The Proteomics Universe:

Delving into World of the

amino acids and
polypeptides
BY: DR. BATO
Clinical Case # 1

 A 6 month-old male infant

suffering from recurrent
fever, otitis media (ear
infection), sore throat, and
URI (upper respiratory
infection)
 Pt grew up inside an
incubator like container
and was only touched
using latex gloves
 Bubble boy
SCID

 Severe combined
immunodeficiency
 Immunoglobulin defect or
adenine deaminase
enzyme defect
 Pt’s only hope was a bone
marrow transplant from
sister but succumbed to an
Epstein-Barr virus (EBV)
infection since the bone
marrow graft from sister was
infected
Clinical Case # 2

 A 5/M with severe joint

pains or arthralgia and
muscle pains or myalgia
associated with a facie of
an old-man
 Progeria

 A defect in Lamin A protein

 Nuclear membrane
cytoskeleton
 Irreversible aging
 fatal
Clinical Case #3

 A 26/M has profound

muscle weakness in the
afternoon associated with
ptosis
 Pt would have no weakness
in the morning
 Myasthenia gravis

 Autoantibodies against
acetylcholine receptor (AchR)
 Acetylcholine could not bind to
site on AchR
Clinical Case # 4

 A 4/M with difficulty

standing associated with
Gower sign
 Hypertrophic calf muscles
Muscular Dystrophy

 Becker or Duchenne
 A defect in the dystrophin
Clinical Case # 5

 A group of female children

and adults with a
neurodegenerative
syndrome
 Difficulty standing without
support
 Prion disease

 Bovine spongiform encephalopathy

 Creutzfeld-Jacob Disease
 Scrapie
 Kuru
Clinical Case # 6

 A 65/M complained of
severe chest pain 10/10
associated with diaphoresis.
(+) Levine sign
Acute Myocardial Infarction

 Coronary artery blockage

causing luminal obstruction
with noted heart muscle
death
 Initially enzymes measured
CK-MB and troponin I
Proteins are made up of amino
acids

 More than 300 amino acids

known in nature
 More or less 20 aas are found
in humans
 Selenocysteine: called the
21st amino acid
 Made from a modification of
the amino acid serine
 Modification occurs during
the time when serine is bound
to its tRNA
 Not a posttranslational
modification
 Amino acids have chirality

 Same as enantiomerism
 The α-carbon is ASYMMETRIC
 Except glycine
 19 out of the 20 aa
 Chiral aa can exist as stereoisomer
 Compound with identical molecular formula but different
configuration or atomic arrangement
 Enantiomers: 2 stereoisomers that are
nonsuperimposable mirror images
 Isoleucine and threonine have 2 chiral carbons
 L and D-aa can be converted to each other using
racemase
Nonpolar, Aliphatic Side
Chains
Polar, Uncharged Side Chains
Polar, Charged Side Chains
Aromatic Side Chains
Sulfur-containing Side Chains
 A Quick Review on Amino Acid
Side Chains
Groupings Amino Acid Three-Letter One-Letter
Abbreviation Abbreviation
Nonpolar Glycine Gly G
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine Ile I
Proline Pro P
Methionine Met M
Phenylalanine Phe F
Polar Serine Ser S
Threonine Thr T
Tyrosine Tyr Y
Asparagine Asn N
Aspartic acid Asp E
Glutamine Gln Q
Glutamic acid Glu D
Cysteine Cys C
Tryptophan Trp W
A Quick Review on Amino Acid Side
Chains
Groupings Amino Acid Three-Letter One-Letter
Abbreviation Abbreviation
Aliphatic Glycine Gly G
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine Ile I
Proline Pro P

Aromatic Phenylalanine Phe F

Tyrosine Tyr Y
Tryptophan Trp W

Alcohol Serine Ser S

Threonine Thr T

Sulfur- Cysteine Cys C

containing Methionine Met M
 A Quick Review on Amino Acid
Side Chains

Groupings Amino Acid Three-Letter One-Letter

Abbreviation Abbreviation
Acidic Aspartic Asp D
acid/Aspartate
Glutamatic Glu E
acid/Glutamate

Basic Histidine His H

Lysine Lys K
Arginine Arg R

Amide Asparagine Asn N

Glutamine Gln Q
 What is Hydropathy Index?

 Relative hydrophilicity or
hydrophobicity of each
amino acid
 High positive values means
highly hydrophobic; high
negative ones are
hydrophilic
 Important determinant in
protein folding
 Amino acids at the center
of the scale might have
differing values
Kyte and Doolittle Scale Mean
Hydrophobicity Profile
Molecular Interactions Between
Amino Acid Side Chains
 pKa

 Amino acids are known weak acids

 Acid strength
 The larger the Ka, the stronger the acid,
because most of the HA has dissociated
into H+ and A–. Conversely, the smaller the
Ka, the less acid has dissociated and,
therefore, the weaker the acid.
 Net charge: alebraic sum of all the (+) and
(-) charged groups present - depends on
the pKa values
 Altering the charge on the aa by varying
the pH facilitates the physical separation
 Net charge of zero found in zwitterions
 pKa is affected by the environment
 A polar environment favors charged form
 Nonpolar one favors uncharged
pI

 pH midway between pKa

values of an isoelectric
species
 zwitterions
 Isoelectric means the
molecule has an equal
number of (+) and (-)
charges
 Neutral
 0
Isoelectric Point
How do Amino Acids bind
together?
What is so Special with the
Peptide Bond?
 Four Orders of Protein Structure

1. Primary
 amino acid sequence

2. Secondary
 folding of 3 to 30- residues forming geometrically
ordered units

3. Tertiary
 assembly of secondary structures into larger functional
units

4. Quaternary
 polypeptide units of oligomeric proteins and spatial
arrangements
Primary Structure

aspartyl, lysyl, glutaminyl, histidinyl, cysteinyl, arginyl,

phenylalanine
Primary Structure
Secondary Structure

Free rotation possible on 1) α-carbon (Cα) to carbonyl carbon (Co) ; 2)

Cα to N
The partial double bond character of Co to α-N requires carbonyl
carbon, carbonyl oxygen and α-nitrogen remain coplanar =
prevents rotation
Phi (Φ) : angle about the Cα - N
Psi (ψ) angle about Cα –Co
Besides glycine, most Φ and ψ are not allowed due to steric
hindrances
Proline is restricted due to absence of Cα – N
Regions of ordered 20 structures arise when aa residues adopt similar
Φ and ψ angles
Most common: α helix and β sheet
 α Helix

Polypeptide backbone twisted

by an equal amount of Φ at -57
degrees and ψ at -47 degrees
1 complete turn = 3.6 aa residues
Pitch per turn = 0.54 nm
Polar R groups face outward
Right-handed α helix are more
stable due to L-amino acids
Represented as cylinders
.
α Helix

Stability due to H bonds between oxygen of the peptide

bond carbonyl and H atom of the peptide bond nitrogen
Maximum number of H bonds coupled with van der Waals
forces provides the thermodynamic driving force
Proline lacks a hydrogen atom and could only be stable
accommodated within the first turn
Glycine induces a bend
Amphipathic helices form interfaces between polar and
nonpolar regions creating a channel or pore
 β Sheet

aa residues form a zigzag or

pleated pattern
R-groups of adjacent residues
point on opposite directions
Peptide backbon highly extended
Stability formed from H bond
between carbonyl oxygen and
amide hydrogen
Parallel: adjacent segments
proceed in the same amino to
carboxyl
Antiparallel: opposite directions
Most β sheets are not perfectly flat
but tend to have right –handed
twist
Many globular proteins
Loops and Bends

Short aa segments joining 2 units of secondary structures

Β turn: 4 aa residues, in which the first residue is H-bonded to the 4th resulting in a 180 degree turn
Proline and glycine
Loops: regions that contain residues beyond the minimum number necessary to connect adjacent regions of
secondary structure
Irregular conformation
Serve key biologic functions
In enzymes: bridge domains which bind substrates and contain aa residues that
participate in catalysis
Lack apparent structural regularity
Exist in a specific conformation stabilized via H bonding, salt bridges, and hydrophobic forces
Helix-loop-helix motifs: provide oligonucleotide-binding portion of DNA-binding proteins such as repressors
and transcription factors
 Loops and Bends

Supersecondary structures:
intermediate between 20 and 30
structures
Epitopes: accessible sites for
antibody recognition and binding
Loops and bends reside on protein
surfaces

Not all protein portions are ordered

Disordered regions: often at
extreme amino or carboxyl terminal
High conformational flexibility
Assume an ordered conformation
upon ligand binding
Enables regions to act as ligand-
controlled switches
 Tertiary Structure

Refers to the entire 3-D conformation of the polypeptide

3-D spatial behavior: helices, sheets, bends, turns, and loops
Domains: protein structure sufficient to perform a particular chemical or physical
task such as substrate or ligand binding
Anchor protein to a membrane
Interact with a regulatory molecule modulating its function
Single domains: triose phosphate isomerase, myoglobin
Two domains: protein kinases
Catalyze transfer of a phophoryl group from ATP to a peptide or
protein
Amino terminus is rich in β sheets and binds ATP
Carboxyl terminus is rich in α helices and binds peptide/protein substrate
Groups that catalyze phosphoryl transfer reside in a loop positioned at the
interface of the 2 domains
Quaternary Structure

Defines the polypeptide

composition of a protein and the
spatial relationships between
subunits or protomers
Proteins are assembled from
more than one polypeptide
Monomer: single polypeptide
chain
Dimer: 2
Homodimer: 2 copies of the
same polypeptide
Heterodimer: 2 copies of different
polypeptide
Quaternary Structure

Greek letters are used to describe the subunits of a

heterooligomeric protein
Α2β2γ
Simplified schematic diagrams used to key features of proteins
tertiary and quaternary structures
Ribbon diagrams trace conformations
Cylinders are helices while arrows are sheets
Line for the polypeptide backbone
Elongation Factor 1-α + GDP
Another look on the 4 Orders of
Protein Structures
What are proteins?

 Polypeptides
 One of the 4 important
biomolecules
 CHON
 Physically and functionally
complex
 Made from amino acids
Protein has various functions
Where does
the body get
proteins?
Where does
the body get
proteins?
How are
dietary
polypeptides
broken down
into
constituent
amino acids?
How are they absorbed?
Different Amino Acid Transport
Systems
Essential vs Nonessential Amino
Acids
Nonessential Amino Acid
Synthesis
 Proteins are Expressions of Genes

Born at translation
Genes to proteins
Matures through posttranslational
modification
Alternates between working
(active) and rest (inactive) states
through regulatory factors
Ages through oxidation,
deamidation
Dies when degraded into
component aa
The Genetic Code: The basis for
Protein Translation
Protein Translation
Posttranslational Modifications:
Carbohydrate Addition
Posttranslational Modification:
Regulation
Posttranslational Modification:
Lipid Addition
Posttranslational Modification
Protein Folding
 How does the body destroy
unwanted or damage proteins?
1. Ubiquitin-Proteasome Proteolytic Pathway
 ATP-dependent
 Cytosol
 Uses ubiquitin
 Chemical tags such as amino acid (PEST sequences) can have an effect
 Depends on the amino acid on the N-terminal
 Serine – 20 hrs half-life; aspartate – 3 mins

 PEST sequences recurring theme on the polypeptide

 Endogenous peptides

2. Lysosomal Degradative Pathway

 ATP-independent
 Lysosomes
 Exogenous peptides
 How does the body destroy
unwanted or damage proteins?

 Ubiquitin (Ub) is an evolutionarily conserved 76-amino acid protein that is

central to multiple cellular functions

 reversible Ub conjugation with target proteins and can be divided into

proteolytic and nonproteolytic (trafficking) pathways:
 Endocytosis

 Intracellular trafficking

 Regulation of histones and transcription

 Cell cycle control

 Autophagy

 Repair of DNA damage

 Cellular signaling

 The Ub molecule contains seven lysine residues, and functional selectivity is

provided by diverse patterns of protein linkage to these amino acids

 Linkage to some lysines leads to passage of the tagged protein to the

proteasome for degradation

 The fate of Ub-conjugated proteins among the several pathways is

determined by the number of Ub moieties conjugated and the site of the
conjugation linkages on the Ub molecule
 How does the body destroy
unwanted or damage proteins?

 A Ub-activating enzyme, E1, binds to Ub and then transfers it to one of dozens of

Ub-conjugating enzymes (E2).

 These act together with one of about 800 different Ub-ligating enzymes (E3) to add
Ub to a lysine onthe doomed protein

 Additional Ub moieties are added to the original Ub, forming a polyubiquitin chain
(at least four Ubs)

 Proteasomes are highly conserved organelles in the cytoplasm and possibly the
nucleus

 barrel-shaped complexes whose main (but not only) function is to digest

polyubiquitinated proteins

 two types of proteasomes: 20S and 26S

 The degradative unit of both is a 20S destruction chamber, to which, in the 26S
proteasome, two 19S “caps” are attached

 The caps at the entrance to the proteolytic core regulate entry

 The 20S proteasomes lack these caps

 The 20S proteasomes are important in degradation of oxidized proteins

 In 26S proteasomes, polyubiquitinated proteins are degraded.

 Mutations that interfere with normal proteasomal function are lethal

Diseases Involving Protein
Degradation Defects
 Proteomics is A Field of Interest

 Study of the proteome

 Entire set of proteins, produced or modified by an organism or system
 Coined by Marc Wilkins in 1994
 Protein and genome
 Large-scale study of proteins: their structures and functions
 Coined in 1997; an analogy to genomics
 Interdisciplinary domain benefitting greatly from the human
genome project
 Experimental analysis using protein purification techniques and
mass spectrophotometry
 Genomics, transcriptomics, metabolomics
Proteomics and the Proteome

Proteomics aim to id the entire complement of proteins

elaborated by a cell under diverse conditions
Many proteins undergo posttranslational modifications during
maturation into functionally competent forms as means or as a
means of regulating their properties
Knowledge of the human genome therefore represents only
the beginning of the task of describing living organisms in
detail and understanding the dynamics of processes such as
growth, aging and disease
Proteins whose appearance or disappearance are associated
with a specific physiologic condition or disease
Determination of proteomes characteristic of each cell type
requires utmost efficiency in the isolation and identification of
individual proteins
Proteomics and the Proteome

Utilizes robotic automation to speed sample preparations and large

2-D gels resolve cellular proteins
Only about a thousand proteins can be resolved on a single gel
Alternative and complementary approach: gene arrays called DNA
chips
Utilizes mRNA that encodes proteins
Gene arrays are more sensitive
Recent advances in bioinformatics permit researchers to compare
amino acid sequences to discover clues to potential properties,
physiologic roles, and mechanism of actioin (eg. Rossmann
nucleotide binding fold, nuclear targeting sequences, EF hands)
Gross Characteristics Classify
Proteins

Solubility
Shape
Presence of nonprotein groups
soluble proteins: extracted using aqueous solutions at physiologic
pH and ionic strength
integral membrane proteins: requires detergent dissolution
globular proteins: compact, spherical with axial ratios  3
fibrous proteins: axial ratios ≥ 10
lipoproteins and glycoproteins: lipid and carbohydrate,
respectively
metalloproteins: tightly-associated metal ions (hemoglobin,
cytochromes)
Homology or similarity in aa sequence and 3-D structure
 Protein Purification

Highly purified protein is needed for determination of physical and

functional protein properties
Isolation of a specific protein in quantities sufficient for analysis
presents a formidable challenge
Classic approaches:
pH (isoelectric precipitation)
Polarity (ethanol or acetone precipitation)
Salt concentration (ammonium sulfate salting)
Chromatography:
Partition molecules between 2 phases: mobile and
stationary such as filter paper or thin layer of cellulose, silica,
or aluminum)
Chromatography

Discovered by Mikhail Tsvet in 1900

Based on thin-layer chromatography of separating plant extracts
Depends on the relative affinity of different proteins for a given
stationary phase and for the mobile phase
Association between each protein and the matrix is weak and
transient
Proteins interacting more strongly with the stationary phase are
retained longer
Length of time that a protein is associated with the stationary phase is
a function of the composition of both stationary and mobile phases
Optimal separation achieved by manipulation of the composition of
the 2 phases
Types of Chromatography

Techniques by chromatographic bed shape

1. Column
2. Planar

Techniques using separation mechanism

1. Size exclusion
2. Ion exchange
3. Hydrophobic interaction
4. Affinity

Techniques based on mobile phase type

1. High-pressure liquid
2. Gas
 Column Chromatography

Stationary phase: column containing

small spherical beads of modified
cellulose, acrylamide, or silica whose
surface has been coated with
chemical functional groups
The matrix interacts with proteins based
on 1) charge 2) hydrophobicity 3)
ligand-binding properties
A protein mixture is applied to the
column
Liquid mobile phase is percolated
through it
Small portions of the mobile phase or
eluant are collected
Planar Chromatography

Separation of mixtures in a filter paper based on

partition of a solute between 2 solvents one of
which is immobilized by the substance paper
 Size Exclusion Chromatography

Also known as gel filtration or gel permeation

Separates proteins based on their Stokes radius
(sphere diameter)
Stoke radius is a function of molecular
mass and shape
A tumbling elongated protein occupies a
larger volume than a spherical protein of
the same mass
Employs porous beads
Proteins with large Stokes radii remain in the
eluent and emerge before proteins that have a
smaller Stokes radii and are able to enter the
porous beads
Proteins emerged via descending order of their
Stokes radii
 Ion Exchange
Chromatography
Proteins interact with the stationary phase via charge-
charge interactions

Proteins with net (+) charge at a given pH adhere to

beads with negatively charged functional groups such as
carboxylates or sulfates (cation exchangers)

Negatively charged proteins adhere to tertiary or

quaternary amines (anion exchangers)

Proteins compete with monvalent ions for binding hence

“ion exchange”

Example: negatively charged proteins bind to

diethylaminoethyl (DEAE) cellulose via replacing the Cl - or
CH3COO-

Bound proteins are selectively displaced by gradually

raising the concentration of the monovalent ions in the
mobile phase

Proteins elute in inverse order of strength of their

interactions with stationary phase

Sequential elution achieved via pH manipulation

Hydrophobic Interaction
Chromatography

Separates via tendency to associate with a stationary phase

matrix coated with hydrophobic groups (phenyl Sepharose,
octyl Sepharose)
Proteins with exposed hydrophobic surfaces adhere to the
matrix via hydrophobic interactions enhanced by a mobile
phase of high ionic strength
Nonadherent proteins are washed first
Polarity of the mobile phase is then decreased by gradually
lowering salt concentration
If interaction between protein and stationary phase is
particularly strong, ethanol or glycerol is added to decrease
polarity and weaken hydrophobic interactions
 Affinity Chromatography

Exploits the high selectivity of most proteins for

their ligands
Enzymes may be purified using immobilized
substrates, products, coenzymes, or inhibitors
In theory, only proteins interacting with
immobilized ligands adhere
Bound proteins eluted either by competition
with soluble ligand or less selectively by
disrupting protein-ligand interactions using
urea, guanidine HCl, mildly acidic pH, high
salt concentration
Stationary phase matrices available
commercially contain ligands such as NAD+ or
ATP analogs
Most powerful and widely applicable affinity
matrices are used for recombinant protein
purifications
Uses a Ni2+ matrix that binds proteins with an
attached polyhistidine tag and a glutathione
matrix that binds a recombinant protein linked to
glutathione S- transferase
High-Pressure Liquid
Chromatography

Employs incompressible silica or alumina microbeads as stationary

phase and pressures of up to a few thousand psi
Incompressible matrices permit both high flow rates and
enhanced resolution
Resolve complex mixtures of lipids or peptides whose properties
differ only slightly
Reversed phase HPLC exploits a hydrophobic stationary phase of
aliphatic polymers 3 -18 carbon atoms in length
Peptide mixtures are eluted using a gradient of a water-miscible
organic solvent such as acetonitrile or methanol
 Polyacrylamide Gel
Electrophoresis (PAGE)

Most widely used method for

determining protein purity
Uses sodium dodecyl sulfate
Electrophoresis separates charged
biomolecules based on the rates at
which they migrate in an applied
electrical field
Acrylamide is polymerized and
cross-linked to form a porous matrix
SDS denatures and binds to
proteins at a ratio of one molecule
of SDS per 2 peptide bonds
 Polyacrylamide Gel
Electrophoresis (PAGE)

2-mercaptoethanol or dithiothreitol
used to reduce or break disulfide
bonds
Large number of anionic SDS
molecules overwhelms the charge
contributions of the aa functional
groups
Charge to mass ratio of each SDS-
polypeptide complex is equal
The physical resistance encountered
by the polypeptide determines the
rate of migration
Large complexes encounter greater
resistance
Polypeptides separate based on their
relative molecular mass (Mr)
Individual polypeptides trapped in the
gel are stained using Coomassie blue
 Isoelectric Focusing

Ionic buffers called ampholytes and

an applied electric field are used to
generate a pH gradient within a
polyacrylamide marix
Applied proteins migrate until they
reach the region of the matrix
where the pH at which a molecule’s
net charge is 0
Used in conjunction with SDS-PAGE
Separates polypeptides based on pI
in one dimension and based on Mr
in the second
Sanger Amino Acid
Sequencing

Frederick Sanger won the Nobel Prize in 1958 for determining

the amino acid sequence of insulin
Insulin: 21 – aa residue A chain and 30 – aa residue B chain
linked by disulfide bonds
Separated both chains by reducing the disulfide bonds and
cleaved with trypsin, chymotrypsin, and pepsin
Resulting peptides isolated and treated with acid to hydrolyze
peptide bonds and generated 2 – 3 aa
Each peptide was reacted with 1-fluoro-2,4 - dinitrobenzene
(Sanger’s reagent)
While the ε-amino group of lysine reacts with Sanger’s reagent, amino-
terminal lysines can be distinguished from other positions because they
react to 2 mol of the Sanger’s reagent
Edman Amino Acid Sequencing

Pehr Edman introduced phenylisothiocyanate (Edman’s reagent)

Phenylthiohydantoin (PTH) derivative can be removed under mild
conditions to generate a new amino terminal residue
Successive rounds of derivatization with Edman’s reagent can be used
to sequence the first 20 – 30 residues
Most polypeptides must be cleaved into smaller peptides prior to
sequencing
Cleavage is necessary to circumvent posttranslational modifications
rendering a proteins α-amino group unreactive to Edman’s reagent
Following cleavage, the peptides are purified by reversed-phase HPLC
and sequenced
Edman Amino Acid
Sequencing
 Hybrid Approach

Edman sequencing provides a partial amino acid sequence

Oligonucleotide primers modeled on this partial sequence are used to ID the
gene and amplify it using PCR
Once the gene is ID, oligonucleotide sequence is determined to infer the
primary structure of the encoded polypeptide
Enhances the speed and efficiency of primary structure analysis
Circumvents obstacles brought about by amino-terminal blocking group or the
lack of key overlap peptide
Hybrid Approach

Only a few segments of the primary structure should be determined using

Edman’s approach
DNA sequencing reveals the order in which amino acids are added but does
not provide posttranslational modification
Proteolytic processing
Methylation
Glycosylation
Phosphorylation
Proline and lysine hydroxylation
Disulfide bond formation
Genomics Elucidates Proteins

Primary structure analysis revolutionized by genomics using the hybrid

approach
Automated oligonucleotide sequencing and computerized data retrieval
and analysis
Identification of the open reading frame (ORF) that encodes the protein
can be accessed from genome databases
A segment of amino acid around 4-5 residues in length is sufficient to identify
the correct ORF
Computerized search algorithms assist the identification of the gene
encoding the protein
Peptide mass profiling: a peptide is digested and introduced into the mass
spec
A computer program is used to find an ORF whose predicted protein
product would produce a set of peptides whose masses match the ones in
the mass spec
 Mass Spectrometry

Replaced Edman’s sequencing

Posttranslational modification of
proteins identified via mass increments
Discriminates molecules based solely
on mass
Can detect subtle physical changes in
proteins that occur during the life cycle
of a cell or organism
A sample in a vacuum is vaporized
under conditions where protonation
can occur, imparting a positive charge
An electrical field propels cations
through a magnetic field which
deflects them at right angles to their
original direction of flight and focuses
them onto the detector
The magnetic force required to deflect
the path of each ionic species onto the
detector measured as the current
applied to the electromagnet is
recorded
Mass Spectrometry

The force of ions with identical net charge is proportionate to their mass
In a time-of-flight mass spectrometer, a briefly applied electric field
accelerates the ions towards a detector that records the time at which
each ion arrives
Molecules with identical charge have velocities inversely proportional to
their mass
Conventional mass specs are used to determine the masses of
molecules of 1000 Da or less
Time-of-flight mass specs are suited for large protein masses
Previously, analysis of peptides and proteins by mass spec was hindered
by difficulties in volatilizing large organic molecules
Matrix-assisted laser-desorption (MALDI) and electrospray dispersion
(e.g. nanospray) permitted determination of large polypeptide masses
( 100,000 Da) at +/- 1 Da
 Mass Spectrometry

Using electrospray dispersion, peptides eluting from a

reversed-phase HPLC column are introduced directly
into the mass spec
Peptides inside the mass spec are broken down into
smaller units by collision with neutral helium (collision-
induced dissociation)
Peptide bond are more labile than carbon –carbon
bonds
Molecular mass of each amino acid is unique except
for leucine and isoleucine
Tandem mass spec:
Complex peptide mixtures can be analyzed without
purification
Employs the equivalent of 2 mass specs linked in series
First mass spec: separates individual peptides based on mass
Second mass spec: fragments single peptide
Used to screen blood samples from newborns (newborn
screening)
Abnormalities in metabolite (phenylketonuria, ethylmalonic
encephalopathy and glutaric acidemia type I)
3-D Structures: Xray
Crystallography

Protein is first precipitated to form crystals

Crystals are mounted into quartz capillaries and irradiated with
monochromatic x rays of 0.15 nm to confirm protein nature
Crystals are frozen in liquid nitrogen
Diffraction patterns recorded
Data analyzed using Fourier synthesis which summates wave
functions
Laue Xray crystallography:
Diffraction of polychromatic X rays
Crystal rotation is avoided
3-D Structures: Nuclear Magnetic
Resonance Spectroscopy

Measures the absorbance of radio frequency electromagnetic

energy by certain atomic nuclei
The frequency or chemical shift at which a particular nucleus
absorbs energy is a function of both the functional group within
which it resides and the proximity of other NMR-active nuclei
2-D NMR permits 3-D representation by determining the proximity of
the nuclei from one another
Analyzes proteins in aqueous solutions therefore conformation
accompanying ligand binding or catalysis is possible
Less than or equal to 30 kDa in size is analyzable
Molecular Modeling

Computer technology determining the 3-D protein structure

Molecular dynamics programs can be used to simulate
conformational dynamics of a protein and the manner in which
factors such as temperature, pH, ionic strength, amino acid
substitutions influence motion
Molecular docking programs simulate interactions between a
ligand-enzyme, etc
Homology modelling: known 3-D structure of a protein is used as a
template to build a model of the probable structure of a related
protein
Molecular Modeling
Introduction to BioEdit
Phylogenetic Mapping using
Protein Distance Method
Introduction to Protein BLAST
Introduction to Protein BLAST