Glycoprotein Analysis Instrumental Techniques

Prafulla Kumar Sahu
M.Pharm (PhD.) Alliance Institute of Advanced Pharmaceutical & Health Sciences

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.

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)

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) 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, spermoocyte), 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 4 platelets

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: • 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. – Small amounts of sample available – Vast structural heterogeneity of the glycans

• Protein analysis allows us to understand the function of the protein based on its structure.

Glycoprotein Analysis
• A variety of methods used in detection, purification, and structural analysis of glycoproteins are:
Periodic acid-Schiff stain

Detects glycoproteins as pink bands 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

Lectin affinity electrophoresis Compositional analysis following acid hydrolysis Mass spectrometry

Resultant shifts in electrophoretic migration help distinguish and characterize glycoforms, i.e. variants of a glycoprotein differing in carbohydrate. Identifies sugars that the glycoprotein contains and their stoichiometry. Provides information on molecular mass, composition, sequence, and sometimes branching of a glycan chain. To identify specific sugars, their sequence, linkages, and the anomeric nature of glycosidic chain.

NMR spectroscopy

Measures the mechanisms underlying the biomolecular interactions, including reaction Dual Polarisation Interferometry rates, affinities and associated conformational changes. Methylation (linkage) analysis Amino acid or cDNA sequencing To determine linkage between sugars. Determination of amino acid sequence.

Glycoprotein Analysis
1. 2. Characterization of intact glycoproteins 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 Release of sugar chains Labelling of glycans Profiling and fractionation of glycans
HPLC techniques Lectin affinity chromatography Capillary electrophoresis


Characterization of glycans
– – –
• • •

– – – – –

Mass mapping Mass spectrometric fragmentation analysis Enzymatic sequencing Linkage analysis Nuclear magnetic resonance spectroscopy

Characterization of intact Glycoproteins
Separation of proteins:
– SDS-PAGE (sodium dodecyl sulfate electrophoresis) – 2-DE (2-dimensional gel electrophoresis) polyacrylamide gel

• 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).


Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis


Scope & Purpose
• Scope:
– Polyacrylamide gel electrophoresis is used for the qualitative characterization of proteins in biological preparations, for control of purity and quantitative determinations. – 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.

• Purpose:

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

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.

Sodium Dodecyl Sulfate



Non-polar Hydrophobic tail Polar Hydrophilic head


• Because it is amphipathic, SDS is a potent detergent

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

SDS and Proteins

• •

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 16 and charge per unit length

Polyacrylamide Gels
• Polyacrilamide is a polymer made of acrylamide (C3H5NO) and bis-acrilamide (N,N’-methylenebis-acrylamide C7H10N2O2)





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

O C CH2 CH NH2 CH2 SO4-.



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


• bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

Polyacrylamide Gels




• bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

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

Polyacrylamide Gels

Lots of bis-acrylamide Little bis-acrylamide


This is a top view of two selected tunnels. All tunnels differ in diameter.

1 2 3


Addition of SDS 2 Protein becomes rodshaped 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.

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).

• Mobility:

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



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).


• 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

• 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.

Stacking Gel

Components of SDS PAGE 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

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):

– – – – –

Protein is added to Loading Buffer (LB) and boiled.

Tracking dye: 0.01% bromphenol blue SDS BME [ß- mercaptoethanol](reduces disulfide bonds) Glycerol (adds density) Stacking gel buffer



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


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


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

2D Gel Electrophoresis


2D Gel Electrophoresis
Yeast Proteome: 50 ug protein loaded, pH 4-8 ampholines, 10% slab gel, silver stain.


2D Gel Electrophoresis

Separation of hundreds of proteins based on -pI -MW Up to 10,000 proteins can be seen using optimized protocols


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)

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

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

• 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. 2mercaptoethanol (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.

Reduction of a disulfide bond by two thioldisulfide exchange reactions involving DTT


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

– 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

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 Requires very high voltages (5000V)and long period of time (10h)

pH 3 10








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 52 reproducibility

• • •

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)


The Second Dimension …Running the Gel


pH 3 4 5 6 7

Negative electrode
8 9 10

IPG strippressed down into the SDSPAGE gel

Similar pI but different mw

Positive electrode

Similar mw but different pI


Different IPG pH ranges yield Different Results
pH 4 pH 4 pH 9 pH 5

pH 5

pH 7


Gel Stains - Summary
Coomassie-type Silver stain Copper stain

Sensitivity (ng/spot)
5-10 1-4 5-15 Simple, fast


Very sensitive, laborious Reversible, 1 reagent negative stain

Zinc stain


Reversible, simple, fast high contrast neg. stain

SYPRO ruby


Very sensitive, fluorescent


2D Gel Results

• •

401 spots (peptides) identified 279 gene products


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


Extracting a Gel Spot
Cut out spot
Trypsin Digestion of Gel spot

Run Mass spec


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



• 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


Improved Sample Throughput- automated spot cutting Improved Sample Throughput


Why alternative approaches?
– 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/SDSPAGE – SDS-PAGE with nano-HPLC

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.

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


Names, Abbreviations, and Properties of The Twenty Amino Acids


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: pK of alpha-NH2 groups: pK of ionizable side chains: 1.8 -2.4 9.0 -10.8 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)


Amino Acid Analysis:
• •

Robust Analytical Methods for Protein Characterization

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 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

Amino-Terminal Sequencing by Edman Degradation:
• •

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,,

Chemical Methods for Protein Characterization


Chemical Methods for Protein Characterization: Proteolysis


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


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 75 by using chromatography or electrophoresis.

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 76 automated to speed up the process

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

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


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

Tosyllysine Chloromethyl Ketone HCl


Combining Analytical Methods for Protein Characterization


Methods suitable for the separation and characterization of glycopeptides


Strategies for the analysis of released glycoprotein-glycans


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 method
– MALDI – Electrospray
(Proteins must be charged and dry)

• Mass analyzer

• MW
– Triple Quadrapole

• AA seq

• Mass spectrum • Database analysis

• AA seq and MW

• AA seq and protein modif.


Generalized Protein Identification by MS
Spot removed from gel Fragmented using trypsin Spectrum of fragments generated



Artificial spectra built

Artificially trypsinated

Database of sequences (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 N -CH2OH HO HO HO MW = 327.1 MW = 197.2 MW = 46.1 L-dopa COOH -CH2CH-NH2 CH3CH2OH Ethanol

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


+ _


Mass Analyzer


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) Detections from multiple laser bursts are averaged. Multiple laser Reflector Time Of Flight tube

time of flight

Tandem MS- MS/MS ion source -separation and identification of compounds in complex mixtures - induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon 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)



time of flight

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 • Resolving power is defined as:


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
High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps


Ion Source

Mass Filter
TOF Quadrupole Ion Trap Mag. Sector FTMS


Data System

Sample Plate Target HPLC GC Solids probe

Microch plate Electron Mult. Hybrid Detec.

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

+ H

CH3 + OH

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 cinnamic acid

Gold tip needle



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

MatrixMatrix-Assisted Laser Desorption Ionization
337 nm UV laser

cyano-hydroxy cinnamic acid


• 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 Laser Analyte

• Absorption of UV radiation by chromophoric matrix and ionization of matrix • 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”)


+ +



• 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-TOF MASS MALDIpeptide mixture embedded in light absorbing chemicals (matrix) pulsed UV or IR laser (3-4 ns) detector

+ + + + + + +

strong electric field



cloud of protonated peptide molecules

Time Of Flight tube

Principle for MALDI-TOF MASS MALDILinear Time Of Flight tube
ion source


time of flight

Reflector Time Of Flight tube
ion source



time of flight

337 nm UV laser

cyano-hydroxy cinnaminic acid





Mass Spectrometer Schematic
High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps


Ion Source

Mass Filter
TOF Quadrupole Ion Trap Mag. Sector FTMS


Data System

Sample Plate Target HPLC GC Solids probe

Microch plate Electron Mult. Hybrid Detec.

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

– Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer
Both separate by MW and AA seq

Magnetic Sector 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

Quadrupole Mass Analyzer

Q-TOF Mass Analyzer





Mass Spec Equation (TOF)
m = z L2
m = mass of ion z = charge of ion V = voltage
2 2Vt

L = drift tube length t = time of travel

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

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

Study protein complexes without gel electrophoresis

Peptides all bind to cation exchange column (1D) Successive elution with increasing salt gradients separates peptides by charge Peptides are separated by hydrophobicity on reverse phase column (2D)

Complex mixture is simplified prior to MS/MS by 2D LC


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 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >Protein 2 acekdfhsadfqea sdfpkivtmeeewe nkdadnfeqwfe >Protein 3 acedfhsadfqeka sdfpkivtmeeewe ndakdnfeqwfe


acedfhsak dfgeasdfpk ivtmeeewendadnfek qwfe acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe acedfhsadfgek asdfpk ivtmeeewendak dnfegwfe



Principles of Fingerprinting
Sequence Mass (M+H) Mass Spectrum

>Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >Protein 2 acekdfhsadfqea sdfpkivtmeeewe nkdadnfeqwfe >Protein 3 acedfhsadfqeka sdfpkivtmeeewe ndakdnfeqwfe




Predicting Peptide Cleavages

Protease Cleavage Rules
Sometimes inhibition occurs

Trypsin Chymotrypsin Lys C Asp N endo CNBr


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: the sum of the exact or accurate masses Mass: 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
12C-12 13C-13.00335, 14C-14.00324


amu 2H-2.014017780 amu

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 R1—NH—CH2—CO—R3
Monoisotopic Mass 1H = 1.007825 12C = 12.00000 14N = 14.00307 16O = 15.99491 Amino acid


Glycine Amino Acid Mass 5xH + 2xC + 2xO + 1xN = 75.032015 amu Glycine Residue Mass 3xH + 2xC + 1xO + 1xN =57.021455 amu

Amino Acid Residue Masses
Monoisotopic Mass Glycine 57.02147 Alanine 71.03712 Serine 87.03203 Proline 97.05277 Valine 99.06842 Threonine 101.04768 Cysteine 103.00919 Isoleucine 113.08407 Leucine 113.08407 Asparagine 114.04293 Aspartic acid Glutamine Lysine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan 115.02695 128.05858 128.09497 129.0426 131.04049 137.05891 147.06842 156.10112 163.06333 186.07932

Amino Acid Residue Masses
Average Mass Glycine 57.0520 Alanine 71.0788 Serine 87.0782 Proline 97.1167 Valine 99.1326 Threonine 101.1051 Cysteine 103.1448 Isoleucine 113.1595 Leucine 113.1595 Asparagine 114.1039 Aspartic acid Glutamine Lysine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan 115.0886 128.1308 128.1742 129.1155 131.1986 137.1412 147.1766 156.1876 163.1760 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

MSMS-MS & Proteomics

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 in series with a collision cell in between Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion Fragmentation of the peptides occur in a predictable fashion, mainly at the peptide bonds The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides… Ser-Glu-Leu-Ile-Arg-Trp

Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu-Ile Ser-Glu-Leu Etc…

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

MSMS-MS & Proteomics
Advantages Disadvantages
• Provides precise sequence- • Requires more handling, specific data refinement and sample manipulation • More informative than PMF methods (>90%) • Can be used for de-novo sequencing (not entirely dependent on databases) • Can be used to ID posttrans. modifications • Requires more expensive and complicated equipment • Requires high level expertise • Slower, not generally high throughput

Different MS-MS Modes MS• 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 tightly to streptavidinagarose resin Reactive group: Thiol-reactive group will bind to Cys

Linker: Heavy version will have deuteriums at * Light version will have hydrogens at *


* *






The ICAT Reagent

How ICAT works?
Affinity isolation on streptavidin beads Lyse & Label

Quantification MS

Identification MS/MS NH2-EACDPLR-COOH

100 MIX



Proteolysis (ie trypsin) 0 550 570 m/z 590 0 200 400 m/z 600

ICAT Quantitation

ICAT Advantages vs. Disadvantages
• Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%) Can be used on complex mixtures of proteins Cys-specific label reduces sample complexity Peptides can be sequenced directly if tandem MS-MS is used • • Yield and non specificity Slight chromatography differences Expensive Tag fragmentation Meaning of relative quantification information No presence of cysteine residues or not accessible by ICAT reagent

• •

Mass Spectrometer Schematic
High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps


Ion Source

Mass Filter
TOF Quadrupole Ion Trap Mag. Sector FTMS


Data System

Sample Plate Target HPLC GC Solids probe

Microch plate Electron Mult. Hybrid Detec.

• 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

MS Detectors

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: Multid Multidimensional Protein Identification Technology (MudPIT)

General Strategy for Proteomics Characterization
Fractionation & Isolation 2-DE Liquid Chromatography


Characterization • Identification • Post Translational modifications • Quantification Mass Spectrometry


Database Search

Overview of Shotgun Proteomics: MudPIT
Protein Mixture Tandem Mass Spectrometer Digestion

2D Chromatography RP SCX

Peptide Mixture

> 1,000 Proteins Identified

MS/MS Spectrum
PySpzS5609 #2438 RT: 66.03 AV: 1 NL: 8.37E6 T: + c d Full m s2 729.75@35.00 [ 190.00-1470.00] 100 95 90 85 80 75 70 65 60 Relative Abundance 55 1031.40 50 45 40 782.23 35 30 25 20 15 10 5 0 200 300 400 500 600 700 800 m /z 900 1000 1100 1200 1300 1400 217.91 317.17 896.29 895.33 546.19 771.24 721.31 431.15 427.27 408.74 399.24 432.40 481.13 559.13 651.14 600.24 669.39 882.07 869.23 1027.22 915.53 986.50 1033.60 1142.43 1123.49 1195.44 801.38 914.34 1241.39 1258.56 1312.35 1028.41 1032.43 913.42 1240.53 658.36 545.31

SEQUEST® DTASelect & Contrast




Trypsin + proteins


Acquiring MS/MS Datasets

2D Chromatography SCX RP

MudPIT Cycle  load sample  wash  salt step  wash  RP gradient  re-equilibration

Tandem MS Spectrum Peptide Sequence is Inferred from Fragment ions

x 3~18

MS/MS of Peptide Mixtures

(MW Profile)

(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.

• Widely used, highly commercialized High resolving power Visual presentation

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

• • •

Limited dynamic range Only good for highly soluble and high abundance proteins Large amount of sample required

• • • •


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