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Table of Contents
Introduction......................................................................................................................................2
Search Strategy............................................................................................................................2
Aims.............................................................................................................................................3
Objectives....................................................................................................................................3
MS Application in Biotechnology...................................................................................................3
Conclusion.....................................................................................................................................10
References......................................................................................................................................11
Introduction
Mass-Spectrometry (MS) refers to the mainstream technique of chemical analysis developed in
1918, by J.J.Thompson, for identifying isotopes of elements (Wasinger, et al., 2013). Even back
to that date the application potential of the technique gained wide recognition it being used by
the Governments for the developing nuclear weapons, and at the 21st century the application
versatility of MS is many it contributing to wide discoveries in fields of biotechnology,
biochemistry, physics and chemistry or in industries for conducting quality control, drug-
discovery and food-safety-protocol. The technique's uniqueness lies with the power of it directly
enabling molecule identification on basis of fragmentation-pattern and mass-charge ratios,
leading to its use both in qualitative analysis of chemicals and in quantitative-molecular-
measurements (Kaltashov, et al., 2012). The technique gained wide recognition with
advancement in micro-electronics and material-sciences due to its ease-of-usage. This followed
transformation of MS stand-alone-instrument towards modern-computer-interfaced MS coupled
to analytical-front-end for analyte confirmation, identification or solving complicated scientific
issues.
The technique works through ionization, determining masses of charged analytes, and in the
present research, several authentic and peer-reviewed academic sources have been traversed to
highlight significant applications of the technique.
Search Strategy
Online search as means of secondary-data-collection has been conducted in present study, in
relevant manner for procurement of updated information, from peer-reviewed, valid journal
manuscripts, books and systematic-reviews corresponding to 10 years time-span, for maintaining
data authenticity, and relevance. Research studies of international repute, conducted during the
same time-frame have also been considered, and search-databases and search-engines like
Google-Scholar, PubMed have been thoroughly searched. Efficient usage of key-words have
been used in the searches namely - Mass-spectrometry, analytical techniques, quantitative MS,
qualitative MS, MS application, metabolomics, enzyme-assays, proteomics, biomarker, clinical-
performance.
Aims
The present study aims "to review significant scientific literatures regarding application of mass-
spectrometry in the field of Biotechnology for understanding measurement techniques on
biotechnology".
Objectives
On the basis of the aforementioned aim, the underlying objectives have been developed in the
study, for conducting research in the right direction aligned with the study aim:
MS Application in Biotechnology
Untargeted-Metabolomics
In the process the now extracted metabolites are further separated by the use of GC/MS or Gas-
Chromatography/Mass-Spectrometry, LC/MS or Liquid-Chromatography/Mass-Spectrometry or
that of CE/MS (Capillary-electrophoresis/Mass-Spectrometry) (Koike, et al., 2014; Yoshida, et
al., 2012; Huan, and Li, 2015). It is however essential to all such MS-approaches that there
occurs efficient absorption and metabolite ionization, and resulting gas-phase-ions may then be
separated via mass-analyzers like time-of-flight, quadrupole, or ion-trap. The ions in the process
are detected by the use of photo-multiplier-tubes and micro-channel-plates. This follows their
further identification via comparative analysis of retention time, exact mass, fragmentation data
following spectral database and standards.
Targeted metabolomics
Often metabolomics approaches are further divided onto untargeted and targeted. The targeted
methods aid in detection and quantification of specified metabolites within sample matrix, and
the particular approach holds the advantage in maximizing MS methods sensitivity and
specificity. These analyses also utilize authentic analytical standards for defining LC or GC
method, for determination of fragmentation pattern of metabolites, and for constructing
calibration curves to conduct absolute quantification (Minati, et al., 2010).
The above Figure depicts, that in A - Chemical probes are used by ABPP for selective profiling
of proteins which are actively bound via cross-linking reactions as well as tagged-peptides'
proteomic analysis. In B - Protein arrays here are used for binding to ligands which can be
subsequently released for identification through MS. In C - the metabolites which are surface
metabolized binds to proteins or aids in detection of enzymatic transformations in respect to
immobilized substrates.
In the methods of GC/MS, CE/MS or LC/MS, limitation exists in form of lost spatial information
which occurs after metabolites are extracted from the homogenized samples. In this aspect, the
metabolite-imaging-techniques can play a role being an alternative to provide information of
metabolites spatial distribution within tissues (Fig. 3). Thus the widely used tissue-imaging MS-
based approach refers to that of MALDI-imaging technique, wherein the MALDI matrix is first
applied to tissue sample typically via spraying or spotting followed by image generation through
laser raster-scanning over sample, providing mass-spectrum in every coordinate (x,y) (Reyzer,
and Caprioli, 2011). Then the images are constructed via distribution mapping and mapping ion
abundance within tissue sample.
Fig.3 Approach of MS-imaging used for spatial distribution based studies of metabolites in tissue
samples.
In the figure it is depicted how desorption and ionization of metabolites occur and mass-
spectrum are obtained, enabling image reconstruction from resulting data, which may be used in
indentifying metabolite-patterns of localization.
There exist two prime factors for driving success in MS in the biomarker-discovery researches.
The former being implementation of rigid experimental-design-constraints which ensures that
discovered biomarkers reflect pathophysiology not being analytical artifacts rather. After
survey of thousand of features in MS analysis, it is often common that false discoveries may
be made, and using contemporary approaches in correction of false-discovery-rates like q-
values only correct finding significance on basis of underlying distribution, tending to
overcorrect older methods namely Bonferroni correction. Thus assurance may be provided
through strict and rigid-experimental-designs, after which decision is made in terms of the
examined matrix. Several options exist but opinion depends largely upon investigator
preference. The options consist of - examining primary tissue of patient, examining patient
urine, examining patient blood-samples, cell-culture, patient-cerebrospinal-fluid, or others.
Most ideal biomarkers are found in patient-blood-samples however, for tumor biomarker
based investigations focus is portrayed on metabolic categorization or protein categorizations
of the patient's tumor tissue. This is based on assumption that metabolite / protein found in
high concentration in patient tumor tissues may secrete onto blood. Biomarker studies may
lack analytical sensitivity for detection of protein isoforms or blood metabolites without
targeted MS analysis. Furthermore, direct tissue analysis via the use of DESI or LAESI as
mechanisms for atmospheric-ionization-detection may be used for experiments on MS
imaging to deliver additional and enriched information regarding spatial resolution, though
lacuna may exist in sensitivity regarding protein-MS- conventional methods.
The specimen being liquid or tissue sample may be analyzed in terms of metabolites or
proteins. Protein-biomarker based discoveries emphasizes upon discovery of protein
isoforms which are disease-specific in nature. For leveraging the contemporary technology,
ETD application as fragmentation technique also helps by improving capacity for detecting
labile modifications in comparison to collision-based activated dissociation. Protein
quantification in general requires labeling in form of isobaric tags - accomplished by the use
of iTRAQ for peptides, but quantification of glycans can be attempted by QUANTITY,
being most recent a method for macromolecular detection, which can justifiably provide
excellent information on glycan modification.
Detection of metabolites may be carried out via targeted approach using MRM library
considering the known authentic standards or through untargeted analysis. While targeted
analysis leverages internal standards requiring consideration of commercial availability and
hypothesis generation in priority basis; untargeted analysis comprises of difficult
considerations in terms of quantification via use of internal;-standard-infusion that aids in
mass accuracy and quantification. The challenge which exists with small molecules-
metabolite based biomarker-discovery is that structure elucidation poses a problem,
considering isobaric compounds' possibilities.
However, with the use of strict-experimental design and advanced enhancements to MS-
biomarker based discovery workflow, potential biomarkers may be generated in abundance
clinically. The ones which will succeed may be vetted through clinicians, biologists,
chemists based on mechanistic likelihood. Finally transition of the same to clinics will
eventually be defined by intended usage and their implementation will be carried out only
after pre-analytical and analytical requisites are defined n clear fashion by standard
laboratories.
Table - 1
Kaltashov, I.A., Bobst, C.E., Abzalimov, R.R., Wang, G., Baykal, B. and Wang, S., 2012.
Advances and challenges in analytical characterization of biotechnology products: mass
spectrometry-based approaches to study properties and behavior of protein
therapeutics. Biotechnology advances, 30(1), pp.210-222.
Yoshida, M., Hatano, N., Nishiumi, S., Irino, Y., Izumi, Y., Takenawa, T. and Azuma, T., 2012.
Diagnosis of gastroenterological diseases by metabolome analysis using gas chromatography–
mass spectrometry. Journal of gastroenterology, 47(1), pp.9-20.
Smart, K.F., Aggio, R.B., Van Houtte, J.R. and Villas-Bôas, S.G., 2010. Analytical platform for
metabolome analysis of microbial cells using methyl chloroformate derivatization followed by
gas chromatography–mass spectrometry. Nature protocols, 5(10), p.1709.
Koike, S., Bundo, M., Iwamoto, K., Suga, M., Kuwabara, H., Ohashi, Y., Shinoda, K., Takano,
Y., Iwashiro, N., Satomura, Y. and Nagai, T., 2014. A snapshot of plasma metabolites in first-
episode schizophrenia: a capillary electrophoresis time-of-flight mass spectrometry
study. Translational psychiatry, 4(4), pp.e379-e379.
Füzéry, A.K., Levin, J., Chan, M.M. and Chan, D.W., 2013. Translation of proteomic biomarkers
into FDA approved cancer diagnostics: issues and challenges. Clinical proteomics, 10(1), p.13.
Dunn, W.B., Erban, A., Weber, R.J., Creek, D.J., Brown, M., Breitling, R., Hankemeier, T.,
Goodacre, R., Neumann, S., Kopka, J. and Viant, M.R., 2013. Mass appeal: metabolite
identification in mass spectrometry-focused untargeted metabolomics. Metabolomics, 9(1),
pp.44-66.
Hrabák, J., Chudáčková, E. and Walková, R., 2013. Matrix-assisted laser desorption ionization–
time of flight (MALDI-TOF) mass spectrometry for detection of antibiotic resistance
mechanisms: from research to routine diagnosis. Clinical Microbiology Reviews, 26(1), pp.103-
114.
Li, D. and Chan, D.W., 2014. Proteomic cancer biomarkers from discovery to approval: it’s
worth the effort.
Minati, L., Aquino, D., Bruzzone, M.G. and Erbetta, A., 2010. Quantitation of normal metabolite
concentrations in six brain regions by in-vivo 1H-MR spectroscopy. Journal of medical
physics/Association of Medical Physicists of India, 35(3), p.154.
Huan, T. and Li, L., 2015. Quantitative metabolome analysis based on chromatographic peak
reconstruction in chemical isotope labeling liquid chromatography mass
spectrometry. Analytical chemistry, 87(14), pp.7011-7016.
Reyzer, M.L. and Caprioli, R.M., 2011. Imaging mass spectrometry. In Detection of Biological
Agents for the Prevention of Bioterrorism (pp. 267-283). Springer, Dordrecht.