data. A clear example of
this is the chapter by Pradhan et al. on methods for high-throughput sequencing. Similarly high-
throughput data streams can also result from the collection of mass spectroscopy data in the iden-
tification of biomolecules and protein:protein interactions, as illustrated by Rajawat et al.
Microbiome data analysis is expounded by Chauhan.
Many believe that the generation of omics data is most useful when it can be translated into
three-dimensional structure. From the experimental perspective this requires careful studies of con-
formation and function in vitro. In this vein, several contributions in the book examine the experi-
mental characterization of structures and interactions, such as that from Banerjee et al., who
describe the use of circular dichroism to determine secondary and tertiary structure; Misra, who
compares various types of fluorescence spectroscopy, and Kumar et al., who describe FTIR.
Furthermore, detailed structures can be derived using NMR as described by Vazquez. A general
chapter on microscopy is contributed by Baliyan and Kumar, covering TEM, SEM, and AFM.
Thermodynamic aspects influence the states living systems find themselves in, and in this regard
basic, underlying principles of isothermal titration calorimetry are explained by Srivastava and
Yadav. The identification of phenotypes and mechanisms in cell-based arrays can be examined
using flow cytometry, as described by Bajgelman.
Techniques such as those described in this book form an integral component of the armory
required to translate molecular biological data into three-dimensional structure and interactions.
Eventually, given enough supercomputing power, these data will be integrated into a fully three-
dimensional computational model of the living cell, melding metabolism and machinery, with which
the efficacy and safety of new drugs will be able to be tested in silico.
Sincerely,

Jeremy C. Smith1,2
1
Center for Molecular Biophysics, Oak Ridge National Laboratory, University of Tennessee, Knoxville, TN, United States,
2
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, United States

xi
 Foreword

The Data Processing Handbook for Complex Biological Sources edited by Dr. Gauri Misra is a collection of
articles representing almost all biophysical techniques currently in use for the analysis of biomolecu-
lar structure and function. Dr. Misra’s extensive experience in editing such volumes is evident in all
the articles included in the volume. Each chapter provides a comprehensive description of one of the
methods and is authored by experts who have extensively used the technique and obtained impor-
tant results as reflected in their publications. The chapters provide not only extensive references to
published literature but also present the type of data obtained by each technique, methods of data
analysis, expected results, and their biological interpretation. Although there are other volumes of
similar content, the present volume is especially useful for several reasons. Students (and often tea-
chers as well) of biology possess limited familiarity of physics and mathematics. The authors of the
present volume have made substantial efforts to cover the theoretical and mathematical background
necessary to understand the techniques. In the background of the theoretical basis of the methods,
the authors present the nature of results obtained, data analysis protocols, their power, and limita-
tions. If all the sophisticated instruments necessary for the experimental procedures covered in the
volume are available in house, experiments can be repeated several times until satisfactory results are
obtained. Due to the high cost of many of the instruments, very frequently it is necessary to use
instruments available in other laboratories. The time available in such laboratories for outside users
is limited and hence performing experiments successfully without repetitions is very essential. The
authors of the various chapters have extensive experience in such efforts. Therefore, first time users of
the techniques and those who do not possess in-depth expertise in physics and mathematics will
find the volume invaluable. It is also a good reference volume to those who already possess expertise
in one or more of these techniques.

M.R.N. Murthy
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India

xiii
 Foreword

With new advanced technologies emerging, enormous biological data is generated. Analysis and
organization of this data is important for gaining valuable information and to create new knowl-
edge. The editor of this book, Gauri Misra, has a clear vision to bridge the gap between theoretical
and practical aspects of biological data processing. Being a biologist she is able to provide a rational
and broad outlook to experimental design and implementation. This is her third accomplishment
as a book editor, which is certainly admirable for a young developing scientist. The emphasis of this
book is on data processing approaches currently implied in a variety of biological systems from col-
lecting raw data, organizing it, and interpreting processed data. No story is complete with informa-
tion from a single technique or a scientific method. Amalgamation of various approaches is
required to address the whole scientific problem. It appealed to me that the experts in different
fields mastering related techniques came together to provide a detailed but clear explanation of vari-
ous techniques are used in modern laboratories using examples from their own research work.
The most captivating part of this book is that it takes the reader with already a brief knowledge
of the principles underlying various techniques to more advanced levels with excellent practical
examples thus enabling this book to open to a wide audience in the scientific community. Also it
is highly important for a student to be able to understand the concepts and the whole chain in
biological data generation and processing to customize them for gaining solutions to their own
research problems. This book certainly provides an excellent framework for both students and
already experienced scientists. There is a lack of available data processing books that deal directly
with the biological systems in the present market. Editor Gauri Misra has successfully filled the
void. I hope this handbook will be a guiding light to scientists who try to use interdisciplinary
approaches to validate their results. Albeit every person is not a master of all scientific domains
and there is always a requirement for a quick handbook on how to analyze the data. This will be
the first-hand resource that can be referred to by scientific personnel. The chapters include not
only classical approaches such as circular dichroism, fluorescence microscopy, NMR, etc., but also
the advances in these fields along with a chapter on more recent microbiome data analysis and
high throughput sequencing. In my opinion, this book will be an asset for the scientific commu-
nity that will help the readers to process and analyze the data generated from their own
experiments.
Sincerely,

Aatto Laaksonen1,2
1
Physical Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden,
2
Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden

xv
 Preface

This is a continuation of the journey I embarked on with my first book on biophysics in 2017. A
seed that was planted by the indefinite curiosity of the students I teach and the young scientists
who try to amalgamate interdisciplinary approaches to enhance the worth of their research endea-
vors. These driving forces bear fruit in the form of the present book. There are various aspects of
data analysis that have been emphasized in this book. The book is an interface that relates in-
depth analysis of data generated from various biological platforms. The enormous amounts of data
generated using several experimental approaches need to be interpreted in simple terms relating
the biology and physics at the atomic level. The book is compiled with a focus on the target audi-
ence consisting of graduate students, young scientific professionals, and technicians from the
industry sector who can use the data processing information for various techniques in this book to
help them process their own data with ease.
The output files generated from various techniques are often not recognized by common user-
friendly programs. When students read research papers they face a lot of difficulty in deriving the
necessary information to process their raw data files. This book includes sample data files and
explains the usage of equations and web servers cited in research articles to extract useful informa-
tion from their own biological data. Details related to raw data files, data processing strategies,
web-based sources relevant for data processing, and examples provide a unique perspective to the
present effort. Unlike the other available books in the market on data analysis, which place more
emphasis on computational and programming aspects, this book deals with biological aspects of
data analysis underpinning the various experimental approaches used in modern biology.
The book is divided into 10 chapters dealing with different experimental techniques, namely
mass spectroscopy, circular dichroism, fluorescence spectroscopy, high throughput sequencing,
nuclear magnetic resonance, Fourier-transform infrared spectroscopy, microscopy, flow cytometry,
isothermal titration calorimetry, and microbiome data analysis. Each chapter is a contribution
from experts in the field thus enhancing the quality of the content and keeping the practical
approach intact. It provides a quick guide for understanding experimental data. Each chapter
begins with an introduction that guides the reader describing the structure, composition, and
application of the chapter with the help of selective examples. The conclusion at the end of each
chapter summarizes the key points and includes suggestions wherever required. However, an expert
looking for a detailed explanation of anyone technique is advised to read further from suggested
references given at the end of each chapter. I firmly believe that the book provides a valuable
resource guide for students to plan and execute their own research experiments with a clear and
simple understanding developed after reading this book.

Gauri Misra, PhD

xvii
 Acknowledgments

Any effort sees the light of the day with the blessings of the Almighty and one’s parents. I acknowl-
edge the hard work of all the contributing authors who have diligently tried to make each chapter
worth reading. Thanks to all the students whose keen interest nurtured the desire to create this
work. Some people leave an everlasting effect on your mind and shape your ideas. Two such peo-
ple I wish to extend my gratitude are Dr. Shekhar C. Mande and Dr. M.R.N. Murthy.
My indebtedness to my mother, Mrs. Kamla Misra; my spiritual teacher, the late Bhaktivedanta
Shri Narayan Swami Maharaj; my grandmother, the late Mrs. Shantidevi Misra; grandfather, the
late Mr. Anand Swaroop Misra; and my school principal Sister Betty Teresa for their love, care, con-
stant support, and freedom of choice to choose and shape my career in science and face the chal-
lenges of life, emerging as a strong person. Support and affection of friends has been a constant
boost in this academic journey. I thank the reviewers for their valuable feedback that has certainly
added weight to the enrichment of this project. The support from all the concerned Elsevier
officials associated with this project is deeply acknowledged. With this I hope to progress in the
future, nurturing new scientific ideas and creating many more masterpieces useful for the general
scientific audience.

xix
 Chapter
Mass spectroscopy
Jyotika Rajawat1 and Gagandeep Jhingan2
1
Molecular & Human Genetics Laboratory, Department of Zoology, University of Lucknow, Lucknow, India, 2Valerian Chem Private Limited, New Delhi, India
1.1 Introduction
Mass spectrometry is an analytical technique that identi-
fies biomolecules or proteins present in biological samples
and also useful for studying protein protein interactions.
The information obtained can then be further employed
to determine protein functions, functional relationships,
and the cellular pathways regulated by the proteins.
Protein protein interactions yield insights on functional rela-
tionships including, enzyme-substrate, substrates, posttransla-
tional modifying enzymes, protein coactivator/corepressor,
and host pathogen interactions [1].
Mass spectroscopy based proteomic workflow consists
of the following steps:
1. Isolation of protein samples from biological samples
and fractionation by 2D gel electrophoresis.
2. Fractionated proteins are tryptic digested and resulting
peptides are further fragmented.
3. Peptides are injected in mass spectrometer (MS) and
subjected to quantitative and qualitative analysis.
4. The dataset generated is analyzed by appropriate
software for identification of the amino acid sequence
and for protein quantification.
5. Protein identity is assigned based on database search.
1.1.1 Principle
Mass spectroscopy determines the characteristic fragments
or ions that arise by organic molecule breakdown. The
basic principle involves the bombarding of organic
compound with a beam of electrons to generate positively
charged ions. Further, the molecular ion is fragmented by
Data Processing Handbook for Complex Biological Data Sources. DOI: https://doi.org/10.1016/B978-0-12-816548-5.00001-0
© 2019 Elsevier Inc. All rights reserved.
|1|
utilizing energy of electrons to break the bonds and yield
positively charged species or fragment ions. Positive ions
or fragment ions formed are further accelerated and
deflected in a circular path using magnetic field and then
focused on the detector or photographic plate according
to their mass and charge. Each ion represents a separate
line on plate and recorded in form of peak intensity. Ion
deflection is based on charge, mass, and velocity, ions
separation is based on mass to charge (m/z) ratio and
detection is proportional to abundance of ions.
1.1.2 Instrumentation
The first MS was designed by JJ Thompson in 1912 and
was mainly used to study atomic weight of elements and
to monitor the abundance of elemental isotopes. Later in
the 1950s, with the advent of techniques to vaporize the
organic compounds, it became possible to analyze biomo-
lecules with a MS. Following are the main components of
a MS as depicted in Fig. 1.1:
1. Ionization source
2. Analyzer
3. Detector
4. Data processor/digitizer
The analyzer and detector are kept in a vacuum so that
the ions produced do not collide with air molecules and
the ion trajectories are maintained at a particular speed.
Once the sample is injected in the instrument through the
inlet, it gets ionized in the ionization chamber. Ionized
species are then extracted into the analyzer, which resolves
the ions based upon their mass to charge ratio. Finally
these ions are detected by detectors and relative
1
 Data Processing Handbook for Complex Biological Data Sources
abundance of each resolved ionic species is recorded in
the form of mass spectra.
The samples can be injected into the instrument either
by direct infusion or through multidimensional chro-
matographic separation. Analytes can be delivered in stag-
gered manner with the use of chromatographic technique.
Gas or liquid chromatography (LC) is commonly used for
sample injection in MS. In gas chromatography (GC) the
mobile phase used is gas such as hydrogen or helium,
which injects the molecules of analytes in the column.
The analyte can be effectively separated from the matrix
by manipulating the column temperature. GC is limited
to compounds that are volatile, heat stable, and lack polar
functional groups. GC-MS is often used for comprehensive
drug screening. LC is nowadays a preferred method for
separation of samples as it is compatible to broad range
of analytes and need less for derivatization than GC. The
mobile phase uses aqueous and organic solvent in a
particular ratio for altering the distribution of analytes
and matrix. LC provides fast and effective separation of
sample prior to MS analysis [2].
1.1.3 Types of ionization techniques
Different types of ionization methods are employed
depending on the type of samples. Volatile samples are
subjected to either electron or chemical ionization.
Nonvolatile samples are ionized using any of the given
techniques:
2
Figure 1.1 Components of mass
spectrometer.
1. Fast atom bombardment
2. Matrix assisted laser desorption ionization (MALDI)
3. Electrospray
1.1.3.1 Electron ionization
The ionizing agent is high energetic electron emitted from
a heated filament and accelerated due to potential differ-
ence of around 70 eV. Sample gets ionized by removal of
an electron and generation of positively charged ion
species.
M 1 e 2 5 M1 1 2e 2
Charged radical cation further gets fragmented into
positive ions. Electron ionization (EI) is widely used for
volatile organic compounds like oils, hydrocarbons, etc.
1.1.3.2 Chemical ionization
The ionizing agent used is electron but the sample is indi-
rectly ionized by reagent gases. Commonly used reagent
gases are methane, isobutane, and ammonia. The vapor-
ized sample along with excess reagent gas is injected into
the spectrometer. Similar to electron ionization, electrons
are generated with heated filament, which ionizes carrier
gas to form primary ions, which further form secondary
ions. These secondary ions then react with the sample and
protonate it to produce protonated species. Chemical ioni-
zation (CI) is also used for volatile organic compounds.

1.1.3.3 Fast atom bombardment
Fast atom bombardment ionization technique is
employed for less volatile large organic compounds like
peptides, proteins, and carbohydrates. Sample is mixed
with nonvolatile matrix usually glycerol or nitrobenzyl
alcohol or crown ethers, and argon or xenon gas is then
used to bombard the immobilized matrix. This bombard-
ment generates charged sample ions, which are then
focused into the analyzer.
1.1.3.4 Matrix assisted laser desorption
ionization
It is similar to fast atom bombardment, wherein the sample
is mixed with the matrix compound, which is capable of
absorbing UV light. Excess fold of matrix is mixed with the
sample using a solvent, which is then evaporated and finally
cocrystallized analytes with crystal matrix are obtained.
Short pulse of UV laser beam is then targeted on solid
matrix resulting into ionization of samples. Due to energy
absorption the matrix gets partially vaporized and hence car-
ries analyte molecules in ionized form along with it in gas
phase. During this process matrix and analytes exchange
protons producing positively charged molecules [3].
1.1.3.5 Electrospray ionization
Electrospray is the softest ionization method, where it
maintains the noncovalent interactions of a macromole-
cule in their gas phase. It is also termed as ion spray, sonic
spray, or nanospray. Sample is passed through a high
potential narrow electrical capillary resulting in an electro-
static spray composed of multiple charged droplets.
Further these droplets pass under a heated capillary where
solvent gets evaporated and gets exploded into microdro-
plets, wherein charged ionized analytes get separated from
the microdrops that escape towards the analyzer. The
major advantage of electrospray techniques is that ions
acquire multiple charges depending on their molecular
mass and hence peptides and proteins get successively
protonated resulting into multiple peak spectra due to
different m/z ratios. It is useful for determining the molec-
ular mass in low resolution spectrometers. Electrospray
ionization (ESI) sensitivity is impoverished by miniatur-
ized ESI sources, whereby the capillary opening size can
be reduced and flow rate can be minimized [4].
1.1.3.6 Fragmentation
Molecular ions formed during the ionization process can
further be fragmented upon bombardment with higher
energy electron beam resulting the formation of
Mass spectroscopy Chapter |1|
fragments. Lowest ionization potential fragments retain
the positive charge. Thus, a molecular ion upon fragmen-
tation produces a radical and a cation; the latter is
detected in MS. This chemical dissociation may be due to
homolytic or heterolytic cleavage of single bond in molec-
ular ion. Fragments resulted from fragmentation of molec-
ular ions provide information about the structure of a
molecule by analyzing the peaks generated in mass spec-
tra. Fragment size and its frequency depend on the bond
energy and the structure of the analyte molecule.
Factors important for fragmentation:
1. Stability of the bonds
2. Energy of the molecular ion and the fragments
3. Steric factors during rearrangement
4. Stevenson’s rule: If the two fragments produced from a
molecular ion compete for cation generation then the
ion with lowest ionization energy will be formed. Also
the largest fragment is lost in a branched radical
cation.
AB 22 22 2 A 1 B1
Or AB 22 22 2 A1 1 B
The fragmentation pattern depends on the functional
group present in the compound.
Types of fragmentation:
1. Collision induced dissociation (CID): Molecules are
allowed to collide with neutral gases leading to
breakage of bonds and fragment ion formation. CID
fragments are produced in triple quadrupole
spectrometer.
2. Electron capture dissociation (ECD): A protonated
molecule is subjected to low energy electrons resulting
into production of an odd electron ion. ECD
fragmented gas phase ions are used in tandem mass
spectroscopic analysis.
3. Electron transfer dissociation (ETD): Fragmentation is
done by transferring electrons to cations, as carried
out for proteins and peptides.
4. Photo dissociation (PD): It is a chemical reaction
where photons induce fragmentation of chemical
compounds. Dissociation can be done using multiple
infrared photons or carbon dioxide laser.
5. Surface induced dissociation (SID): Molecular ions in
gas phase undergo fragmentation due to collision with
a surface under high vacuum.
6. Charge remote fragmentation (CRF): A gas phase ion
undergoes covalent bond breaking and cleaved bond
is not next to the charge atom. Such fragmentation is
used in tandem mass spectrometry.
7. High energy C-trap dissociation (HCD): This
technique is applied in peptide modification analysis.
HCD modification generates immonium ions.
3
 Data Processing Handbook for Complex Biological Data Sources
1.1.4 Analyzers
Analyzers resolve and differentiate the ions based on their
m/z ratio and this separation is driven by electric or mag-
netic field. All mass resolved ions are focused on a single
focal point. Desirable features of analyzer are:
1. Mass range is maximum permissible m/z ratio.
2. Mass accuracy is expressed as parts per million (ppm)
and determines how close is the measured mass to the
accurate mass.
3. Resolution of MS is its ability to resolve molecular
species with similar but distinct masses. Resolution is
calculated by dividing the peak value of m/z with its
width at half maximum intensity.
4. Efficiency is the transmission multiplied by the duty
cycle.
5. Sensitivity is the minimum concentration detected by
the spectrometer.
6. Linear dynamic range.
The instruments can have two analyzers coupled
together for mass analysis and such spectrometers are
termed as tandem MSs, which we will discuss in the fol-
lowing section.
Types of analyzers:
1. Time of flight (TOF)
2. Quadrupole (Q)
3. Ion trap (LIT & QIT)
4. Fourier transform ion cyclotron resistance (FT-ICR)
5. Orbitrap
6. Tandem MS
The analyzers vary in terms of mass range, sensitivity,
resolution, dynamics, and performing tandem mass spec-
troscopy experiments. In the following section we will dis-
cuss these analyzers and tandem instruments.
1.1.4.1 Time of flight
It is the simplest mass analyzer, which consists of a flight
tube placed in high vacuum. Ionized molecules fly
through the flight tube with different velocities depending
upon their mass, where mass is inversely proportional to
the velocity. In a TOF analyzer, time of the flight to the
detector is measured, where ions with lower mass will
reach the detector earlier than higher mass ions. Flight
time may vary from 1 to 50 μs. The TOF analyzer has sev-
eral advantages: (1) all ions will reach the detector eventu-
ally depending on the TOF and (2) it can detect ions with
very high mass range. The major drawback of TOF is the
varying resolution due to different kinetic energies of ions
entering the linear tube. To overcome this problem an
electrostatic ion mirror, also known as a reflectron, is used
4
in TOF analyzers, which improves the resolution by nar-
rowing down the kinetic energy distribution of ions due
to deeper penetration of faster ions. Resolution is also
improved by increasing the flight time with increase in
tube length.
The majority of the TOF spectrometers are linked to the
MALDI for source of ions. The current MALDI-TOF instru-
ments can achieve peak mass resolution of 25,000 FWHM
order or detect macromolecules with mass range of mega
dalton. This technique is suitable for peptides, proteins,
polynucleotides, lipids, and polysaccharides [3,4].
1.1.4.2 Quadrupole
It consists of four rods parallelly arranged in a hyperbolic
section, whereby two opposite rods are connected to
direct current (DC) and other two opposite rods are
linked to radio frequency (RF) alternate current (AC).
Opposite rods have DC voltage and other two have RF
voltage 180 degrees out of phase thus generating an elec-
trically oscillating field. When ions are pulsed from the
ionization chamber toward the quadrupole, the positive
ions will move toward the negative rod but due to chang-
ing polarity, the ions take up a complex trajectory. Thus
trajectory of ions is regulated by varying the DC/Rf volt-
age. The ions within a narrow range of m/z will take up
stable trajectory in the quadrupole ultimately reaching the
detector. Ions with improper trajectory collide with
the rods and will not reach the detector. By manipulating
the voltage and frequency, the ions with different m/z
ratios can be transmitted to the detector. In the RF mode
where DC is zero, most ions acquire stable trajectory and
pass through the quadrupole. In the scanning mode RF
and DC simultaneously vary to permit ions with different
m/z ratio to pass through the quadrupole sequentially.
The third mode is to stabilize the voltages thus permitting
stable trajectory for ions with predetermined m/z ratio.
Major disadvantages of the quadrupole are the limited
mass range up to 4000 Da, lower resolution and decreased
ion transmission with increased m/z ratio. The quadru-
pole’s ability for MS/MS analysis can be enhanced by
attaching another analyzer to it. Quadrupole when cou-
pled with ESI ion source determines the molecular weight
of proteins and nucleic acids.
1.1.4.3 Ion trap analyzer
As the name indicates, ions are trapped in a long tube and
can be accumulated during the time of operations in the
instrument. The conventional ion trap analyzers are quad-
rupole ion trap (QIT) or 3D IT, comprising of a ring elec-
trode and two endcap electrodes. These three electrodes
are arranged in a manner to create a cavity for trapping

ions and analyzing them. RF voltage and electrodes are
designed in a way to generate stable trajectories of ions,
which remain entrapped in instrument for seconds to
hours. Increasing the RF voltage destabilizes the ion
trajectory and ions are ejected to the detector. Besides
MALDI-TOF, QIT is the most common MS instrument for
proteomic studies. When coupled to ESI source QIT has
high sensitivity for peptide analysis and is popularly used
in proteomics. Generally with QIT both full scan and
product scan are carried out at high scan speed and low
resolution. Nevertheless good resolution can be obtained
at lower scan speed when only short m/z is to be consid-
ered. A disadvantage of QIT mass analyzers is low resolu-
tion, limited ion trapping capacity, and low sensitivity due
to space charging effect resulting in lack of accuracy in
mass measurement [3,5].
Linear ion trap (LIT) mass analyzers have been developed
recently to overcome the drawbacks of QIT, expanding the
sensitivity and dynamic range of the ion trapping tech-
nique. LIT or 2D ITs have higher ion trapping capacity
where 50 times more number of ions can be trapped, and
have higher trapping efficiency of one order higher magni-
tude than QIT. LIT is comprised of quadrupole rods with
RF voltages and end with DC arranged to form a cavity for
trapping ions. These instruments have higher resolution
with incorporation of optional slow scanning mode. Triple
quadrupole instruments with a second analyzer substituted
with LIT results in a Q-Q-LIT MS/MS instrument, which has
higher scanning speed and sensitivity and is also capable of
posttranslational modifications like phosphorylation.
1.1.4.4 Fourier transform ion cyclotron
resistance
The ICR MS is composed of four electrodes placed in a
magnetic field such that a Penning trap is created where the
electric field is perpendicular to the magnetic field. The ions
get trapped in the Penning trap and oscillate with cyclotron
frequency. Cyclotron frequency is inversely proportional to
m/z ratio and directly proportional to magnetic field inten-
sity. The signal is detected in form of image current on the
detector plates and this signal is termed as free induction
decay (FID), which has frequency equal to the cyclotron fre-
quency of ions. Useful information or data is extracted
from this signal by using a mathematical equation known
as Fourier transform (FT), thus yielding mass spectra. FT-
ICR can detect molecules with low ppm to sub ppm range
thus improving on mass accuracy measurement. Currently
it is the only MS with highest resolution of approximately
106 values and thus intact undigested proteins can be stud-
ied. Higher resolution also corresponds to increased peak
capacity and hence more signals can be detected.
Mass spectroscopy Chapter |1|
1.1.4.5 Orbitrap analyzer
Orbitrap analyzer works on the principle where ion sepa-
ration occurs in an oscillating electric field. Orbitrap mass
analyzer has inherited key features of previous analyzers,
like electrostatic field adapted from TOF, ion trapping
within the electrodes from ion trap analyzers, and image
current measurement from FT-ICR. Mass accuracy mea-
surement and resolution of orbitrap analyzer is similar to
FT-ICR but overcomes the limitation of expensive super-
conducting magnet. Orbitrap analyzer is comprised of
three electrodes, where the outer two electrodes face each
other in the form of cups and are separated by a dielectric
central ring. The central electrode is spindle shaped and
holds the trap and aligns it towards the dielectric end
spacers. The radial electric field between the outer and
central electrode and the centrifugal force pushes the ion
to take up harmonic axial oscillations. Outer electrodes
serve as receiver plates, which detect axial oscillations in
form of image current detection, and then further signal is
transformed similar to FT-ICR into mass spectra [6].
1.1.4.6 Tandem mass spectrometer
When two or more than two mass analyzers are connected
in sequence to reveal in-depth information about the
molecular structure of analyte then such instrument is
termed as tandem MS or MS/MS instrument. Mass
analysis of analyte is carried out in two consecutive stages,
wherein the first analyzer performs ion isolation, then
fragmentation induced by CID in collision cell, and finally
followed by separation of fragmented ions based on m/z
ratio in the final analyzer. Thus there are two scanning
modes: the first is parent scan mode in the first analyzer,
which detects the precursor ion of interest; and the second
is product ion scan or MS/MS scan or fragmentation scan
where fragmented ion is detected. Tandem MS can be of
two types:
1. Tandem-in-space
2. Tandem-in-time
Tandem-in-space are MS/MS instruments where analysis
is performed in different space, that is, mass analyzers.
Triple quadrupole (QqQ or TQ) is the common tandem-
in-space MS/MS instrument where Q denotes two quadru-
pole analyzers and q denotes the collision cell. These
quadrupoles mostly operate in rf mode, specifically the
collision cell q. The third analyzer in TQ when replaced
with other mass analyzers results in hybrid tandem MS
like Q-TOF and Q-LIT MSs. Q-TOF combines the positive
features of two analyzers: the scanning power of quadru-
pole and resolving capacity of TOF analyzer. The advan-
tage with Q-TOF analyzer is simultaneous monitoring of
5
 Data Processing Handbook for Complex Biological Data Sources
several ions, in contrast to single ion checking in TQ.
Thus Q-TOF provides higher sensitivity and resolution of
one order higher magnitude than the TQ instrument.
Tandem MS is mostly coupled to ESI or MADLI for ioniza-
tion source. Such instruments provide good quality of
fragmentation spectra resulting in protein identification.
Tandem MS instruments are very useful when protein
cannot be identified by peptide mass fingerprinting
(PMF). QTRAP spectrometer has been developed for espe-
cially for analysis of posttranslational modifications in
proteins.
Tandem-in-time are trapping MS instruments that eject
all ions except for one m/z that gets fragmented in same
space. Ion trap quadrupole and FT-ICR MS is such an
instrument that performs multiple MS experiments (MSn)
in a single analyzer.
1.1.5 Detectors
Basically there are four types of detectors used in MSs:
1. Faraday cup
2. Electron multiplier
3. Photomultiplier
4. Microchannel plate
1.1.5.1 Faraday cup
The dynode consists of secondary emitting materials such
as GaP, BeO, and CsSb. The principle of the Faraday cup
is that when incident ions or ion beam strikes the dynode,
electrons are emitted that induce current, which is ampli-
fied and recorded. Such detectors are suitable for isotope
analysis.
1.1.5.2 Electron multipliers
Dynodes are made up of copper-beryllium. Electron
multipliers are especially useful when both positive and
negative ions have to be detected. The principle involves
the emission of electron from first dynode upon trans-
ducing initial ion current, which is then focused to the
next dynode and so on. This cascade of current is finally
amplified a million times and recorded.
1.1.5.3 Photomultipliers
The dynode is made up of a substance called the scintilla-
tor, which emits photons. Light emitted from the dynode
is converted into electric current by a photomultiplier
tube and current is recorded. Photomultiplier detectors
are suitable to study metastable ions.
6
1.1.5.4 Microchannel plate
It comprises of an array of miniature electron multipliers,
which act as a continuous dynode. The channels are com-
prised of a special glass with coating of high resistance
semiconductor and these semiconductors function as
electron multipliers. The multiple electrons emitted from
the channel produce a pulse equivalent to 106 electrons,
which is emitted from a small spot at the rear end of
microchannel plate and generated as the output and
ultimately recorded.
The overall workflow of a MS is depicted in Fig. 1.2.
1.1.6 Mass interpretation
Based on mass spectra obtained from peptide analysis,
protein identification is done by mainly three approaches:
1. Peptide mass fingerprinting (PMF)
2. Peptide fragmentation fingerprinting (PFF)
3. De novo peptide sequencing
Before studying the details of these approaches for pep-
tide identification we will have a brief look into the peaks
obtained in mass spectrum and the fragmentation rules.
1.1.6.1 Types of peaks
1. Molecular ion peak: It represents the molecular ion
produced when sample is bombarded with 9 15 eV
energy electrons.
2. Fragment ion peak: Molecular ions, when further
bombarded with high energy electron of up to 70 eV,
result in fragment ions, which correspond to fragment
ion peak.
3. Rearrangement ion peak: it is the representation of
recombination of fragment ions.
4. Metastable ion peak: decomposition of analyte
between source and magnetic analyzer results in
formation of metastable ions, which correspond to
broad peaks known as metastable ion peaks.
5. Multicharged ion: Some ions are formed with more
than one charge and are represented as multicharge
ion peaks.
6. Base peak: the largest and most intense peak in the
mass spectrum is known as the base peak.
7. Negative ion peak: capture of electron by a molecule
during electron bombardment results in negative ion
formation, which forms negative ion peak in the
spectrum.

1.1.6.2 Fragmentation rules
There are nine rules to analyze fragmentation peaks in the
mass spectrum.
Rule 1: Increase in degree of branching results in
decrease in M 1 peak height.
Rule 2: Increase in molecular weight decreases M 1
peak height.
Rule 3: M 1 peak is stabilized by aromatic rings, cyclic
structures, and double bonds.
Rule 4: Allylic cleavage favored by double bond results
in resonance stabilized cation.
Rule 5: Cleavage occurs at alkyl substituted carbon
resulting in carbocation formation,
Rule 6: Alkyl side chain in saturated ring is removed
while retrodiels alder reaction occurs in unsaturated rings.
Rule 7: Aromatic compounds are preferably cleaved at
β-bond resulting in resonance stabilized benzyl ion.
Rule 8: C C bonds next to hetero atom (O, NH, or S)
are cleaved frequently, producing charged hetero atom,
which is stabilized by resonance.
Rule 9: There has been elimination of small neutral mole-
cules during cleavage of bonds along with rearrangements of
atoms. This is known as McLafferty rearrangement.
1.1.6.3 Peptide mass fingerprinting
PMF is the most commonly used strategy to identify proteins
and protein expression pattern in biological samples. The
Mass spectroscopy Chapter |1|
Figure 1.2 Outline depicting the
workflow of mass spectrometer.
mass profile obtained after MALDI-TOF MS is compared
with the predicted mass values obtained from the in silico
digestion of all proteins present in the database. Common
search programs used in PMF are MASCOT, PROFOUND,
MSFIT, and ALDENTE, which can be easily accessed through
the Internet. Four or five peptide mass matches are sufficient
to identify the correct protein hit, covering 15% of the
sequence and mass accuracy better than 30 ppm. Successful
protein identification depends on the following factors:
1. Peak mass accuracy
2. Assigned and unassigned peaks relation in the
spectrum
3. Database size for analysis
Drawbacks to PMF:
1. Masses of the experimental peak might not match
with the peptide masses of the theoretical proteins
2. Not all peptide masses are generated theoretically
upon in silico digestion in the database [3,7].
1.1.6.4 Peptide fragmentation
fingerprinting
This approach uses low energy collision induced fragmen-
tation (CID) for peptide ions fragmentation in gas phase
in MS/MS instrument. Such fragmentation occurs mainly
at peptide amide bonds and yield two types of fragments:
7
 Data Processing Handbook for Complex Biological Data Sources
β ion, which preserves N-terminus, and γ ion with C-
terminus. The fragmentation spectra obtained is compared
with the search programs containing the predicted spectra
of peptides after in silico digestion of all available proteins
in the database. MASCOT and SEQUEST are the programs
used to analyze the CID fragmentation spectra and iden-
tify the peptides and proteins. A single peptide can iden-
tify a protein match but a greater number of peptide
matches with one protein increases the sequence coverage
and increased probability of correct match. Unfortunately
the disadvantage for peptide fragmentation spectra analy-
sis is that this approach is not applicable if there is any
mass difference because of certain modifications or if
there is no match to the peptide in the database as the
protein is not reported in that particular database.
1.1.6.5 De novo peptide sequencing
De novo peptide sequencing is reconstitution of the
peptide sequence from the fragmentation spectra based on
peptide fragmentation rules. It is a straightforward
approach for protein identification when protein identifi-
cation becomes elusive. This task was traditionally done
by Edman’s sequencing but with the advent of the latest
technologies and bioinformatics tools, the data produced
is searched for protein homology using BLAST tool.
In our next section we have discussed raw data proces-
sing, focusing mainly on proteins.
1.2 Raw data
The last 10 years has seen a significant growth in proteo-
mics study. The credit goes to the development of compel-
ling protein profiling technologies, which has led to
research in protein biomarker discoveries of various illness
and diseases thereby making a remarkable improvement
in the diagnostic and therapeutic field. Proteomics, in a
broader sense, involves study of a specific proteome,
validation of information about protein abundances, its
modifications along with protein protein interactions,
and its interacting partners. These kinds of studies come
under discovery proteomics, which involves two kinds of
approaches, namely, top-down and bottom-up
approaches. The former involves protein characterization
by MS without undergoing proteolysis, thus retaining
mass ranges to a greater extent in the case of PTMs, detec-
tion of “native” molecular mass of proteins, and greater
sequence coverage. Unlike top-down, bottom-up
approach involves proteolytic digestion of crude protein
samples before running in MS. The profiles of these runs
are acquired simultaneously by a PC coupled with MS.
8
The method of data acquisition can be classified into
data-dependent acquisition and data-independent acquisi-
tion. Data-dependent acquisition involves selection of cer-
tain peptides to be fragmented at the MS/MS level based
on specified standards. Data-independent acquisition
involves selection of all peptides within a certain mass
range at MS/MS level. After data acquisition, raw files are
taken for protein/peptide identification and quantification
using software, like TPP, which employs search engines
like SEQUEST, OMSSA, Mascot, etc., and the quantified
proteins are statistically analyzed using statistical software
like R, Perseus, etc.
We will now discuss the nuances of Nano-ESI LC-MS/
MS with Orbitrap technology followed by data-dependent
acquisition, Trans-Proteomic Pipeline (open-source for
protein/peptide identification and quantification), label-
free quantification, and various Annotation Software
tools.
Nano-ESI LC-MS/MS: It is a powerful setup that com-
prises of nanoscale LC paired with tandem mass spec-
trometry. unlike the traditional LC-MS/MS techniques,
this setup provides high sensitivity and nanoflow UHPLC
performance.
1.2.1 NanoLC principle
The term “nanoLC” refers to flow rates in the nL min 1
range (as opposed to high μL min 1 to low mL min 1
range for regular HPLC). NanoSpray mass spectrometry
with nanoLC flow rate and concentration-sensitive detec-
tors usually separates a minute amount of samples at
microscale. A typical nanoLC/MS/MS setup will be a 75-
μm I.D. column at a flow rate of 200 350 nL min 1. In
recent times, gel-free analytical approaches based on LC
and nanoLC separations have been developed, leading to
faster and more extensive proteomics-based studies than
the classical 2D gel electrophoresis approach.
It is here that the biological samples, which are proteo-
lytically digested using an enzyme and broken down to
peptides, are injected into liquid chromatography
(HPLC), strategically forming a gradient along with the
injected sample and getting eluted into the MS.
1.2.2 Gradient
Gradient method is employed for those samples that have
wide “k” range and hence cannot be separated using
isocratic methods. Gradient in reversed-phase HPLC is
specified by three essential parameters: initial %B, final %
B, and gradient time (tG) over which the transition in
eluotropic strength will be achieved. The most frequent
questions in gradient HPLC are asked about how to

Figure 1.3 Diagrammatic representation of different steps involved in the gradient elution.
decide upon the optimum values for these three para-
meters, and it is conventional to begin method develop-
ment with a “scouting gradient” as the eluotropic strength
increases from a low to high value over a set period,
typically 5% 95% B over 10 or 20 minutes. The optimum
operating conditions can then be assessed by the analyte
elution behavior during this scan (Fig. 1.3).
After forming a gradient, the sample reaches the MS and
undergoes three main stages:
1. Ionization: An ESI source acts as a junction to couple
LC with MS. The sample or analyte mixed with
solvent, in its liquid state pushes through a capillary
to form a jet of aerosol (miniature droplets of 10 μm),
which is further nebulized by nebulizer gas like
nitrogen. During nebulization, the solvent evaporates
and the analyte ions happen to undergo fission, and
these ions enter the MS to be separated by their m/z
ratio. For nanoLC, nanospray ionization (NSI) source
is used where the sample elutes from a nano-ESI
emitter.
2. MS1 scan, where m/z ratios are measured and
abundances are calculated. Here, a specific m/z range
is selected from the precursor ion.
3. MS2 scan, where fragmentation of ions takes place and
m/z ratios are measured and detected, yielding
fragment ion spectra. Here, product ions are captured
for detection.
1.2.3 Orbitrap technology
LTQ Orbitrap and LTQ Orbitrap Velos represent a consoli-
dation of two mass analyzers, a LIT and an Orbitrap mass
analyzer. It offers up to three fragmentation approaches:
CID or ETD in the bifold LIT, or HCD in its devoted
collision cell [8]. The fragment ions generated by CID or
Mass spectroscopy Chapter |1|
ETD are normally analyzed in the ion trap at low resolu-
tion, along with parallel acquisition of the MS transient.
Nonetheless, they can also be conveyed to the C-Trap and
registered in the Orbitrap analyzer. HCD fragments are
always analyzed in the Orbitrap analyzer [9].
1.2.4 Data-dependent acquisition
This is the basic mode of data acquisition used in LC-MS/
MS related experiments across various biological samples,
for protein identification and relative quantification. Here
peaks are identified based on preset values from a survey
scan and are further taken in for MS/MS analysis.
After MS1 survey scan, we get main base peak with max-
imum intensity, which corresponds to the protein of inter-
est. As shown in Fig. 1.4, the peak with m/z value of
1424.6 is the base peak (mother peak). Peptide masses are
determined from the LC-MS spectrum, and further, for
MS/MS analysis or MS2 scan, the precursor ion of desired
peak (each peptide) is subjected to fragmentation. Ions
with certain m/z values are only further subjected to frag-
mentation. There is a list of m/z values in-built in the soft-
ware on the basis of which certain ion peaks will be
eliminated from further analysis. Fragment ion spectra are
obtained resulting in daughter peaks, where the last peak
corresponds to m/z value of base peak 1424.6, as seen in
Fig. 1.5. Peptide fragment ion mass is determined for each
peak obtained from MS/MS spectrum. Peptide fragment
ion mass is then coupled with the peptide mass of LC-MS
spectrum and subjected to peptide scoring through data-
base search. Protein is then identified from the correctly
identified peptide scoring.
In this chapter we have also discussed interpreting the
mass spectrum of phosphoprotein. Below is an example
of mass spectrum of a nonphosphopeptide and a phos-
phopeptide. Peptide mixture of Mycobacterium kinase
9
 Data Processing Handbook for Complex Biological Data Sources

Figure 1.4 Mother peak obtained after mass spectrometry (MS1 scan).

Figure 1.5 Daughter peak obtained after MS/MS fragmentation analysis.

10
 Mass spectroscopy Chapter |1|

Figure 1.6 MS/MS spectrum of nonphosphopeptide during HCD fragmentation.

Figure 1.7 Peptide (nonphosphopeptide) summary and fragment matches using database.

was resolved and MS data acquired. Figs. 1.6 and 1.8 rep-
resent the mass spectrum of nonphosphopeptide and
phosphopeptide respectively. MS data was acquired using
a data-dependent top 10 method dynamically choosing
the most abundant precursor ions from the survey scan.
The raw file was then analyzed with Proteome Discoverer
1.8 (Thermo) against the Uniprot Mycobacterium
Tuberculosis database. For MS Amanda search, the precur-
sor and fragment mass tolerances were set at 10 ppm and
0.5 Da, respectively. The protease used to generate

11
 Data Processing Handbook for Complex Biological Data Sources
Figure 1.8 MS/MS spectrum of a phosphopeptide.
Figure 1.9 Peptide summary and fragment matches of a phosphopeptide using database.
peptides, that is, enzyme specificity was set for trypsin/P
(cleavage at the C-terminus of “K/R: unless followed by P”)
along with maximum missed cleavages value of two.
Carbamidomethyl on cysteine as fixed modification and
phosphorylation at S, T, Y, oxidation of methionine and N-
terminal acetylation were considered as variable modifica-
tions for database search. Both peptide spectrum match and
protein false discovery rate were set to 0.01 FDR (Fig. 1.7).
12
Fig. 1.8 represents MS/MS spectrum of precursor m/z
765.71613 (13) and MH1 2295.15 Da, of the semitryptic
phosphopeptide VLTDAER (pT) SLLSSAAGNLSGPR,
where pT indicates the site of phosphorylation. The
unambiguous location of the intact phosphate group was
determined by the presence of the “B” and “Y” ion series
containing B9 12, B16, B18 and Y15, Y18, Y20, Z18
(Fig. 1.9).

Figure 1.10 Trans-Proteomic Pipeline workflow.
http://www.socr.umich.edu/CSCD/html/Cores/Macore2/Macore2_2015.html.
1.3 Data processing
1.3.1 Trans-proteomic pipeline
This online software provides a global platform to per-
form various tasks from MS/MS raw file conversion
using MSConvert GUI to peptide and protein-level
identification and quantitation. It is open source and
provides tools that can be run individually or can be
used as a conduit to perform various tasks, and are easy
to follow for analysis and validation of the dataset
(Fig. 1.10). http://tools.proteomecenter.org/wiki/index.
php?title 5 TPP:5.1.0_Release_Notes
1.3.2 Installation
A newer version of TPP 5.1.0 is available for downloading,
for Windows users, at https://sourceforge.net/projects/
sashimi/files/. This version unlike one of its older
versions, namely TPP 4.8.1, does not require dependencies.
As for the Windows users, the older version of TPP has to
be uninstalled to install the newer version. TPP is available
for OSX users as well, but is not official yet. For Linux
users, follow the link for installation: http://tools.proteo-
mecenter.org/wiki/index.php?title 5 TPP:5.1_Installation
Steps for installation:
• Install the latest version of ActivePerl, preferably 5.9
or higher (www.activestate.com/activeperl/
downloads)
• Download the latest version of TPP and Run the .exe
file.
• Accept the license agreement and select a location to
install TPP and its components. “C:\TPP\” is set as
default and can be changed according to preference.
Mass spectroscopy Chapter |1|
• When asked for the port number, set default 10401.
• Select the driver location for the data.
• Choose all extra components to install:
• Apache web server
• ProteoWizard
• Download and install dependencies for
ProteoWizard
• Strawberry Perl
• Reboot after completing TPP Setup Wizard.

The whole installation process takes around 10 30
minutes. After installation, TPP Petunia interface will be
available on the Desktop or Start menu. Launch TPP icon
and log in when the browser window opens. For detailed
tutorial on TPP, follow the link: http://tools.proteomecen-
ter.org/wiki/index.php?title 5 TPP_Demo2009
The following details will shed some light on the work-
flow components and tools of TPP and its analysis. Also,
to get a better idea of how to perform the analysis, we
have included a demo tutorial analysis, which contains
the raw files and files of other formats derived out of TPP
tools ( data from sourceforgenet ). Log in to TPP
GUI and add the mzML files CONTROL.mzML and
TREATED.mzML.
1.3.3 Raw file conversion
File format conversion and further analysis of data can be
well understood by the flow diagram (Fig. 1.11).
Depending on the instrument type, the formats of raw
files generated from MSs have to be first converted to an
open format mzXML or mzML. TPP uses the MSConvert
tool for this purpose.
Tutorial: Add the raw files downloaded from sourcefor-
genet to the subdirectory, which directs to the drive where
the analysis folder will be located.
13
 Data Processing Handbook for Complex Biological Data Sources

Figure 1.11 Schematic workflow of “Downstream processing” file format conversion.

1.3.4 Search engine support
The MS/MS raw files can be processed initially by match-
ing spectra (hypothetical spectra estimated from sequence
database) with extracted peptide sequences. TPP supports
search engines like X!Tandem, Mascot, OMSSA, SEQUEST,
ProbID, and Phenyx, of which only X!Tandem is available
with TPP. The rest need to be installed individually. The
raw files can also be processed by matching spectra using
spectral libraries (existing spectral library), which is the

14
 Mass spectroscopy Chapter |1|

preferred method to obtain a good result, in comparison 1.3.5 PeptideProphet for

with hypothetical spectra match. corroboration
Some of the open source database search algorithms are
given below: PeptideProphet is a software component developed to per-
form validation of peptides allocated to MS/MS
1. Andromeda (part of MaxQuant) spectra. One of its features includes employing estimation
2. Comet maximization algorithm to calculate PSM probability.
3. Tide (rewrite of Crux)
Additionally, it can also provide information on mass
4. Greylag
deviation, missed cleavages, and retention time. In other
5. InsPecT
words, it assigns probabilities that peptides assigned to
6. MassMatrix
7. MassWiz
MS/MS spectra are true. This option can be set to avoid
8. MS-GF 1 information on decoy for separate evaluation at the end
9. MyriMatch result. The resulting pepXML format file can be analyzed
10. OMSSA with PepXMLViewer Application. http://peptideprophet.
11. pFind sourceforge.net/
12. ProbID Tutorial: Go to “Analyze Peptides” option in TPP tools
13. ProLuCID to add the mzML files and set the default options avail-
14. Protein Prospector able for PeptideProphet and then run analysis.
15. SIMS Final result of PeptideProphet can be viewed using
16. SimTandem PepXML viewer as shown below.
17. SQID
18. X!Tandem

Tutorial: For this tutorial, we have used Comet Search 1.3.6 Pep3D to view chromatogram
Engine coupled with Human database, which has been To view the quality of sample run in MS, Pep3D
provided in the directory. While carrying out search, the allows the user to view chromatograms from various
process will look like this: LC runs.

15
 Data Processing Handbook for Complex Biological Data Sources
Tutorial: PepXML Viewer has a feature to view the chro-
matogram of sample. Go to Other actions menu, the last
option located in the viewer and click on “Generate 3D”
option with defaults set as it is.
1.3.7 iProphet for peptide-level
corroboration
iProphet provides the rightly identified peptide sequences
with high posterior probability and FDR by assimilating
16
same documented peptide sequences of the same data
searched via various search engines. It also couples the
information collected about the same peptide spectra
across various experiments, other diverse spectra, precur-
sor ion charge, and modified states, thereby creating a
multifaceted model to corroborate the peptide. This
further increases the confidence at which the peptide is
being scored with high confidence. It is because of
such profound analysis done at peptide level that this soft-
ware tool is considered to be more effective than
PeptideProphet or Percolator [10].
 Mass spectroscopy Chapter |1|

Tutorial: To further validate the peptides statistically, go
to “Refine/Combine Analysis” option in TPP tools menu
and uncheck all other Search parameters except that of
iProphet and upload the pepXML files. As a result a
detailed and much refined group of peptides are obtained
in this process. The result can be viewed using PepXML
viewer. As PSMs are calculated for each peptide, the
quality of individual ion spectra can be viewed when
clicking on “ions” of each peptide, as shown below:
1.3.8 XPRESS/ASAP ratio for peptide
quantitation
XPRESS tool quantifies ratios of differentially labeled
proteins computing relative abundance of proteins. The
precursor ions are remodeled from light and heavy elution
profiles, where the area of each peak is found. It also cites
labeled residues and determines mass difference between
two isotope labels.

17
 Data Processing Handbook for Complex Biological Data Sources
ASAP Ratio (Automated Statistical Analysis on Protein
Ratio) on the other hand is a tool that uses a refined way
to measure and accumulate peptide ions at the peptide
and protein level, thus generating a list of proteins based
on their relative abundance. It also sets another list of
proteins that will show PTMs. These two tools are used in
case of heavy- and light-labeling techniques like ICAT or
SILAC. https://omictools.com/xpress-tool
However in the case quantitation to be performed from
isobaric labeling techniques like iTRAQ, relative abundances
have to be calculated using relative peak intensities of the
reporter ion. Another tool called Libra performs quantita-
tion for analysis of this kind. Some of the open source MS/
MS peptide quantitation software is given below:
1. MassChroQ
2. MaxQuant
3. OpenMS/TOPP
4. ProtMax
5. Skyline
6. BACIQ
1.3.9 Protein prophet for protein
interpretation and corroboration
Protein Prophet, like PeptideProphet, confirms that the
proteins based on peptides assigned to MS/MS spectra are
true. It mainly directs to less-likely “single hit” proteins and
their corresponding peptides and looks upon the
peptide probabilities to group the proteins accordingly.
Also, it helps the peptides to be allocated to their respective
18
proteins in case of multiple databases used. The result is
produced in protXML files, which are in HTML format.
Tutorial: After deriving result from iProphet in pepXML
format, this is used further to group proteins corresponding
to peptides to which they belong. For this, go to “Analyze
Proteins” option in the TPP tools menu and add pepXML
files to be analyzed. Check on the “Input is from iProphet”
option in ProteinProphet and run Protein Analysis. The
result will be of protXML format, which can be viewed
using View option. The final result looks like this:

After deriving protXML file, it can be further used to
perform Label-free quantification using “Quantify Label
free (MS2)” option in TPP tools menu. Label-free quantifi-
cation is discussed further in this chapter under Protein
quantification.
1.4 Protein quantification and
identification
Recent progress on quantitative proteomics technology
has enabled researchers to observe quantitative differences
in different protein profiles under different experimental
conditions and to understand molecular functions of pro-
teins along with their modifications.
1.4.1 Label-free quantification
Label-free approach typically carries out detection of pep-
tides, peptide matching across multiple LC-MS data, and
Another random document with
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I’m sure. And while we are on the subject of room, Miss Fewkes, will
you let me know how much you will charge me for the bedroom only,
as I shan’t be wanting the sitting-room after this week.”
“Oh, but I don’t particularly care to let the bedroom by itself,” Miss
Fewkes objected. “I haven’t another bedroom vacant, and what use
would the sitting-room be to anybody by itself? Perhaps you’d prefer
to give up both rooms?”
Nancy hesitated. Then she plunged.
“Certainly, Miss Fewkes. I really wanted to give them up some
time ago. They’re very expensive and very uncomfortable, and not
overclean.”
“Well, I shan’t argue about it, Miss O’Finn,” said Miss Fewkes
haughtily. “Because I wouldn’t soil my lips by saying what I think of a
person who behaves like you do. But I do know a little about the
prerfession, having been in it myself, and if you are what you pretend
to be, which I don’t think, all I can say is the prerfession has changed
for the worse since my day.”
With this she snapped out of the room, as a little wooden cuckoo
snaps back into his clock.
Nancy sat down and wrote to Mrs. Pottage.

5 Blackboy Passage,
Soho.
Friday.
Dear Mrs. Pottage,
I’m rather worried what to do with Letizia while I am
looking for work. I wonder if you’d look after her for a week
or two? I have a very unpleasant landlady here who hates
children, and so I must get a new room until I find an
engagement. Don’t ask me to come to Greenwich too,
because, dearest Mrs. Pottage, I simply couldn’t. But
Letizia’s different, and if you wouldn’t mind having her with
you for a little while it would be such a weight off my mind.
You must please charge me whatever you think is fair. I
 haven’t the least idea, so I must leave that to you. I leave
here on Monday, and if you will let me know by then what
time will suit you I will bring Letizia to Greenwich station, if
you’d be kind enough to meet us. I haven’t told Letizia
herself that she may be going to stay with you, because I
don’t want to disappoint her if you can’t manage it, and of
course I will perfectly understand if you can’t.
Yours affectionately,
Nancy O’Finn.

Nancy felt more cheerful when she had posted this letter.
Early on Sunday morning the Kinos left Blackboy Passage.
“You won’t change your mind at the last minute and let us take the
kid?” Mr. Kino asked.
Nancy shook her head.
“It would be a weight off your shoulders, wouldn’t it?” he pleaded.
“Yes, but it would be a terrible weight on my mind,” said Nancy.
“Dear Mr. Kino, I couldn’t let her be adopted, I couldn’t really.”
Yet when the Kinos had gone, and Nancy was sitting by the
window, listening to the church bells and to the occasional footsteps
of people clinking along the frozen Sabbath streets and to the
emptiness of Soho without the distant roar of traffic, she began to
wonder if she ought not to have accepted the Kinos’ offer. She tried
to make up her mind to put on her things and take Letizia to the late
Mass in the Soho Square church; but the dejection reacted on her
energy, and she felt incapable of getting up from her seat, of doing
anything except stare out of the window at the grey March sky or
look with a listless resentment at Miss Fewkes’s pictures of girls in
sunbonnets cuddling donkeys over gates or of girls in furs feeding
robins in the snow. She even lacked the energy, when the morning
had passed and it was nearly two o’clock, to ring and ask when Miss
Fewkes proposed to serve dinner. And when dinner did arrive, with
everything cooked so badly as to make it nearly inedible, she did not
feel that she could be bothered to protest.
 “Muvver,” said Letizia, “why has my gravy got spots of soap in it?”
“Because it’s getting cold, my dear. So eat it up quickly before it
gets any colder.”
“But, muvver, when I put it into my mouf, it all sticks to the top of it
and won’t come off.”
“Don’t go on grumbling, there’s a good little girl. If you don’t like it,
don’t eat it.”
“Well, I won’t,” said Letizia decidedly.
A rice-pudding, which tasted like a dry sponge wrapped up in old
leaves, caused Letizia many sighs before she could swallow even a
mouthful, and some bananas which looked as if the greengrocer had
tried to reshape them after they had been driven over by the traffic of
Covent Garden all day, did nothing to help matters. As for the coffee,
it might have been smeared on a boy’s fingers to stop him from
biting his nails, but it was never meant to be drunk.
One of the reasons for Miss Fewkes’s perpetual bad temper was
an inclination on the part of visitors to the lower basement of the
house to ring Miss Fewkes’s bell and so fetch her downstairs
unnecessarily to open the front door. This being the second Sunday
of the month, Louisa had been allowed to go out, and Miss Fewkes
was in her tiny little bedroom in the roof of the house when her bell
rang twice. The idea of going all the way downstairs only to find that
a visitor had arrived for the people in the basement did not appeal to
the little woman. So she opened the bedroom window and, peering
out over the sill, perceived upon the steps below an exceedingly
bright cerise bonnet belonging to what was apparently a respectable
middle-aged woman.
“Who are you ringing for?” Miss Fewkes called down in her
rasping voice.
The cerise bonnet bobbed about for a while until at last it
discovered from what window it was being addressed, when it
looked up and shouted back:
 “What’s it got to do with you who I’m ringing for? If you’re the
servant here, just you come down and open the door the same as
what I would if anybody rung my front bell.”
“Do you want Mr. and Mrs. Blanchit?” Miss Fewkes called down.
“Because if you do, it’s the broken bell by the area gate and kindly
ring that.”
“Do I want who?” the cerise bonnet shouted back.
“Mr. and Mrs. Blanchit!”
“No, I don’t, you saucy old outandabout! What next are you going
to ask? You just come down and open the door the same as I should
myself.”
Miss Fewkes slammed her window down and left the cerise
bonnet on the steps. After ringing about a dozen times, it went down
the steps again and standing in the middle of the pavement shouted
“Hi!” several times in rapid succession. A small boy blowing a mouth-
organ stared at the cerise bonnet for a moment, stopped his tune,
and asked it if it had lost anything.
“What ’ud I be staring up at the top story of a house for, you saucy
little image, if I’d have lost anything—unless I’d dropped my umbrella
out of a balloon, and which I haven’t?... Hi!”
On hearing the cerise bonnet begin to shout again, the small boy
put the mouth-organ in his pocket and looking up in the air shouted
“Hi!” too. Two little girls dragging behind them a child of doubtful sex
smeared with barley sugar stopped to gaze, and then three more
small boys arrived on the scene and proceeded to augment the duet
of “Hi!”
A policeman, who had been lured from Dean Street into Blackboy
Passage by the noise, inquired of the cerise bonnet what its need
was.
“Can’t you get into your house, mum?”
“No, I can’t. I want to visit a lady friend of mine who lives at 5
Blackboy Passage, and when I rung the bell a female like a potted
shrimp poked her head out of a top-floor window and asked me if I
wanted Mr. and Mrs. Blanchit.”
“Mr. Blanchit lives at number five,” one of the small boys
volunteered. “Down in the basement, he lives.”
“Well, what’s that got to do with you, you pushing little eel? I don’t
want the man. I want to see my lady friend.”
“Perhaps you’ve got the wrong number,” the policeman suggested.
“Wrong number be ... well, I won’t say what I was thinking,
because it doesn’t always do.”
It was at this moment that Letizia, who had been trying vainly for
an hour after dinner to make her mother play with the gilded
antelopes, decided to look out of the window.
“Muvver! Muvver!” she shouted, clapping joyful hands. “I can see
Mrs. Porridge in the street, and she’s talking to a policeman.”
Nancy jumped up, and ran to the window.
“Why, so it is! Dear, dear Mrs. Pottage! I’ll go down and open the
door.”
“There you are!” Mrs. Pottage exclaimed triumphantly to the
policeman after she had embraced Nancy. “Didn’t I tell you my lady
friend lived here?”
The policeman strode off with a good-natured smile: the small boy
took the mouth-organ out of his pocket and, after watching the
policeman safely through the archway of the Tavern, resumed his
interrupted tune. The two little girls, without looking to see if the
sugar-smeared neutral was ready to be dragged on again, moved
forward on their way. The three other small boys discovered a new
method of wearing out boots and set off to practise it. Mrs. Pottage
and Nancy retired into Number Five. Blackboy Passage was once
more abandoned to its Sabbath emptiness and silence.
“Well, you do live in a Punch and Judy show and no mistake,” Mrs.
Pottage declared, as she followed Nancy up the stairs, the jet bugles
of her best bonnet tinkling and lisping as she moved.
 “My landlady doesn’t like being called down to open the door; and
the girl’s out,” Nancy explained.
“Landlady you call her? Skylady I should call her. That is if I called
her a lady at all, and which I most certainly never shouldn’t not if I
lived to be as old as Methussalem.”
Letizia was waiting at the head of the stairs to welcome Mrs.
Pottage, into whose outspread arms she flung herself in a rapture of
welcome.
“Mrs. Porridge! Mrs. Porridge!”
“My heart’s jool!”
“Oh, Mrs. Porridge, where have you been? I didn’t know where
you could be, and I went to a circus and touched an ephelant on his
trunk and it was all hot and I saw a little friend who the baby Jesus
liked and I saw my big grannie and the wolf didn’t eat her at all and I
saw two aunts and they smelt all funny like the inside of a dirty-
cloves basket and we had rice-pudding for dinner and it sticked my
teef togevver, and I’ve got two golden auntylopes and they eat
apples made of soap.”
“My good gracious, if you haven’t been going it,” Mrs. Pottage
declared, with a critical glance round the sitting-room of the lodgings.
“Poky! Very poky! And not at all clean. Why, that grate don’t look as if
it had been swept since the fire of London, and, oh, dear, oh, dear,
just look at the dust on those pictures! If a water pipe burst in this
house you’d have weeds growing on the frames. Well, I suppose I
haven’t got to tell you who I’ve come to fetch?”
Nancy smiled.
“I knew you wouldn’t fail me.”
“Yes, but wait a minute. I didn’t at all like the tone of that letter you
wrote me.”
Nancy looked worried.
“Not at all I didn’t like it. Yes, I see myself sending in a bill for that
blessed infant’s keep. Why, you might as well ask me to charge you
for the sun shining in at your windows.”
Nancy saw that she had genuinely hurt the good soul by
mentioning money in connection with Letizia’s visit.
“Dear Mrs. Pottage, you could hardly expect me to plant her down
on you without at least offering to pay, but I won’t offend you by
arguing further. You know exactly how I feel about your kindness, my
dear soul.”
“Kindness be ... oh, dear, now that’s twice in the last half hour I’ve
nearly said that word. It comes of keeping company with Mr. Currie.
Let me see, you won’t have heard of him, because he’s only been
courting me since I gave the go-by to Watcher and Hopkins. He’s a
very hasty-tempered man, and his language is a bit of a coloured
supplement. Mrs. Bugbird passed the remark to me I’d really have to
mind my p’s and q’s, and I said it wasn’t my p’s and q’s I had to look
out for, it was my b’s and d’s. Of course, Mrs. Bugbird herself didn’t
mind. Oh, no, she’s a very broad-minded woman. In fact her father,
so she’s often told me, used to preach regularly at street corners
against any kind of religion at all. But I shan’t keep this Currie
hanging around much longer. No, he gets me into bad habits, and
the next time he proposes marriage will be the last. Besides, even if I
liked him, I don’t like his business which is fried fish. Fancy me in a
fried-fish shop for the rest of my life! Why, I’d sooner marry an
engine driver and live in a railway station. Well, an engine driver did
propose to me once. But I saw he had the habit of driving too much,
and that would never have suited me. Why, even of a Sunday
afternoon he wasn’t happy if he couldn’t walk me round Greenwich
Park at sixty miles an hour. I remember once just for a joke I started
whistling the same as an engine might, and everybody stopped and
begun staring, and which made him a bit annoyed. In fact he thought
I was touched in my head, and that Sunday was his last. Well, he’s
the only one of all my many who didn’t wait for me to say ‘no’
definite, but went and hooked it himself. And going back to the
subject of my language this last month, it wouldn’t do at all if
Letichia’s coming home with me, so I think I’ll drop him a p.c. and not
wait for the third time of asking.”
 “Am I coming home with you, Mrs. Porridge?” Letizia asked,
clapping her hands.
“You’re coming home with me this blessed afternoon just as soon
as your dear ma’s packed up your tiddlies. Your friend Mrs. Bugbird
will be popping in, and we’ll have a sprat tea together. And dear
Aggie Wilkinson’s dancing about on her pore crutches, because
you’re coming home to your Mrs. Porridge.” She took Nancy aside,
and continued in a lower voice. “I read between the lines of your
letter, dearie, and I knew you didn’t want to come near Greenwich.
So I just skipped into my Sunday best and come along to fetch her.
She can stay as long as you like. I’d say she could stay for ever.
Only she wants a better bringing-up than what a woman like me
could give her.”
“Oh, but I’m sure to get an engagement very soon, Mrs. Pottage,
and then of course she’ll go on tour again with me. I wouldn’t have
bothered you now, if I hadn’t thought you’d be glad to have her for a
while, and if I hadn’t wanted to leave these rooms as soon as
possible.”
“And I don’t blame you. I’d sooner live in a dustbin. But where are
you going when you leave here?”
“Oh, I shall find somewhere to-morrow. I’m so glad you did come
to-day for Letizia. It will make it ever so much easier for me. The only
thing I’m worrying about is the luggage.”
“Well, why don’t you let me take what luggage you don’t want
down to Greenwich, and then when I bring you Letichia, I can bring
you your luggage at the same time. There’s no sense in travelling a
lot of luggage round with you like a peacock’s tail. We can just pop
what you don’t want into a four-wheeler and take it to London
Bridge.”
Nancy hesitated. She was wondering if she had enough money
left to pay the cab now, and Miss Fewkes’s bill to-morrow morning.
However, if she hadn’t, she could visit the pawnbroker early and
pledge some odds and ends, so she decided to accept Mrs.
Pottage’s offer.
 “Now who’s going to fetch the four-wheeler?” Mrs. Pottage wanted
to know when the packing was finished. “Shall I give a holler to Her
Landladyship upstairs?”
“Ask Miss Fewkes to fetch a cab?” Nancy exclaimed. “Why,
she’d....” Words failed her to express what Miss Fewkes would do.
“But what is this Miss Fewkes?” demanded Mrs. Pottage
indignantly. “Three ha’porth of nothing from what I could make out of
her. Still, rather than create a row on a Sunday afternoon I’ll go and
fetch the four-wheeler myself. I’ll stand in Shaftesbury Avenue till
one comes along. There’s one thing, the police won’t be so likely to
take me for a kerbstone fairy as what they would Lady Fewkes. Oh,
dear, oh, dear! Well, I’m bothered if some people nowadays don’t
give theirselves as much airs as if they was Margate, Ramsgate, and
Brighton all rolled into one.”
In about ten minutes Mrs. Pottage returned, followed by a burly old
cab-driver in a dark blue beaver coat with treble capes and a shiny
bowler hat.
“I’ve brought a most obliging driver along with me,” she
proclaimed. “The first cab I got, the fellow wouldn’t leave his horse at
the corner to come and help down with the luggage. Afraid of his
horse, he said. ‘I suppose you’re afraid it’ll fall down and never stand
up again if you left go of the reins?’ I said. ‘Never heard of a horse
running away, I suppose,’ he answers back very sarcastic. ‘What?’
said I, ‘that pore skelington run away? Why, it couldn’t walk away. It
might fade away, yes. And if it didn’t run away of itself, I’m sure
nobody wouldn’t ever run away with it. Not even a cats meat man,
and they’ll run away with anything as looks a little bit like flesh and
blood. But that horse of yours don’t. That horse of yours looks more
like a clothes-horse than a real animal. Only I’d be very afraid to
hang a towel on its back for fear it might break in half under the
weight.’ And with that I walked on and found this driver who’s been
most obliging, I’m sure.”
The cabman touched his hat in acknowledgment of the flattery,
and asked which piece they wanted down first.
 But now a greater obstacle to the departure of the luggage than an
unwilling cab-driver presented itself, for Miss Fewkes appeared, her
tow-coloured hair elaborately done as it always was on a Sunday
afternoon to resemble a brand-new yacht’s fender from which state it
gradually wore away during the stress of the week.
“And what is the meaning of this?” she demanded, folding her
arms.
Nancy explained why her luggage was going away this afternoon.
“Then perhaps you’ll pay my weekly bill, Miss O’Finn, before you
remove your boxes?” said Miss Fewkes.
“My bill will be paid to-morrow morning before I leave.”
“Yes, but I’m not in the habit of permitting my lodgers to remove
their luggage until their bills are paid,” Miss Fewkes insisted.
Mrs. Pottage gasped.
“Well, of all the impudence I ever did hear! Well, I passed the
remark to the policeman that you looked like a potted shrimp, but
shrimp sauce is more what you ought to be called.”
“It’s easy to see what you are,” Miss Fewkes spat out venomously.
“The sort of woman you are is plain to any one who’s sharp and has
eyes.”
At this point, the burly cab-driver, who was evidently afraid of
being involved in this feminine dispute, retired downstairs until the
matter was settled.
“It is easy to see what I am,” Mrs. Pottage agreed. “Because I’m a
decent-made woman. But it’s far from easy to see what you are, let
me tell you, very far from easy, because you aren’t as big as a
second helping of underdone mutton at an eating-house. You may
have eyes. So’s a needle. You may be sharp. So’s a needle. And I
wouldn’t care to look for you in a haystack any more than what I
would a needle, and that’s the solid truth I’m telling you. You asked
for it, ma’am, and now you’ve had it, and if you’ll kindly stand on one
side you won’t get carried out with the luggage like a speck of dust
off of your own dusty banisters.”
 “This luggage don’t leave my house before my account’s been
settled,” Miss Fewkes shrilled. “Not if I have to fetch in a policeman
to you.”
“Fetch a policeman?” Mrs. Pottage jeered. “Well, for a woman who
looks like last night’s buttonhole or a sucked sweet as a kid’s spat
out on the pavement, you’ve got a tidy nerve.”
Nancy thought that it was time to interfere, because she did not
want Letizia to be frightened by the quarrel.
“I’m quite willing to pay your bill, Miss Fewkes, if you suspect that
I’m trying to give you the slip,” she said.
“Not at all,” Mrs. Pottage interposed. “It’s beyond reason giving in
to such as she. Let her call this policeman we’ve heard so much
about, and it’s my opinion he’ll laugh in her face, that is if he could
tell it was her face, which I don’t think.”
“You vulgar, impertinent woman,” Miss Fewkes ejaculated.
“Yes, thank goodness I am a woman,” Mrs. Pottage retorted. “And
thank goodness you can reckonise me as such, which is more than
what I could reckonise you, not if I was looking at you with two
telescopes at once. Why, if I was you I’d be afraid to go out alone in
case I got took by a showman for a performing flea. It’s a nine days’
wonder you never got pecked up by a sparrow; but there, I suppose
even a sparrow knows what isn’t good for him.”
To what heights of invective Mrs. Pottage might have risen was
never to be known, because Nancy insisted on paying Miss Fewkes
her bill, which enabled her to retreat to her own room and cease to
oppose the departure of the luggage.
“But there, perhaps it’s as well,” conceded Mrs. Pottage. “Or I
might have been tempted to say something a bit rude.”
With the aid of the good-natured cabman the luggage was put on
the four-wheeler; and an hour later Nancy waved farewell to Letizia
and her hostess at London Bridge Station.
 CHAPTER XVII
THE TWO ROADS
On Monday morning with a lighter heart than she had known for
many weeks Nancy left Miss Fewkes. She had ten shillings and a
few odd coppers when she stepped out of the tall thin house in
Blackboy Passage, carrying her dressing-case in her hand; but she
had not to worry about Letizia at present, and the removal of this
anxiety had revived her confidence in being soon able to get a
“shop.” Meanwhile, she had to find a cheap room somewhere. This
proved to be much less easy than she had expected. At first all the
owners of the houses announcing apartments seemed to regard her
with equal suspicion.
“I don’t keep the kind of room you want,” said one.
“I wouldn’t mind taking you in myself,” said another. “But my
husband don’t like having women in the house.”
“If you’re looking for gay rooms,” said a third with brutal directness,
“you’d better try the other side of Oxford Street. You won’t find
anything to suit you round here. We have to be too careful of the
police.”
When at last Nancy did reach a quarter where landladies
appeared less dismayed by the prospect of letting to a single
woman, she found that the most exorbitant prices were asked in
every house.
“Two pounds a week for a bedroom only,” said one. “Or if you have
a latchkey, three pounds.”
“But why should I pay a pound a week for a latchkey?” Nancy
asked in astonishment.
“Well, if you have your own latchkey, I shouldn’t make any extra
charge for the gentlemen you brought home. Otherwise I’d have to
charge you five shillings a head.”
Nancy laughed.
“But I don’t want to bring gentlemen home with me. I’m on the
stage,” she explained.
The stolid countenance of the woman with whom she was
negotiating did not change its expression.
“If you don’t want to bring men back, you don’t want a room in my
house.”
With this she slammed the door in Nancy’s face, obviously
annoyed at the waste of her time.
Another landlady was quite distressed by the suggestion that
Nancy should have a bedroom for ten shillings a week.
“A nice-looking girl like you doesn’t want to come down to that,”
she exclaimed. “You trust your luck a bit, my dear. Why don’t you
take my two nice rooms on the ground floor and cheer up? They’ve
always been lucky rooms to girls like you. The last one who had
them got off with a wine-merchant somewhere up North, and he’s
fitted her out with a lovely little flat of her own. He only comes up to
London for a day or two every month, so she has a nice easy time of
it. I’m sure I don’t know whether it’s me or my rooms, but certainly
I’ve seen a lot of luck come the way of girls like you.”
At last after a peregrination of various apparently economical
quarters Nancy found a tiny garret at the top of a tumbledown house
in Unicorn Street, which joined Red Lion Square to Theobalds Road.
This was the third time in succession that she had taken lodgings in
a thoroughfare for foot passengers only, and superstition began to
suggest a hidden significance in this collocation. The third time? It
might be from here that she would discover the main thoroughfare of
her future life.
Unicorn Street was dark and narrow, and the upper portions of
several of the houses overhung the pavement so far as almost to
meet. These relics of London before the Great Fire had by this date
already been condemned, although they were not actually pulled
down for another ten years. The majority of the shops belonged to
second-hand booksellers, whose wares seemed as tattered and
decrepit as the mouldering old houses above. Their trade was mostly
done from shelves outside the shops containing books labelled at
various prices from one penny to a shilling. There were of course
other books inside, but these were usually stacked anyhow in
tottering heaps and simply served to replenish the shelves and
boxes on the contents of which, when the weather allowed it, seedy
men of various ages browsed slowly, humping their backs from time
to time like caterpillars when they thought they had caught sight of a
rarity. Mr. Askin, the owner of the shop high above which Nancy
found her cheap room, resembled the English idea of an elderly
German professor before the war destroyed that pleasantly
sentimental conception. His lanky white hair hung over his collar like
greasy icicles; he wore blue glasses, carpet slippers, and a frock
coat; he even smoked a long china pipe. The prospect of seeing his
shop pulled down to make way for blocks of eligible offices did not
disturb him, because he had made up his mind that within two years
he was going to be drowned. As he apparently never moved a yard
away from his shop, Nancy was puzzled by this confident belief, and
ventured to ask him on what it was based.
“Have you studied the effects of the moon?” he inquired
contemptuously.
Nancy admitted that she never had.
Whereupon he put his forefinger against his nose and said very
solemnly:
“Then don’t meddle in what you don’t understand. If I say I’m going
to be drowned before two years are out, then it means I’ve studied
the question and come to my own conclusions and resigned myself
to what must be. And that’s that, isn’t it? So try and not talk so silly,
young lady.”
Mr. Askin had bought enough books, according to his calculations,
to outlast him and leave a trifle over for his widow. These had at one
time filled every room in the house; but as soon as they were sold
the empty rooms were furnished with a few odds and ends and let.
The top stories were now completely void of books, which was how
Nancy managed to rent one of the garrets in the roof for the sum of
seven shillings a week. The other garret was inhabited by Maudie
Pridgeon, the Askins’ maid-of-all-work, who could not do enough for
Nancy once she heard she was a real actress.
“Oh, Miss O’Finn,” she begged. “I wonder if you’d be kind enough
to hear me recite The Lighthouse-Keeper’s Daughter some
afternoon, and tell me if I’ve got a chance to get on the stage myself.
It’s the dream of my life. I may not be a Sarah Burnhard or an Elling
Terry, but it’s in me, Miss O’Finn. I feel shore it’s in me. Sometimes I
feel I could burst with what’s in me. I was afraid it might be wind for a
time after I’d been reading about some medicine or other. But it ain’t,
Miss O’Finn, it’s acting. It is reelly. So some time, when we have a
moment to ourselves, I do wish you’d hear me recite and give me a
bit of good advice. And of course I can rely on you not to say a word
to Mrs. Askin about my ambishing or she might pass some nasty
remark about it. She never moves out of the back room behind the
shop herself, and she’d never believe as I might be a star hiding my
light under a bushel.”
The reason why Mrs. Askin never moved out of that back room
was her profound conviction that all men were thieves, and collectors
of old books the greatest. So, day in day out, she sat in a flocculent
armchair which at night was turned into her bedstead, watching with
a suspicious eye the behaviour of prospective customers. She was a
dark unwieldy woman with a hairy chin, a profusion of tufted moles,
and what was almost a heavy moustache. It was agreed when
Nancy took the garret that she was not to expect any cooking to be
done for her; and when she saw the Askins’ meals being prepared in
that back room and Mr. and Mrs. Askin and Maudie each eating a
disgusting plateful balanced on different heaps of incredibly dusty
books, she did not regret the arrangement. She managed to make
her own garret fairly clean; and though it was perishingly cold up
there under the ancient roof, though the bed was hard and the rats
scampered round inside the raw-boned plaster walls, she had the
satisfaction of feeling perfectly sure that nowhere in London could
she be lodged more cheaply. The solitude of the long, long evenings
when she used to go to bed at eight o’clock in order to keep warm
was immense; and yet she liked it, for she seemed, high up in this
garret, to be as near to Bram as she could reach on earth. There
was no blind to the decayed window of the dormer and, blowing out
her candle, Nancy used to lie for hours staring out at the tawny
London sky, while beneath her pillow Bram’s watch was always
ticking, his watch that she had never allowed to run down. And once
in sleep he held her in his arms, and once she woke with his kisses
warm upon her lips; but mostly when she dreamed of that beloved
lost one it was of running with him along endless platforms to catch
fantastic and unattainable trains, and of acting with him in nightmare
plays without having studied the part in which she was being
suddenly called upon to appear. Meanwhile, it seemed that the
tangible and visible world was fast dissolving into an unstable dream
when Nancy, after three weeks of pawnshops and agents’ offices
and of apparently being as far away as ever from any engagement,
was persuaded by Maudie to hear her recite The Lighthouse-
Keeper’s Daughter and was asked at the end of it to advise her
about a dramatic future.
“I think you said it very well, Maudie,” Nancy assured the little
maid, whose cheeks were flushed and whose eyes were flashing
with excitement. “But I must really advise you to give up all idea of
the stage as a profession. Look at me. I am an experienced actress
and yet I can’t get an engagement. I’ve been trying ever since
January, and now it’s nearly April. All the managers say I’m too tall;
and you know, Maudie dear, you’re just as much the other way,
aren’t you? You and I want special parts written for us, that’s the
trouble.”
The little maid’s eyes filled with tears.
“Oh, Miss O’Finn,” she sobbed, “you’ve been and gone and
shattered my life’s ambishings with them words. You see, when I’m
reciting I feel as if my head was going through the ceiling, but of
course what you feels and what you is ain’t the same, is they? Still,
it’s always the darkest hour before the dawn, they say, and I’ve still
got my young man. When I see him on Sunday night I’ll tell him as
I’ve given up my life’s ambishings, and he can start saving up for
merridge as soon as he likes. It’s broke my heart, but he’ll be happy.
He was always afraid he’d lose me, Miss O’Finn. He never could
believe I’d remain a simple milkman’s wife when I become famous,
and on’y last week he let a lovely double-bed go by because he
didn’t want to have it on his hands and me out of his reach.”
A few days after Nancy had destroyed Maudie’s dramatic
ambitions she received a letter from Mrs. Pottage.

3 Starboard Alley,
Greenwich.
April 1st
!
My dear Mrs. Fuller,
Its’ a nice day to choose to write a letter to any one but
there you won’t get it till April 2nd so you won’t think any
one’s sending you a live mouse or any silly joke like that.
Well, here we are as well as we can be thank God—
Letitsha is in the pink there’s no doubt about it and so am I
but this is not what I am writing about. Last week we had
the Lights of Home company at the Royal and Mr. Plimmer
who was acting in it was lodging with me and this week
they finished and he’s staying on with me because he
says he’s never been so comfortable in his life but he’s
took a great fancy to Letitchia and that’s a fact—He raves
about her and I won’t say I’m surprised because she’s
been on the Top of her Form and making us all laugh fit to
Bust. Mrs. B. says she’s laughed a lot in her life and which
is a fact but she don’t think she ever laughed so much as
what she has this week. She split her stays one afternoon
—They went off like a Cannon—Talk about a royal Salute
—And Mr. Plimmer says she’s a born actress and ought to
be on the boards without delay—well, he’s taking out a
company himself in a drama he’s written something after
the stile of East Lynne well about the same as far as I can
see only a bit more East in it from what I can make out
and he wants Letissia for the child and you for the Mother.
 He’ll write plenty of stuff for her because he says She’ll
Knock Them. Well, I’m bound to say I think she will and
Mrs. B’s convinsed of it: So I gave him your address and
he’s going to pop up to London to-morrow if you’ll make
arrangements to be in I’ve given him your address. What a
voice that Kid has he said to me Mrs. Pottage. Good God
it would reach to the back row of the gallery in any theatre.
Well I hope this’ll be the end of all your troubles and which
I think it will dearie—
Letitsia sends her love and so do I and I hope this is an
end of all your worries even if it is April Fools Day. Mrs. B.
sends all the best and so do I.
Your loving old
Johanna Pottage.

Here was a most unmistakable turning out of this long lane, Nancy
thought, a turning at so sharp an angle that the prospect of taking it
alarmed her imagination, so far did it seem likely to lead Letizia and
herself away from the direction in which Bram and she had been
travelling together. Nancy’s mind went back to her own appearance
at the age of six in Green Bushes. Her mother was no longer alive to
witness that first performance of a squeaky-voiced little boy in the
old-fashioned melodrama, of which she could remember nothing
except the hazy picture of the heroine dressed in a Fenimore Cooper
get-up as she came running down the bank, gun in hand. Her father
had made arrangements for her to live with the baggage man and
his wife during that tour. She had liked Mr. Ballard, a big fat man with
a very much waxed moustache, but little Mrs. Ballard with her cold
hooked nose, pink and half-transparent at the tip, had been
antipathetic. She could see her now sighing and sewing all day. If
Letizia did go on the stage as a child, she should not act away from
her mother at any rate. It would always have to be a joint
engagement.
Maudie interrupted Nancy’s pictures of the past by coming up to
say that a gentleman was down in the shop and wanted to see her.
 “I didn’t know if you’d have liked me to have brought him up here,
Miss O’Finn? I hope I done right in asking him to wait a minute in the
shop?”
“Good gracious, yes, Maudie! He couldn’t come up here. I’ll be
down very soon.”
Nancy looked at the card: Rodney Plimmer. “Custody of the Child”
Company. Evidently that was the play he was presently going to take
out on tour. Nancy put on her hat and coat, for if she was going to
talk business with Mr. Plimmer they would certainly have to talk
elsewhere than in Unicorn Street.
The actor was turning over the pages of one of Mrs. Askin’s
tattered folios when she came down into the shop.
“Now don’t tell me you’ve got another appointment, Miss O’Finn,”
he said. “I’ve been hoping you would come out to lunch with me.”
“Oh, no, I haven’t any appointment, and I’ll be delighted to lunch
with you.”
“Capital! Then, if you’re ready, shall we wend our way toward
some little place where we can talk far from the madding crowd?”
There was nothing remarkable about Mr. Plimmer’s appearance.
The clean-shaven face, the full mobile lips, the tendency toward
sleekness, the suggestion that his clothes were being worn with a
little too much of an air, the moist impressionable eyes, all these
traits were sufficiently familiar to Nancy among the men of her
profession.
“Now, have you any prejudices on the subject of restaurants?” Mr.
Plimmer inquired with rich voice and elaborate manner.
“None whatever.”
“You don’t pine for music and such like gaieties?”
She shook her head.
“Then, let me see.” He paused with such dramatic abruptness in
the middle of the pavement that an errand-boy who was just behind
bumped into his broad back. “Why don’t you look where you’re
going, my lad?” he asked with exaggerated dignity.
“Why don’t you look where you’re stopping?” the errand-boy
retorted and hurried on, whistling indignantly.
“Self-possession is nine points of the law,” said Mr. Plimmer. “By
the way, that’s not bad, eh, Miss O’Finn? I think I’ll note that down as
rather a good line.” He took out a small pocket-book, and entered the
remark. “A word in the hand is worth two in the head,” he observed
with a smile; and as he did not bother to enter this line under the
other Nancy supposed that he used it frequently.
“Then, let me see,” said Mr. Plimmer, returning to the original
attitude which had provoked this diversion. “I have it! Kettner’s.
You’ve no prejudice against Kettner’s?”
“None whatever. I’ve never been there,” Nancy replied.
“Never been to Kettner’s? Oh, then of course we must go to
Kettner’s. No music at Kettner’s. And if there’s one thing I hate it’s
chops and sonata sauce.”
Mr. Plimmer blinked his moist eyes as if he were dazzled by the
brilliancy of his own wit.
“And now what about a hansom?”
The drive from the corner of Theobalds Road to Kettner’s was a
strain on Nancy, because Mr. Plimmer was evidently extremely
nervous in hansoms and talked all the time of the close shaves he
had had when driving in them. If ever their driver showed the least
audacity in passing another vehicle, Mr. Plimmer would draw in his
breath with a hiss, or put his hand out over the apron as if he would
seize the too urgent horse by the tail and stop his going too fast.
However, Kettner’s was reached in safety, and Mr. Plimmer was no
sooner on the pavement than he recovered all his suave composure
so that he entered the restaurant with the air of knowing exactly
where to go and what to order, whenever he should choose to eat in
London.