Glycoprotein Analysis

Instrumental Techniques

Prafulla Kumar Sahu

M.Pharm (PhD.)
Alliance Institute of Advanced Pharmaceutical & Health Sciences
www.allianceinstitute.org 1
 What is Glycoprotein?
• Glycoproteins are proteins that contain
oligosaccharide chains (glycans) covalently
attached to polypeptide side-chains.
• This process is known as glycosylation. The
carbohydrate is attached to the protein during
the following modifications:
– Cotranslational modification
– Posttranslational modification
• In proteins that have segments extending
extracellularly, the extracellular segments are
often glycosylated.
2
 Types of glycoproteins
There are two types of glycoproteins:
1. N-glycosylation:
The addition of sugar chains at the amide
nitrogen of the amino acid (asparagine).
2. O-glycosylation:
The addition of sugar chains on the hydroxyl
oxygen side chain of the amino acid.
(hydroxylysine, hydroxyproline, serine, or
threonine)

3
 Functions
Structural molecule
Lubricant and protective agent
Transport molecule
Immunologic molecule
Hormone
Enzyme
Cell attachment-recognition site
Antifreeze
Interact with specific carbohydrates
Receptor
Affect folding of certain proteins
Regulation of development
Hemostasis (and thrombosis)
Glycoproteins
Collagens
Mucins
Transferrin, ceruloplasmin
Immunoglobins, histocompatibility antigens
Human Chorionic Gonadotropin (HCG),
Thyroid-Stimulating Hormone (TSH)
Various, eg, alkaline phosphatase
Various proteins involved in cell-cell (eg, sperm-
oocyte), virus-cell, bacterium-cell, and hormone cell
interactions
Certain plasma proteins of coldwater fish
Lectins, selectins (cell adhesion lectins), antibodies
Various proteins involved in hormone and drug
action
Calnexin, calreticulin
Notch and its analogs, key proteins in development
Specific glycoproteins on the surface membranes of
platelets 4
 Why do we analyse Glycoproteins?
• Glycoproteins are known to exhibit multiple biological functions.
• In order to assign distinct functional properties to defined structural
features, detailed information on the respective carbohydrate
moieties is required.
• Chemical and biochemical analyses, however, are often not possible
for the following reasons:
– Small amounts of sample available
– Vast structural heterogeneity of the glycans
• Thus highly sensitive and efficient methods for detection,
separation and structural investigation are required.
• glycoprotein analysis tries to explore:
– Suitable strategies for characterization of glycosylation at the levels of
intact proteins, glycopeptides and free oligosaccharides.
– Methods commonly used for isolation, fractionation and carbohydrate
structure analysis of liberated glycoprotein glycans with potential
applications in glycoproteomics.
• Protein analysis allows us to understand the function of the protein
based on its structure.
5
 Glycoprotein Analysis
• A variety of methods used in detection, purification,
and structural analysis of glycoproteins are:
Method Use
Detects glycoproteins as pink bands
Periodic acid-Schiff stain
after electrophoretic separation.
Incubation of cultured cells with
Leads to detection of a radioactive
glycoproteins as radioactive decay
sugar after electrophoretic separation.
bands
Resultant shifts in electrophoretic
migration help distinguish among
Treatment with appropriate endo- or
proteins with N-glycan, O-glycan, or GPI
exoglycosidase or phospholipases
linkages and also between high
mannose and complex N-glycans.
Agarose-lectin column chromatography, To purify glycoproteins or glycopeptides
lectin affinity chromatography that bind the particular lectin used. 6
 Method Use
Resultant shifts in electrophoretic migration help
distinguish and characterize glycoforms, i.e.
Lectin affinity electrophoresis
variants of a glycoprotein differing in
carbohydrate.
Compositional analysis Identifies sugars that the glycoprotein contains
following acid hydrolysis and their stoichiometry.
Provides information on molecular mass,
Mass spectrometry composition, sequence, and sometimes branching
of a glycan chain.
To identify specific sugars, their sequence,
NMR spectroscopy linkages, and the anomeric nature of glycosidic
chain.
Measures the mechanisms underlying the
biomolecular interactions, including reaction
Dual Polarisation Interferometry
rates, affinities and associated conformational
changes.
Methylation (linkage) analysis To determine linkage between sugars.
Amino acid or cDNA sequencing Determination of amino acid sequence.
7
 Glycoprotein Analysis
1. Characterization of intact glycoproteins
2. Characterization of glycopeptides
– Enrichment and capturing of glycopeptides
– Separation and selective detection of glycopeptides
– Analysis of N- and O-glycopeptides
– Analysis of O-GlcNAc peptides
3. Characterization of glycans
– Release of sugar chains
– Labelling of glycans
– Profiling and fractionation of glycans
• HPLC techniques
• Lectin affinity chromatography
• Capillary electrophoresis
– Mass mapping
– Mass spectrometric fragmentation analysis
– Enzymatic sequencing
– Linkage analysis
– Nuclear magnetic resonance spectroscopy
8
Characterization of intact Glycoproteins
Separation of proteins:
– SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis)
– 2-DE (2-dimensional gel electrophoresis)
• SDS-PAGE: is a technique widely used to separate proteins
according to their electrophoretic mobility (a function of
length of polypeptide chain or molecular weight). SDS gel
electrophoresis of samples having identical charge per unit
mass due to binding of SDS results in fractionation by size.
• Glycoprotein bands observed are often broad due to the
heterogeneous glycosylation pattern, thus making a
complete separation of different glycoforms difficult.
• 2D-E or 2-D electrophoresis: is a form of gel
electrophoresis where mixtures of proteins are separated
by two properties in two dimensions on 2D gels (isoelectric
point, protein mass).
9
 SDS-PAGE

Sodium Dodecyl Sulfate

PolyAcrylamide Gel Electrophoresis

10
 Scope & Purpose
• Scope:
– Polyacrylamide gel electrophoresis is used for the
qualitative characterization of proteins in biological
preparations, for control of purity and quantitative
determinations.
• Purpose:
– To identify and to assess the homogeneity of proteins in
pharmaceutical preparations
– Routine application for the estimation of protein subunit
molecular masses and for
– Determination of subunit compositions of purified
proteins.
– To separate proteins according to their size, and no other
physical feature.
11
 Sodium Dodecyl Sulfate
• SDS is a common ingredient in detergents
• Other names for SDS include laurel sulfate and
sodium laurel sulfate
• As a detergent SDS destroys protein secondary,
tertiary and quaternary structure
• This makes proteins rod shaped
• SDS also sticks to proteins in a ratio of
approximately 1.4 g of SDS for each gram of
protein
• Negative charge on the sulfate groups of SDS
mask any charge on the protein 12
 Sodium Dodecyl Sulfate
• Therefore, if a cell is incubated with SDS, the
membranes will be dissolved, all the proteins will
be soluablized by the detergent, plus all the
proteins will be covered with many negative
charges.
• The end result has two important features:
1. All proteins retain only their primary structure and
2. All proteins have a large negative charge which
means they will all migrate towards the positive
pole when placed in an electric field.
13
 SDS
Sodium Dodecyl Sulfate
C12H25NaO4S

HHHHHHHHHHHH O
H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+
HHHHHHHHHHHH O
Non-polar Polar
Hydrophobic tail Hydrophilic head

• Because it is amphipathic, SDS is a potent detergent

14
SDS and Proteins

SDS

Protein

15
 SDS and Proteins
• SDS nonpolar chains arrange themselves on proteins and
destroy secondary tertiary and quarternary structrure
• Thus shape is no longer an issue as the protein SDS
complex becomes rod shaped

• In aqueous solutions, SDS polarizes releasing Na+ and

retaining a negative charge on the sulfate head
• So much SDS binds to proteins that the negative charge
on the SDS drowns out any net charge on protein side
chains
• In the presence of SDS all proteins have uniform shape
and charge per unit length 16
 Polyacrylamide Gels
• Polyacrilamide is a polymer made of acrylamide
(C3H5NO) and bis-acrilamide (N,N’-methylene-
bis-acrylamide C7H10N2O2)

CH2 CH Acrylamide
O
O C NH2
C NH2
CH2
CH2 CH
O C NH2
Acrylamide
Acrylamide CH2 CH

bis-Acrylamide
17
 Polyacrylamide Gels
• Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate

O O

C NH2 C NH2
CH2 CH CH2 CH
SO4-.

18
 Polyacrylamide Gels
1. Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate
2. TMED (N,N,N’,N’-tetramethylethylenediamine)
serves as a catalyst in the reaction
O O O O

C NH2 C NH2 C NH2 C NH2

CH2 CH CH2 CH CH2 CH CH2 CH

SO4-.
19
 Polyacrylamide Gels
• bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
O O O

C NH2 C NH2 C NH2

CH2 CH CH2 CH CH2 CH CH2 CH

O C NH2

CH2 O O O

O C NH2 C NH2 C NH2 C NH2

CH2 CH CH2 CH CH2 CH CH2 CH
bis-Acrylamide
20
 Polyacrylamide Gels
• bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains

21
 Polyacrylamide Gels
• Pore size in gels can be varied by varying the ratio of
acrylamide to bis-acrylamide

Protein separations typically use a 29:1 or 37.5:1
acrylamide to bis ratio

Lots of bis-acrylamide
Little bis-acrylamide
22
 PAGE

This is a top view of two selected

tunnels.
All tunnels differ in diameter.

23
 SDS-PAGE
1 2 3

3
Addition
2 of SDS
1 Protein becomes rod-
shaped with uniform
charge distribution 24
 Since all the proteins have
strong negative charges,
they will all move in the
direction the arrow is
pointing (run to red+).

• Now take the mixture of denatured proteins to the gel and

apply the current.
• All the proteins enter the gel at the same time and have the
same force pulling them towards the other end
• Small molecules can manuver through the polyacrylamide
forest faster than big molecules.
• Because of their small size, they move through the forest
faster since they have access to more of the paths in the
forest while biggers are limited to only the larger paths. 25
• The collection of proteins of any given size tend to move through the gel
at the same rate, even if they do not take exactly the same tunnels to get
through.
• Proteins tend to move through a gel in bunches, or bands, since there are
so many copies of each protein and they are all the same shape and size.
• When running an SDS-PAGE, we never let the proteins electrophorese
(run) so long that they actually reach the other side of the gel. We turn off
the current and then stain the proteins and see how far they moved
through the gel (until we stain them, they are colorless and thus invisible).
• Notice that the actual bands are equal in size, but the proteins within each
band are of different sizes. 26
• You must always keep in mind…….
• SDS-PAGE separates proteins based on their:
– primary structure or size
– but not amino acid sequence.

we would not be able to use SDS-PAGE to separate

two proteins of the same molecular weight from
each other.

27
 Electrophoresis Principle
Separation of charged molecules in electric field is a
function of:
• Relative mobility of charged species (related to
frictional resistance which is related to size).
• Charge on the species.
• If < pH > then proteins are charged.
• Will migrate toward cathode (-) or anode (+).
• Separation occurs due to different rates of
migration due to magnitude of charge and
frictional resistance (related to size).
28
• Mobility:

Where,
Z = charge on molecule
E = Voltage applied (driving force)
f = frictional resistance
• Rf is measured by:

29
30
Factors influencing f:
• PAGE gel is a lattice or mesh with pores of defined size. Gel acts as a
sieve.
• Size of pore is inversely proportional to % acrylamide (the higher %
acrylamide, the smaller the pore).
• Increasing the % acrylamide in gel decreases pore size, increasing f
(frictional resistance).

• Rate of migration inversely proportional to molecular wt or mass of

protein.
• The larger the molecular, the slower it migrates in gel at constant voltage
(opposite of behavior on SEC column!) and charge.

• Problem in direction of movement is determined by Z:

if Z < 0, then  +
if Z > 0, then  -
if Z = 0, then  no movement

• How can you control Z?

– pH of the buffer
– Uniformly coating the protein with negative charge using Sodium
Dodecyl Sulfate (SDS). 31
• In addition to coating the protein with negative
charge, SDS also helps denature protein,
exposing hydrophobic groups to solvent.
• Statistically: 1 SDS / 2 amino acids.
• So, all proteins are negatively charged  they
will migrate to ANODE (+)
AND
• The (Z / mass) ratio for all proteins will be same.
• Because of this, the Rf for proteins will only be
dependent on the mass (f, frictional coefficient).
• Remember Rf ~ 1/mass
32
• When the (Z / mass) ratio
is the same (+SDS), the
proteins separate ONLY
based on MASS,
• Geometry having no
effect since the protein
has been denatured.
• Note: rate and order of
migration is opposite that
of SEC.

33
 Components of SDS PAGE Gel
Stacking Gel
Is prepared Tris/HCL buffer pH 6.8,
~2pH units lower than running
buffer. Large pore polyacrylamide
used to align and create a thin
starting zone of the protein of apx.
19um on top of the resolving gel.
• Lower % Acrylamide
Resolving Gel
Small pore polyacrylamide gel (3 -
30% acrylamide monomer) typically
made using a pH 8.8 Tris/HCl buffer.
• Higher % Acrylamide
Resolves protein ~24 – 205 kDa
Running Buffer
Tris/Glycine: Glycine(pKa=9.69) is a trailing ion (or slow ion). In other words
it runs through the gel slower then the slowest protein at a pH above 8.0. 34
 Stacking Gel Interactions:
• The upper portion is called the STACKING gel where the protein
bands get squeezed down to a very thin layer migrating toward
the anode. Stacking occurs due to differential migration of ionic
species that carry the electrical current through the gel.
• When an electrical current is applied to gel, ions carry the current
to the anode (+).
• Cl- ions, having the highest charge/mass ratio migrate faster, being
depleted at cathode end and concentrated at anode end.
• Glycine from electrophoresis buffer enters gel at pH 6.8 and
becomes primarily zwitterionic (charge zero) moving slowly.
• Protein, coated with SDS has a higher charge/mass ratio than
glycine  so moves fast, but slower than Cl-.
• When protein encounters resolving gel it slows down due to
increased frictional resistance (smaller pore size), allowing
following protein to “catch up” or stack.
• As protein is depleted from cathode end, glycine must carry
current so begins to migrate behind protein, in essence
concentrating the proteins further at stacking gel/resolving gel
interface. 35
• The stacking gel is a low concentration polyacrylamide gel
with a pH that is lower than the pH of the running buffer.
With this low pH, the glycine ions become zwitterions
(charge zero) that will be able to enter but they will not be
able to run through the stacking gel. Since the number of
charged ions in the stacking decreases when glycine
becomes a zwitterion, the voltage in the stacking will
increase and therefore the proteins will run very fast
through the stacking and will compact on the front of the
separating gel. Here, the pH is higher and, when the glycine
eventually reaches the separating, it will restore the voltage
in the stacking but it won't change it in the separating. In
the separating the proteins will be separated according to
their size

36
 Resolving Gel Interactions
• When glycine reaches resolving gel it becomes anionic and
migrates much faster than protein due to higher charge/mass
ratio. (pH is higher)
• Now proteins are sole carrier of current and separate according to
their molecular mass due to sieving effect of pores in gel.
• NOTE: in order for the proteins to behave in this manner, SDS
performs two important functions: Denaturing protein so
geometry is not a factor AND coating the protein UNIFORMLY with
negative charge!!!!!!!
• SDS is present in all of the buffers used AND is used to pretreat
the protein prior to loading onto gel.
Loading Buffer (LB):
– Tracking dye: 0.01% bromphenol blue
– SDS
– BME [ß- mercaptoethanol](reduces disulfide bonds)
– Glycerol (adds density)
– Stacking gel buffer
Protein is added to Loading Buffer (LB) and boiled. 37
38
 Staining Polyacrylamide Gels
• Coomassie Blue Stain- can usually detect a 10-50 ng protein per band
• Blue Safe Stains -Similar to Coomassie. Destaining optional or water rinse (8
ng/band)
– BioSafe Blue
– SimplyBlue
– GelCode
– Instant Blue- destaining not recommended
• Silver Staining- 50 times more sensitive than Coomassie Blue. (0.3ng/BAND)
– Fixation [Acetic acid-methanol]
– Sensitize gel with sodium thiosulfate
– Stain with silver solution
– Rinse with water
– Develop with formaldehyde and carbonate followed by stopping with Glacial acetic
• Fluorescent Stains –almost as sensitive as Silver but requires excitation
source
– Flamingo Fluorescent Gel Stain
– Deep Purple* Total Protein Stain
– SYPRO* Ruby Protein Gel Stain
– Krypton Protein Stain
– IR stains

39
 Coomassie –Protein Binding
Sulfonic acid group interacts with positively charged amine R
groups.
Basic amino acids including arginine, lysine and histidine but
weakly with histidine, tyrosine, tryptophan and phenylalanine

Interactions in its anionic form [-]

• Electrostatic
• Ionic
• Vander Waals
• Hydrophobic

40
 Destaining Gels
• Most gels require destaining to see banding and to eliminate
background stain for high resolution.
• Gels with abundant protein need not be destained when
using certain SafeBlue stains such as Instant Blue.
• Coomassie blue destaining:
– Usually requires acetic acid , methanol, and water
• Safe Blue destaining:
– Usually requires water rinse
• Silver Stains:
– Some methods use Potassium Ferricynide -Sodium Thiosulfate
solutions
– Some methods use Sodium chloride -Cupric sulfate -Sodium
thiosulfate pentahydrate.
– Some destaining may require a stop solution including 10% Acetic acid
41
2D Gel Electrophoresis

42
2D Gel Electrophoresis

Yeast Proteome:
50 ug protein loaded, pH
4-8 ampholines, 10% slab
gel, silver stain.

43
 2D Gel Electrophoresis

Separation of hundreds
of proteins based on

-pI
-MW

Up to 10,000 proteins can be

seen using optimized
protocols

44
 Why 2D Gels

Oldest method for large scale protein separation (since 1975)

Popular method for protein display and proteomics-one spot

at a time

Can be used in conjunction with Mass Spec

Permits simultaneous detection, display, purification,

identification, quantification, pI, and MW.

Robust, reproducible, simple, cost effective, scalable

Provides differential quantification using Differential 2D Gel

Electrophoresis (DIGE)
45
Processes involved in 2D gel electrophoresis
Protein isolation and quantification

Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)

Visualization of proteins spots with Dye

Identification of protein spots with Mass Spec

Bioinformatics
46
 Sample Preparation
• Sample preparation is key to successful 2D gel experiments
• Must select appropriate method to get selected proteins from
cellular compartment of interest
• Membrane proteins, nuclear proteins, and mitochodrial proteins
require special steps
• Must break all non-covalent protein-protein, protein-DNA,
protein-lipid interactions, disrupt S-S bonds
• Must prevent proteolysis, accidental phosphorylation, oxidation,
cleavage, ect..
• Must remove substances that might interfere with separation
process such as salts, polar detergents (SDS), lipids,
polysaccharides, nucleic acids
• Must try to keep proteins soluble during both phases of
electrophoresis process
• Must quantify protein

47
 Reduction
• DTT Treatment:
• Dithiothreitol (DTT) is a reducing agent typically used to
break down the disulfide bonds contributing to tertiary
structure which SDS was unable to affect, further
denaturing the protein to deemphasize the role of protein
shape in PAGE. DTT reduces a disulfide bond by two
sequential thiol-disulfide exchange reactions resulting in
DTT becoming a six-member ring structure. 2-
mercaptoethanol (ME), another disulfide reducing agent, is
commonly used in lieu of DTT. Furthermore, it should be
noted that while both DTT and ME sufficiently reduce
disulfide bridges in proteins, there is a propensity for them
to reform. Thus the protein sample is commonly treated
with 2-Iodoacetamide, an alkylating sulfhydryl reagent,
which binds covalently to free sulfides and prevents
disulfide bridges from reforming.
48
Reduction of a disulfide bond by two thiol-
disulfide exchange reactions involving DTT

49
 Protein Solubilization
• 2-20 mM Tris base (Carrier ampholytic buffer)
• 5-20 mM DTT (to reduce disulfide bonds)
• 8 M Urea (neutral chaotrope)
– Increases the solubility of some proteins
– Chaotropic agents interfere with stabilizing non-covalent
forces (hydrogen bonds, van der Waals forces, and
hydrophobic)
• 4% CHAPS Detergent (3-[(3-Cholamidopropyl)dimethylammonio]-1-
propanesulfonate)
– pH of 5-7
– Zwitterionic detergent (electronically neutral-has a both Neg
and Pos useful for varible charged peptides )
– Protects the native state of proteins
– Better when downstream apps include IEF because no affect
on pH gradients
50
 IEF and IPG (immobilized pH Gradient)
Strip of paper Made by covalently
integrating acrylamide and
variable pH ampholytes.
Separation on basis of pI, not MW
Available in different pH ranges
3-10
4-8
5-7 pH 3
10
4 5 6 7 8 9

Requires very high voltages

(5000V)and long period of time
(10h)

51
 Isoelectric Focusing
• In a pH gradient, under an electric field, a protein will move to the position in
the gradient where its net charge is zero.
• An immobilized pH gradient is created in a polyacrylamide gel strip by
incorporating a gradient of acidic and basic buffering groups when the gel is
cast.
• Proteins are denatured, reduced, and alkylated, and loaded in a visible dye.
• The sample is soaked into the gel along its entire length before the field is
applied.
• Resolution is determined by the slope of the pH gradient and the field
strength.

Immobilized pH
gradient gel
strips

Many can be run in

parallel for greater
reproducibility 52
 IPG Strips Contain Ampholytes

Ampholytes are molecules that contain both acidic and basic groups
Protein will migrate in the Matrix and will find their pH equilibrium (pI)

53
The Second Dimension …Running the Gel

54
 SDS Gel
Negative electrode
pH 3 4 5 6 7 8 9 10 IPG strip-
pressed down
into the SDS-
PAGE gel

Similar pI but
different mw
Similar mw but
Positive electrode different pI

55
 Different IPG pH ranges yield Different Results
pH 4 pH 5

pH 4 pH 9

pH 5 pH 7

56
 Gel Stains - Summary

Stain Sensitivity (ng/spot) Advantages

Coomassie-type 5-10 Simple, fast

Silver stain 1-4 Very sensitive, laborious

Copper stain 5-15 Reversible, 1 reagent

negative stain

Zinc stain 5-15 Reversible, simple, fast

high contrast neg. stain

SYPRO ruby 1-10 Very sensitive, fluorescent

57
 2D Gel Results

• 401 spots (peptides) identified

• 279 gene products

58
 2D Gel Post Analysis

Compare gel images and determine what bands/spots are different

Requires software to compare gels

Apparent
difference- Need
to extract spot
for MS

59
 Extracting a Gel Spot
Run Mass spec
Cut out spot Trypsin Digestion of Gel
spot

60
 Differential 2D Gel Electrophoresis [DIGE]

Allows you to mix samples and run a single 2d gel for comparative and
quantitative purposes

Fluorescent stain

Cy3-- Normal liver

Cy5--Tumor
Both

61
 Conclusions
• 2D gel electrophoresis is a popular method for
protein display, separation, visualization, and
quantitation
• A good precursor to MS, but not required
• 2D gels provide pI, MW data, and
photodocumentation
• Web tools are now available that permit partial
analysis and comparison of 2D gels using
software and simulators
• 2D gels are fun to run
62
63
Improved Sample Throughput- automated spot cutting
Improved Sample Throughput

64
 Why alternative approaches?
Drawbacks:
– The frequent under-representation of membrane
(glyco)proteins in common 2D-GE due to a low solubilizing
power of the non-ionic and zwitterionic detergents used.
– Many of them are only weakly stained by conventional
dyes due to the high carbohydrate content.
• Therefore, alternative approaches have to be applied
for isolation, purification and/or characterization of
membrane (glyco)proteins:
– Blue native electrophoresis (BN-PAGE):
Coomassie Brilliant Blue dye provides the necessary
charges to the protein complexes for the electrophoretic
separation.
– 2D benzyldimethyl-n-hexadecylammoniumchloride/SDS-
PAGE
– SDS-PAGE with nano-HPLC
65
 Why alternative approaches?
• A sensitive detection and characterization of the
glycosylation pattern of electroblotted proteins may be
achieved by carbohydrate-specific lectins.
• Using a panel of lectins with different specificities,
glycoproteins can be probed for defined oligosaccharide
epitopes.
• Additional information on the type of glycans attached can
be obtained by combining gel electrophoretic separation
and/or lectin probing with treatment by specific exo- and
endo-glycosidases as well as respective amidases.
• Using this approach, an initial assessment of the
glycosylation properties of a given glycoprotein may be
achieved.

66
 Robust Analytical Methods for
Protein Characterization
• Chemical derivatization:
• The ionization state of amino acids changes
with pH

67
Names, Abbreviations, and Properties of
The Twenty Amino Acids

68
 Acid-Base Chemistry in Protein
Characterization
• The net charge of a peptide or protein at any pH depends on the combined
pK values for its amino acids and terminal groups.
pH = pK + log [A-]/[HA]
pK of alpha-COOH groups: 1.8 -2.4
pK of alpha-NH2 groups: 9.0 -10.8
pK of ionizable side chains: 3.9 -12.5

– The isoelectric point is the pH at which there is no net charge.

It is important to remember how protein and peptide pK values
affect chemistry and separations:
Chemical Modification (e.g. Reduction / Alkylation)
Proteolysis (e.g. specificity of Glu-C)
Chromatography (e.g. Ion Exchange)
2D Gel Separations (Isoelectric Focusing)
Ionization for Mass Spectrometry (e.g. MALDI-TOFMS) 69
 Robust Analytical Methods for Protein
Characterization
Amino Acid Analysis:
Acid Hydrolysis followed by derivatization and HPLC
• Determines the precise molar ratios of amino acids present
• Can also be used to accurately determine concentration
Asp/Asn and Glu/Gln are not distinguished
Cysteine and Tryptophan are problematic in some methods
Amino-Terminal Sequencing by Edman Degradation:
• Very sensitive
• Standard method-still best approach to NH2-terminus
Very steep learning curve to do it well
Blocked proteins cannot be analyzed
Mixtures are challenging
Peptides with long repeats are problematic
PTMs (Post Translational Modifications)are often missed but can be dealt with
Often not competitive with MS for internal sequence 70
 Robust Analytical Methods for Protein
Characterization
Polyacrylamide Gel Electrophoresis
A crude measure of molecular weight and purity
• Analytical or preparative separations
• Coupled with Blotting-sensitive & selective detection
Isoelectric Focusing
Analytical or preparative separations
Used for mapping disease markers (e.g. Chronic granulomatous diseases)
Variety of pH gradients
Automated, high throughput instruments
Two Dimensional IEF –PAGE
• Orthogonal separations-large separation space
• Detection of small changes in complex samples
Separation of post-translationally modified proteins
Dynamic Range problems due to sample loading capacity,,
71
Chemical Methods for Protein Characterization

72
Chemical Methods for Protein Characterization: Proteolysis

73
 Edman degradation
• Edman degradation, developed by Pehr
Edman, is a method of sequencing amino
acids in a peptide. In this method, the amino-
terminal residue is labeled and cleaved from
the peptide without disrupting the peptide
bonds between other amino acid residues.

74
 Edman degradation: Mechanism

1. Phenylisothiocyanate is reacted with an uncharged terminal amino

group, under mildly alkaline conditions, to form a cyclical
phenylthiocarbamoyl derivative.
2. Then, under acidic conditions, this derivative of the terminal amino acid
is cleaved as a thiazolinone derivative.
3. The thiazolinone amino acid is then selectively extracted into an organic
solvent and treated with acid to form the more stable
phenylthiohydantoin (PTH)- amino acid derivative that can be identified
by using chromatography or electrophoresis. 75
 Edman degradation: Mechanism
• This procedure can then be repeated again to identify the
next amino acid.
• Drawback: to this technique is that the peptides being
sequenced in this manner cannot have more than 50 to 60
residues (and in practice, under 30).
• The peptide length is limited due to the cyclical derivitization
not always going to completion.
• The derivitization problem can be resolved by cleaving large
peptides into smaller peptides before proceeding with the
reaction. It is able to accurately sequence up to 30 amino
acids with modern machines capable of over 99% efficiency
per amino acid.
• Advantage: It only uses 10 - 100 picomoles of peptide for the
sequencing process. Edman degradation reaction is
automated to speed up the process 76
Chemical Methods for Protein Characterization:
A Basic Protocol for Denaturation & Proteolysis

Trichloro-
acetic acid

(Pellet should be formed from whitish, fluffy ppt.)

77
Chemical Methods for Protein Characterization:
A Basic Protocol for Denaturation & Proteolysis

Tosyllysine Chloromethyl Ketone HCl

78
Combining Analytical Methods for Protein Characterization

79
 Methods suitable for the separation
and characterization of glycopeptides

80
Strategies for the analysis of released
glycoprotein-glycans

81
Mass Spectrometry in
Proteomics
Methods & Theory
 Proteomics Tools

• Molecular Biology Tools

• Separation & Display Tools
• Protein Identification Tools
• Protein Structure Tools
 Mass Spectrometry Needs
• Ionization-how the protein is injected in to the MS
machine
• Separation-Mass and Charge is determined
• Activation-protein are broken into smaller fragments
(peptides/AAs)
• Mass Determination-m/z ratios are determined for
the ionized protein fragments/peptides
 Mass Spectrometry (MS)
• Introduce sample to the instrument
• Generate ions in the gas phase
• Separate ions on the basis of differences in m/z
with a mass analyzer
• Detect ions
 How does a mass spectrometer work?

Create ions Separate ions Detect ions

• Ionization • Mass analyzer • Mass

– MALDI-TOF spectrum
method • MW
• Database
– MALDI – Triple Quadrapole
• AA seq analysis
– Electrospray – MALDI-QqTOF
(Proteins must be charged
and dry) • AA seq and
MW
– QqTOF
• AA seq and
protein modif.
86
 Generalized Protein Identification by MS
Spectrum of
Spot removed Fragmented
fragments
from gel using trypsin
Library generated

MATCH

Database of
Artificial Artificially
sequences
spectra built trypsinated
(i.e. SwissProt)
 Methods for
protein
identification
 MS Principles
• Analytical method to measure the
molecular or atomic weight of samples
• Different compounds can be uniquely
identified by their mass
Butorphanol L-dopa Ethanol

N -CH2-
OH COOH
HO -CH2CH-NH2 CH3CH2OH

HO
HO

MW = 327.1 MW = 197.2 MW = 46.1

 Mass Spectrometry

• For small organic molecules the MW can be

determined to within 5 ppm or 0.0005% which is
sufficiently accurate to confirm the molecular
formula from mass alone

• For large biomolecules the MW can be determined

within an accuracy of 0.01% (i.e. within 5 Da for a 50
kD protein)

• Recall 1 dalton = 1 atomic mass unit (1 amu)

 MS Principles
• Find a way to “charge” an atom or molecule
(ionization)

• Place charged atom or molecule in a magnetic

field or subject it to an electric field and measure
its speed or radius of curvature relative to its
mass-to-charge ratio (mass analyzer)

• Detect ions using microchannel plate or

photomultiplier tube
 Mass Spec Principles

Sample

+
_

Ionizer Mass Analyzer Detector

 Mass spectrometers
Linear Time Of Flight tube
• Time of flight (TOF) (MALDI)
– Measures the time required for ions to fly ion source
down the length of a chamber.
– Often combined with MALDI (MALDI-TOF) detector

Detections from multiple laser bursts are

averaged. Multiple laser time of flight

Reflector Time Of Flight tube

• Tandem MS- MS/MS ion source

-separation and identification of compounds in
complex mixtures detector
reflector
- induce fragmentation and mass analyze the
fragment ions.
- Uses two or more mass analyzers/filters
separated by a collision cell filled with Argon time of flight

or Xenon

• Different MS-MS configurations

– Quadrupole-quadrupole (low energy)
– Magnetic sector-quadrupole (high)
– Quadrupole-time-of-flight (low energy)
– Time-of-flight-time-of-flight (low energy)
All proteins are sorted based on a
mass to charge ratio (m/z)

m/z ratio:

Molecular weight divided by the

charge on this protein
 Typical Mass Spectrum
Relative
Abundance

aspirin

120 m/z-for singly charged ion this is the mass

 Resolution & Resolving Power
• Width of peak indicates the resolution of the MS
instrument

• The better the resolution or resolving power, the

better the instrument and the better the mass
accuracy
DM
• Resolving power is defined as: M
M is the mass number of the observed mass (DM) is
the difference between two masses that can be
separated
Resolution in MS
 Different Types of MS
• GC-MS - Gas Chromatography MS
– separates volatile compounds in gas column and ID’s by mass

• LC-MS - Liquid Chromatography MS

– separates delicate compounds in HPLC column and ID’s by mass

• MS-MS - Tandem Mass Spectrometry

– separates compound fragments by magnetic field and ID’s by
mass

• LC/LC-MS/MS-Tandem LC and Tandem MS

– Separates by HPLC, ID’s by mass and AA sequence
 Mass Spectrometer Schematic
Turbo pumps
High Vacuum System Diffusion pumps
Rough pumps
Rotary pumps

Ion Mass Data

Inlet Detector
Source Filter System

Sample Plate MALDI TOF Microch plate PC’s

Target ESI Quadrupole Electron Mult. UNIX
HPLC IonSpray Ion Trap Hybrid Detec. Mac
GC FAB Mag. Sector
Solids probe LSIMS FTMS
EI/CI
 Different Ionization Methods
• Electron Impact (EI - Hard method)
– small molecules, 1-1000 Daltons, structure

• Fast Atom Bombardment (FAB – Semi-hard)

– peptides, sugars, up to 6000 Daltons

• Electrospray Ionization (ESI - Soft)

– peptides, proteins, up to 200,000 Daltons

• Matrix Assisted Laser Desorption (MALDI-Soft)

– peptides, proteins, DNA, up to 500 kD
 Electron Impact Ionization
• Sample introduced into instrument by heating it
until it evaporates

• Gas phase sample is bombarded with electrons

coming from rhenium or tungsten filament
(energy = 70 eV)

• Molecule is “shattered” into fragments (70 eV >>

5 eV bonds)

• Fragments sent to mass analyzer

EI Fragmentation of CH3OH

CH3OH CH3OH+
CH3OH CH2O=H+ + H
CH3OH + CH3 + OH
CH2O=H+ CHO=H+ + H
Why wouldn’t Electron Impact be suitable
for analyzing proteins?
 Why You Can’t Use EI For
Analyzing Proteins
• EI shatters chemical bonds

• Any given protein contains 20 different amino

acids

• EI would shatter the protein into not only into

amino acids but also amino acid sub-fragments
and even peptides of 2,3,4… amino acids

• Result is 10,000’s of different signals from a

single protein -- too complex to analyze
 Soft Ionization Methods

337 nm UV laser

Fluid (no salt)

+
_

cyano-hydroxy Gold tip needle

cinnamic acid

MALDI ESI
 Soft Ionization
• Soft ionization techniques keep the molecule of
interest fully intact

• Electro-spray ionization first conceived in 1960’s

by Malcolm Dole but put into practice in 1980’s
by John Fenn (Yale)

• MALDI first introduced in 1985 by Franz

Hillenkamp and Michael Karas (Frankfurt)

• Made it possible to analyze large molecules via

inexpensive mass analyzers such as quadrupole,
ion trap and TOF
 Ionization methods
• Electrospray mass spectrometry (ESI-MS):
– Liquid containing analyte is forced through a steel capillary at
high voltage to electrostatically disperse analyte. Charge
imparted from rapidly evaporating liquid.

• Matrix-assisted laser desorption ionization (MALDI):

– Analyte (protein) is mixed with large excess of matrix (small
organic molecule)
– Irradiated with short pulse of laser light. Wavelength of laser is
the same as absorbance max of matrix.
 Electrospray Ionization
• Sample dissolved in polar, volatile buffer (no
salts) and pumped through a stainless steel
capillary (70 - 150 mm) at a rate of 10-100 mL/min

• Strong voltage (3-4 kV) applied at tip along with

flow of nebulizing gas causes the sample to
“nebulize” or aerosolize

• Aerosol is directed through regions of higher

vacuum until droplets evaporate to near atomic
size (still carrying charges)
Electrospray (Detail)
 Electrospray Ionization
• Can be modified to “nanospray” system with flow < 1 mL/min

• Very sensitive technique, requires less than a picomole of

material

• Strongly affected by salts & detergents

• Positive ion mode measures (M + H)+ (add formic acid to

solvent)

• Negative ion mode measures (M - H)- (add ammonia to solvent)

 Positive or Negative Ion Mode?
• If the sample has functional groups that readily
accept H+ (such as amide and amino groups
found in peptides and proteins) then positive ion
detection is used-PROTEINS

• If a sample has functional groups that readily

lose a proton (such as carboxylic acids and
hydroxyls as found in nucleic acids and sugars)
then negative ion detection is used-DNA
Matrix--Assisted Laser Desorption
Matrix
Ionization

337 nm UV laser

cyano-hydroxy
cinnamic acid

MALDI
 MALDI
• Sample is ionized by bombarding sample with
laser light

• Sample is mixed with a UV absorbant matrix

(sinapinic acid for proteins, 4-hydroxycinnaminic
acid for peptides)

• Light wavelength matches that of absorbance

maximum of matrix so that the matrix transfers
some of its energy to the analyte (leads to ion
sputtering)
HT Spotting on a MALDI Plate
 MALDI Ionization
+ Matrix
+ - • Absorption of UV radiation by
+ - Laser chromophoric matrix and
-
+ ionization of matrix
Analyte

+ • Dissociation of matrix, phase

+ +-
+ + --+ change to super-compressed gas,
-
+ charge transfer to analyte
molecule
+
+
+ • Expansion of matrix at
+
supersonic velocity, analyte
+ trapped in expanding matrix
plume (explosion/”popping”)
 MALDI
• Unlike ESI, MALDI generates spectra that have just a singly
charged ion

• Positive mode generates ions of M + H

• Negative mode generates ions of M - H

• Generally more robust than ESI (tolerates salts and

nonvolatile components)

• Easier to use and maintain, capable of higher throughput

• Requires 10 mL of 1 pmol/mL sample

 Principle for MALDI-
MALDI-TOF MASS
peptide mixture
embedded in
light absorbing pulsed
UV or IR laser detector
chemicals (matrix)
(3-4 ns)

vacuum
+ +
+
+ + + +
+ + +

strong
electric
field
cloud of
Time Of Flight tube
Vacc protonated
peptide molecules
Principle for MALDI-
MALDI-TOF MASS
Linear Time Of Flight tube

ion source

detector

time of flight

Reflector Time Of Flight tube

ion source

detector
reflector

time of flight
 MALDI = SELDI

337 nm UV laser

cyano-hydroxy
cinnaminic acid

MALDI SELDI
MALDI//SELDI Spectra
MALDI
Normal

Tumor
 Mass Spectrometer Schematic

Turbo pumps
High Vacuum System Diffusion pumps
Rough pumps
Rotary pumps

Ion Mass Data

Inlet Detector
Source Filter System

Sample Plate MALDI TOF Microch plate PC’s

Target ESI Quadrupole Electron Mult. UNIX
HPLC IonSpray Ion Trap Hybrid Detec. Mac
GC FAB Mag. Sector
Solids probe LSIMS FTMS
EI/CI
 Different Mass Analyzers
• Magnetic Sector Analyzer (MSA)
– High resolution, exact mass, original MA

• Quadrupole Analyzer (Q)

– Low (1 amu) resolution, fast, cheap

• Time-of-Flight Analyzer (TOF)

– No upper m/z limit, high throughput

• Ion Trap Mass Analyzer (QSTAR)

– Good resolution, all-in-one mass analyzer

• Ion Cyclotron Resonance (FT-ICR)

– Highest resolution, exact mass, costly
 Different Types of MS

• ESI-QTOF
– Electrospray ionization source + quadrupole mass
filter + time-of-flight mass analyzer

• MALDI-QTOF
– Matrix-assisted laser desorption ionization +
quadrupole + time-of-flight mass analyzer

Both separate by MW and AA seq

Magnetic Sector Analyzer
 Quadrupole Mass Analyzer
• A quadrupole mass filter consists of four parallel
metal rods with different charges

• Two opposite rods have an applied + potential

and the other two rods have a - potential

• The applied voltages affect the trajectory of ions

traveling down the flight path

• For given dc and ac voltages, only ions of a

certain mass-to-charge ratio pass through the
quadrupole filter and all other ions are thrown
out of their original path
Quadrupole Mass Analyzer
 Q-TOF Mass Analyzer

NANOSPRAY MCP
TIP DETECTOR

PUSHER

HEXAPOLE
HEXAPOLE
COLLISION
CELL TOF
QUADRUPOLE
SKIMMER REFLECTRON
ION HEXAPOLE
SOURCE
 Mass Spec Equation (TOF)

m 2Vt2
=
z L2

m = mass of ion L = drift tube length

z = charge of ion t = time of travel
V = voltage
 Ion Trap Mass Analyzer
• Ion traps are ion trapping
devices that make use of a
three-dimensional
quadrupole field to trap and
mass-analyze ions

• invented by Wolfgang Paul

(Nobel Prize1989)

• Offer good mass resolving

power
 FT-ICR
Fourier-transform ion cyclotron resonance

• Uses powerful magnet (5-10 Tesla) to create a

miniature cyclotron

• Originally developed in Canada (UBC) by A.G.

Marshal in 1974

• FT approach allows many ion masses to be

determined simultaneously (efficient)

• Has higher mass resolution than any other MS

analyzer available
FT-Ion Cyclotron Analzyer
 Current Mass Spec Technologies
• Proteome profiling/separation
– 2D SDS PAGE - identify proteins
– 2-D LC/LC - high throughput analysis of lysates
(LC = Liquid Chromatography)
– 2-D LC/MS (MS= Mass spectrometry)

• Protein identification
– Peptide mass fingerprint
– Tandem Mass Spectrometry (MS/MS)

• Quantative proteomics
– ICAT (isotope-coded affinity tag)
– ITRAQ
 2D - LC/LC
Peptides all bind
to cation
(trypsin)
exchange
Study protein column (1D)
complexes without gel
electrophoresis Successive elution
with increasing
salt gradients
separates peptides
by charge

Peptides are
Complex mixture is separated by
simplified prior to hydrophobicity
MS/MS by 2D LC on reverse phase
column (2D)
 2D -
LC/MS
Peptide Mass Fingerprinting (PMF)
• Used to identify protein spots on gels or protein peaks from
an HPLC run

• Depends of the fact that if a peptide is cut up or fragmented

in a known way, the resulting fragments (and resulting
masses) are unique enough to identify the protein

• Requires a database of known sequences

• Uses software to compare observed masses

with masses calculated from database
 Principles of Fingerprinting
Sequence Mass (M+H) Tryptic Fragments
>Protein 1 acedfhsak
acedfhsakdfqea dfgeasdfpk
4842.05
sdfpkivtmeeewe ivtmeeewendadnfek
ndadnfekqwfe qwfe

>Protein 2 acek
acekdfhsadfqea dfhsadfgeasdfpk
4842.05
sdfpkivtmeeewe ivtmeeewenk
nkdadnfeqwfe dadnfeqwfe

>Protein 3 acedfhsadfgek
acedfhsadfqeka asdfpk
4842.05
sdfpkivtmeeewe ivtmeeewendak
ndakdnfeqwfe dnfegwfe
 Principles of Fingerprinting
Sequence Mass (M+H) Mass Spectrum
>Protein 1
acedfhsakdfqea
4842.05
sdfpkivtmeeewe
ndadnfekqwfe

>Protein 2
acekdfhsadfqea
4842.05
sdfpkivtmeeewe
nkdadnfeqwfe

>Protein 3
acedfhsadfqeka
4842.05
sdfpkivtmeeewe
ndakdnfeqwfe
Predicting Peptide Cleavages

http://ca.expasy.org/tools/peptidecutter/
http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps
 Protease Cleavage Rules
Sometimes
inhibition occurs

Trypsin XXX[KR]--[!P]XXX
Chymotrypsin XX[FYW]--[!P]XXX
Lys C XXXXXK-- XXXXX
Asp N endo XXXXXD-- XXXXX
CNBr XXXXXM--XXXXX
K-Lysine, R-Arginine, F-Phenylalanine, Y-Tyrosine,
W-Tryptophan,D-Aspartic Acid, M-Methionine, P-Proline
 Why Trypsin?
• Trypsin is the digestion enzyme
– Highly specific
– Cuts after K(Lysine) & R(Arginine) except if followed by
P(Proline)
• Robust, stable enzyme
• Works over a range of pH values & Temp.
• Quite specific and consistent in cleavage
• Cuts frequently to produce “ideal” MW peptides
• Inexpensive, easily available/purified
• Does produce “autolysis” peaks (which can be used in
MS calibrations)
– 1045.56, 1106.03, 1126.03, 1940.94, 2211.10, 2225.12, 2283.18,
2299.18
 Calculating Peptide Masses
• Sum the monoisotopic residue masses
Monoisotopic Mass
Mass:: the sum of the exact or accurate masses
of the lightest stable isotope of the atoms in a molecule
• Add mass of H2O (18.01056)
• Add mass of H+ (1.00785 to get M+H)
• If Met is oxidized add 15.99491
• If Cys has acrylamide adduct add 71.0371
• If Cys is iodoacetylated add 58.0071
• Other modifications are listed at
– http://prowl.rockefeller.edu/aainfo/deltamassv2.html

1H-1.007828503 amu 12C-12

2H-2.014017780 amu 13C-13.00335, 14C-14.00324
Masses in MS

• Monoisotopic mass is
the mass determined
using the masses of
the most abundant
isotopes

• Average mass is the

abundance weighted
mass of all isotopic
components
 Mass Calculation (Glycine)

NH2—CH2—COOH Amino acid

R1—NH—CH2—CO—R3 Residue

Glycine Amino Acid Mass

Monoisotopic Mass 5xH + 2xC + 2xO + 1xN
1H = 1.007825
= 75.032015 amu
12C = 12.00000
Glycine Residue Mass
14N = 14.00307
3xH + 2xC + 1xO + 1xN
16O = 15.99491
=57.021455 amu
 Amino Acid Residue Masses
Monoisotopic Mass

Glycine 57.02147 Aspartic acid 115.02695

Alanine 71.03712 Glutamine 128.05858
Serine 87.03203 Lysine 128.09497
Proline 97.05277 Glutamic acid 129.0426
Valine 99.06842 Methionine 131.04049
Threonine 101.04768 Histidine 137.05891
Cysteine 103.00919 Phenylalanine 147.06842
Isoleucine 113.08407 Arginine 156.10112
Leucine 113.08407 Tyrosine 163.06333
Asparagine 114.04293 Tryptophan 186.07932
 Amino Acid Residue Masses
Average Mass

Glycine 57.0520 Aspartic acid 115.0886

Alanine 71.0788 Glutamine 128.1308
Serine 87.0782 Lysine 128.1742
Proline 97.1167 Glutamic acid 129.1155
Valine 99.1326 Methionine 131.1986
Threonine 101.1051 Histidine 137.1412
Cysteine 103.1448 Phenylalanine 147.1766
Isoleucine 113.1595 Arginine 156.1876
Leucine 113.1595 Tyrosine 163.1760
Asparagine 114.1039 Tryptophan 186.2133
 Advantages of PMF
• Uses a “robust” & inexpensive form of MS (MALDI)

• Doesn’t require too much sample optimization

• Can be done by a moderately skilled operator (don’t need to

be an MS expert)

• Widely supported by web servers

• Improves as DB’s get larger & instrumentation gets better

• Very amenable to high throughput robotics (up to 500 samples

a day)
 Limitations With PMF
• Requires that the protein of interest already be
in a sequence database

• Spurious or missing critical mass peaks always

lead to problems

• Mass resolution/accuracy is critical, best to have

<20 ppm mass resolution

• Generally found to only be about 40% effective

in positively identifying gel spots
 Tandem Mass Spectrometry
• Purpose is to fragment ions from parent ion to
provide structural information about a molecule

• Also allows mass separation and AA identification

of compounds in complex mixtures

• Uses two or more mass analyzers/filters separated

by a collision cell filled with Argon or Xenon

• Collision cell is where selected ions are sent for

further fragmentation
MS--MS & Proteomics
MS
 Tandem Mass Spectrometry

• Different MS-MS configurations

– Quadrupole-quadrupole (low energy)
– Magnetic sector-quadrupole (high)
– Quadrupole-time-of-flight (low energy)
– Time-of-flight-time-of-flight (low energy)
 How Tandem MS
sequencing works
• Use Tandem MS: two mass analyzers Ser-Glu-Leu-Ile-Arg-Trp
in series with a collision cell in
between

• Collision cell: a region where the ions Collision Cell

collide with a gas (He, Ne, Ar) resulting
in fragmentation of the ion
Ser-Glu-Leu-Ile-Arg
• Fragmentation of the peptides occur in
a predictable fashion, mainly at the
peptide bonds Ser-Glu-Leu-Ile

Ser-Glu-Leu
• The resulting daughter ions have
masses that are consistent with known
molecular weights of dipeptides, Etc…
tripeptides, tetrapeptides…
 Advantages of Tandem Mass Spec
FAST
No Gels
Determines MW and AA sequence
Can be used on complex mixtures-including low copy #
Can detect post-translational modif.-ICAT
High-thoughput capability

Disadvantages of Tandem Mass Spec

Very expensive-Campus
Hardware: $1000
Setup: $300
1 run: $1000

Requires sequence databases for analysis

 MS--MS & Proteomics
MS
Advantages Disadvantages
• Provides precise sequence- • Requires more handling,
specific data refinement and sample
manipulation
• More informative than
PMF methods (>90%) • Requires more expensive and
complicated equipment
• Can be used for de-novo
sequencing (not entirely • Requires high level expertise
dependent on databases)
• Slower, not generally high
• Can be used to ID post- throughput
trans. modifications
 Different MS-
MS-MS Modes
• Product or Daughter Ion Scanning
– first analyzer selects ion for further fragmentation
– most often used for peptide sequencing

• Precursor or Parent Ion Scanning

– no first filtering, used for glycosylation studies

• Neutral Loss Scanning

– selects for ions of one chemical type (COOH, OH)

• Selected/Multiple Reaction Monitoring

– selects for known, well characterized ions only
ISOTOPE-CODED AFFINITY TAG (ICAT): a
quantitative method
• Label protein samples with heavy and light reagent
• Reagent contains affinity tag and heavy or light isotopes

Chemically reactive group: forms a covalent bond to

the protein or peptide

Isotope-labeled linker: heavy or light, depending on

which isotope is used

Affinity tag: enables the protein or peptide bearing an

ICAT to be isolated by affinity chromatography in a
single step
 Example of an ICAT Reagent
Biotin Affinity tag: Binds Reactive group: Thiol-reactive group will
tightly to streptavidin- bind to Cys
agarose resin

O
Linker: Heavy version will have
deuteriums at *
HN Light version will have hydrogens
NH at *

H H
N * O O * N
I
* O *

S O
O
The ICAT Reagent
 How ICAT works?
Affinity isolation on
streptavidin beads

Lyse & Quantification Identification

Label MS MS/MS

NH2-EACDPLR-COOH
Light
100
100
MIX
Heavy

Proteolysis
(ie trypsin)
0 0
550 570 590 200 400 600
m/z m/z
ICAT Quantitation
 ICAT
Advantages vs. Disadvantages
• Estimates relative protein • Yield and non specificity
levels between samples with a
reasonable level of accuracy • Slight chromatography
(within 10%) differences

• Can be used on complex • Expensive

mixtures of proteins
• Cys-specific label reduces
sample complexity
• Tag fragmentation
• Meaning of relative
quantification information

• Peptides can be sequenced

• No presence of cysteine
directly if tandem MS-MS is
residues or not accessible by
used ICAT reagent
 Mass Spectrometer Schematic
Turbo pumps
High Vacuum System Diffusion pumps
Rough pumps
Rotary pumps

Ion Mass Data

Inlet Detector
Source Filter System

Sample Plate MALDI TOF Microch plate PC’s

Target ESI Quadrupole Electron Mult. UNIX
HPLC IonSpray Ion Trap Hybrid Detec. Mac
GC FAB Mag. Sector
Solids probe LSIMS FTMS
EI/CI
 MS Detectors
• Early detectors used photographic film

• Today’s detectors (ion channel and electron

multipliers) produce electronic signals via 2o
electronic emission when struck by an ion

• Timing mechanisms integrate these signals with

scanning voltages to allow the instrument to report
which m/z has struck the detector

• Need constant and regular calibration

Mass Detectors

Electron Multiplier (Dynode)

Limitations of Proteomics

-solubility of indiv. protein differs

-2D gels unable to resolve all proteins at a given time
-most proteins are not abundant (ie kinases)
-proteins not in the database cannot be identified
-multiple runs can be expensive
-proteins are fragile and can be degraded easily
-proteins exist in multiple isoforms
-no protein equivalent of PCR exists for amplification
of small samples
Shotgun Proteomics:
Multi
Mu ltid
dimensional Protein
Identification Technology
(MudPIT)
General Strategy for Proteomics Characterization

Fractionation &
Isolation

2-DE Liquid
Chromatography

Peptides

Characterization
MALDI-TOF MS
Mass Spectrometry
• Identification -(LC)-ESI-MS/MS
• Post Translational modifications
• Quantification
Database Search
Overview of Shotgun Proteomics: MudPIT
Protein Mixture

Tandem Mass Digestion

Spectrometer

Peptide
Mixture
2D Chromatography

RP SCX
> 1,000 Proteins
Identified

SEQUEST®
DTASelect &
MS/MS Spectrum
Contrast
PySpzS5609 #2438 RT: 66.03 AV: 1 NL: 8.37E6
T: + c d Full m s2 729.75@35.00 [ 190.00-1470.00]
545.31
100

95

90

85

80

75

658.36
70

65 900.36

60
Relative Abundance

55
1031.40
50

45

913.42
40
1240.53
782.23
35 896.29

895.33 1032.43
30
546.19 771.24
1028.41
25
721.31

20
431.15 801.38

15 914.34 1241.39
427.27 559.13
317.17 1258.56
669.39 1033.60
10 408.74 651.14 1027.22 1312.35
1142.43
432.40 882.07 915.53
399.24 600.24
5 217.91 986.50 1123.49
481.13 869.23 1195.44 1356.10

0
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m /z
 MudPIT
IEX-HPLC RP-HPLC

Trypsin
+ proteins

p53
 Acquiring MS/MS Datasets

2D Chromatography

SCX RP

Tandem MS Spectrum
MudPIT Cycle
Peptide Sequence is Inferred from Fragment ions
 load sample
 wash
 salt step
 wash x 3~18
 RP gradient
 re-equilibration
 MS/MS of Peptide Mixtures

LC

MS
(MW Profile)

MS/MS
(AA Identity)
 Summary of MudPIT
 It is an automated and high throughput technology.

 It is a totally unbias method for protein identification.

 It identifies proteins missed by gel-based methods (i.e. (low

abundance, membrane proteins etc.)

 Post translational modification information of proteins can be

obtained, thus allowing their functional activities to be derived or
inferred.
 2-DE
• Widely used, highly
commercialized
• High resolving power
• Visual presentation
• Limited dynamic range
• Only good for highly soluble and
high abundance proteins
• Large amount of sample required
vs MudPIT
• Highly automated process
• Identified proteins with extreme
pI values, low abundance and
those from membrane
• Thousands of proteins can be
identified
• Not yet commercialized
• Expensive
• Computationally intensive
• Quantitation
THE END