Glycoprotein AnalysisGlycoprotein AnalysisInstrumental TechniquesInstrumental TechniquesInstrumental TechniquesInstrumental Techniques

Prafulla Kumar SahuM.Pharm (PhD.)

Alliance Institute of Advanced Pharmaceutical & Health Sciences

www.allianceinstitute.org1

What is Glycoprotein?

• Glycoproteins are proteins that containoligosaccharide chains (glycans) covalentlyattached to polypeptide side-chains.

• This process is known as glycosylation. Thecarbohydrate is attached to the protein duringcarbohydrate is attached to the protein duringthe following modifications:– Cotranslational modification– Posttranslational modification

• In proteins that have segments extendingextracellularly, the extracellular segments areoften glycosylated.

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Types of glycoproteins

There are two types of glycoproteins:1. N-glycosylation:

The addition of sugar chains at the amidenitrogen of the amino acid (asparagine).nitrogen of the amino acid (asparagine).

2. O-glycosylation: The addition of sugar chains on the hydroxyloxygen side chain of the amino acid.(hydroxylysine, hydroxyproline, serine, orthreonine)

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FunctionsFunctions GlycoproteinsGlycoproteinsStructural molecule Collagens

Lubricant and protective agent Mucins

Transport molecule Transferrin, ceruloplasmin

Immunologic molecule Immunoglobins, histocompatibility antigens

Hormone Human Chorionic Gonadotropin (HCG),Thyroid-Stimulating Hormone (TSH)

Enzyme Various, eg, alkaline phosphatase

Cell attachment-recognition siteVarious proteins involved in cell-cell (eg, sperm-oocyte), virus-cell, bacterium-cell, and hormone cellCell attachment-recognition site oocyte), virus-cell, bacterium-cell, and hormone cellinteractions

Antifreeze Certain plasma proteins of coldwater fish

Interact with specific carbohydrates Lectins, selectins (cell adhesion lectins), antibodies

Receptor Various proteins involved in hormone and drugaction

Affect folding of certain proteins Calnexin, calreticulin

Regulation of development Notch and its analogs, key proteins in development

Hemostasis (and thrombosis) Specific glycoproteins on the surface membranes ofplatelets 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 carbohydratemoieties is required.

• Chemical and biochemical analyses, however, are often not possiblefor the following reasons:– Small amounts of sample available– Vast structural heterogeneity of the glycans

• Thus highly sensitive and efficient methods for detection,• 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 potentialapplications in glycoproteomics.

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

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Glycoprotein Analysis• A variety of methods used in detection, purification,

and structural analysis of glycoproteins are:

MethodMethod UseUse

Periodic acid-Schiff stain Detects glycoproteins as pink bandsafter electrophoretic separation.

Incubation of cultured cells withIncubation of cultured cells withglycoproteins as radioactive decaybands

Leads to detection of a radioactivesugar after electrophoretic separation.

Treatment with appropriate endo- orexoglycosidase or phospholipases

Resultant shifts in electrophoreticmigration help distinguish amongproteins with N-glycan, O-glycan, or GPIlinkages and also between highmannose and complex N-glycans.

Agarose-lectin column chromatography,lectin affinity chromatography

To purify glycoproteins or glycopeptidesthat bind the particular lectin used. 6

MethodMethod UseUse

Lectin affinity electrophoresis

Resultant shifts in electrophoretic migration helpdistinguish and characterize glycoforms, i.e.variants of a glycoprotein differing incarbohydrate.

Compositional analysis following acid hydrolysis

Identifies sugars that the glycoprotein containsand their stoichiometry.

Mass spectrometryProvides information on molecular mass,composition, sequence, and sometimes branchingof a glycan chain.of a glycan chain.

NMR spectroscopyTo identify specific sugars, their sequence,linkages, and the anomeric nature of glycosidicchain.

Dual Polarisation Interferometry

Measures the mechanisms underlying thebiomolecular interactions, including reactionrates, affinities and associated conformationalchanges.

Methylation (linkage) analysis To determine linkage between sugars.

Amino acid or cDNA sequencing Determination of amino acid sequence.7

1. Characterization of intact glycoproteins2. 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

Glycoprotein Analysis

– 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

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Characterization of intact GlycoproteinsSeparation of proteins:

– SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis)

– 2-DE (2-dimensional gel electrophoresis)• SDS-PAGE: is a technique widely used to separate proteins

according to their electrophoretic mobility (a function oflength of polypeptide chain or molecular weight). SDS gelelectrophoresis of samples having identical charge per unitmass due to binding of SDS results in fractionation by size.electrophoresis of samples having identical charge per unitmass due to binding of SDS results in fractionation by size.

• Glycoprotein bands observed are often broad due to theheterogeneous glycosylation pattern, thus making acomplete separation of different glycoforms difficult.

• 2D-E or 2-D electrophoresis: is a form of gelelectrophoresis where mixtures of proteins are separatedby two properties in two dimensions on 2D gels (isoelectricpoint, protein mass).

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SDS-PAGE

Sodium Dodecyl SulfatePolyAcrylamide Gel ElectrophoresisPolyAcrylamide Gel Electrophoresis

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• Scope:– Polyacrylamide gel electrophoresis is used for the

qualitative characterization of proteins in biologicalpreparations, for control of purity and quantitativedeterminations.

• Purpose:

Scope & Purpose

• 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 structuretertiary 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 ofprotein

• Negative charge on the sulfate groups of SDSmask 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 willbe soluablized by the detergent, plus all theproteins will be covered with many negativecharges.charges.

• The end result has two important features:1. All proteins retain only their primary structure and2. All proteins have a large negative charge which

means they will all migrate towards the positivepole when placed in an electric field.

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SDSSodium Dodecyl Sulfate

H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+

HHHH HHHHHHH H O

C12H25NaO4S

PolarHydrophilic head

Non-polarHydrophobic tail

• Because it is amphipathic, SDS is a potent detergent

HHHH HHHHHHH H O

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SDS and Proteins

SDS

Protein

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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

Acrylamide

Polyacrylamide Gels• Polyacrilamide is a polymer made of acrylamide

(C3H5NO) and bis-acrilamide (N,N’-methylene-bis-acrylamide C7H10N2O2)

CHCH2O

Acrylamide

Acrylamide

O

CH2

NH2C

O

CHCH2

NH2C

bis-Acrylamide

O

CHCH2

NH2C

Acrylamide

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Polyacrylamide Gels

OO

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

O

CHCH2

NH2C

O

CHCH2

NH2C

SO4-.

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Polyacrylamide Gels1. Acrylamide polymerizes in the presence of free

radicals typically supplied by ammonium persulfate

OOOO

2. TMED (N,N,N’,N’-tetramethylethylenediamine)serves as a catalyst in the reaction

SO4-.

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

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Polyacrylamide Gels• bis-Acrylamide polymerizes along with acrylamide

forming cross-links between acrylamide chainsO

CHCH2

NH2C

O

CHCH2

NH2C

O

CHCH2

NH2C

CHCH2CHCH2

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

CHCH2CHCH2

bis-Acrylamide

O

CH

CH2

NH2C

O

CHCH2

NH2C

CH2

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Polyacrylamide Gels• bis-Acrylamide polymerizes along with acrylamide

forming cross-links between acrylamide chains

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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-acrylamideLittle bis-acrylamide

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PAGE

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

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1 2 3

SDS-PAGE

Addition of SDS23

1 Protein becomes rod-shaped with uniform charge distribution

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• Now take the mixture of denatured proteins to the gel andapply the current.

Since all the proteins havestrong negative charges,they will all move in thedirection the arrow ispointing (run to red+).

Now take the mixture of denatured proteins to the gel andapply the current.

• All the proteins enter the gel at the same time and have thesame force pulling them towards the other end

• Small molecules can manuver through the polyacrylamideforest faster than big molecules.

• Because of their small size, they move through the forestfaster since they have access to more of the paths in theforest while biggers are limited to only the larger paths.

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• The collection of proteins of any given size tend to move through the gelat the same rate, even if they do not take exactly the same tunnels to getat the same rate, even if they do not take exactly the same tunnels to getthrough.

• Proteins tend to move through a gel in bunches, or bands, since there areso 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 offthe current and then stain the proteins and see how far they movedthrough 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 eachband are of different sizes.

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• You must always keep in mind…….• SDS-PAGE separates proteins based on their:

– primary structure or size– but not amino acid sequence.– but not amino acid sequence.

we would not be able to use SDS-PAGE to separatetwo proteins of the same molecular weight fromeach other.

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Electrophoresis Principle

Separation of charged molecules in electric field is afunction of:

• Relative mobility of charged species (related tofrictional resistance which is related to size).Charge on the species.• 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 andfrictional resistance (related to size).

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• Mobility:

Where, Z = charge on moleculeE = Voltage applied (driving force)E = Voltage applied (driving force)f = frictional resistance

• Rf is measured by:

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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.(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 negativecharge, 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 (+)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/mass32

• When the (Z / mass) ratiois the same (+SDS), theproteins separate ONLYbased on MASS,

• Geometry having noeffect since the proteineffect since the proteinhas been denatured.

• Note: rate and order ofmigration is opposite thatof SEC.

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Stacking Gel

Is prepared Tris/HCL buffer pH 6.8,~2pH units lower than runningbuffer. Large pore polyacrylamideused to align and create a thinstarting zone of the protein of apx.19um on top of the resolving gel.

• Lower % Acrylamide

Resolving Gel

Components of SDS PAGE Gel

Resolving Gel

Small pore polyacrylamide gel (3 -30% acrylamide monomer) typicallymade using a pH 8.8 Tris/HCl buffer.

• Higher % Acrylamide

Resolves protein ~24 – 205 kDaRunning Buffer

Tris/Glycine: Glycine(pKa=9.69) is a trailing ion (or slow ion). In other wordsit 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 towardthe anode. Stacking occurs due to differential migration of ionicspecies that carry the electrical current through the gel.

• When an electrical current is applied to gel, ions carry the currentto the anode (+).

• Cl- ions, having the highest charge/mass ratio migrate faster, beingdepleted at cathode end and concentrated at anode end.

• Glycine from electrophoresis buffer enters gel at pH 6.8 and• Glycine from electrophoresis buffer enters gel at pH 6.8 andbecomes primarily zwitterionic (charge zero) moving slowly.

• Protein, coated with SDS has a higher charge/mass ratio thanglycine so moves fast, but slower than Cl-.

• When protein encounters resolving gel it slows down due toincreased frictional resistance (smaller pore size), allowingfollowing protein to “catch up” or stack.

• As protein is depleted from cathode end, glycine must carrycurrent so begins to migrate behind protein, in essenceconcentrating the proteins further at stacking gel/resolving gelinterface.

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• The stacking gel is a low concentration polyacrylamide gelwith 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 beable to run through the stacking gel. Since the number ofcharged ions in the stacking decreases when glycinebecomes a zwitterion, the voltage in the stacking willcharged ions in the stacking decreases when glycinebecomes a zwitterion, the voltage in the stacking willincrease and therefore the proteins will run very fastthrough the stacking and will compact on the front of theseparating gel. Here, the pH is higher and, when the glycineeventually reaches the separating, it will restore the voltagein the stacking but it won't change it in the separating. Inthe separating the proteins will be separated according totheir size

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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!!!!!!!negative charge!!!!!!!

• SDS is present in all of the buffers used AND is used to pretreatthe 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

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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– 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

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Coomassie –Protein BindingSulfonic 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• Electrostatic• Ionic• Vander Waals• Hydrophobic

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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

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2D Gel Electrophoresis

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2D Gel Electrophoresis

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

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2D Gel Electrophoresis

Separation of hundredsof proteins based on

-pI-MW-MW

Up to 10,000 proteins can be seen using optimized protocols

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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 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)

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Processes involved in 2D gel electrophoresisProtein isolation and quantification

Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)

Visualization of proteins spots with Dye

Identification of protein spots with Mass Spec

Bioinformatics46

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,• 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 ofelectrophoresis process

• Must quantify protein

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Reduction• DTT Treatment:• Dithiothreitol (DTT) is a reducing agent typically used to

break down the disulfide bonds contributing to tertiarystructure which SDS was unable to affect, furtherdenaturing the protein to deemphasize the role of proteinshape in PAGE. DTT reduces a disulfide bond by twosequential thiol-disulfide exchange reactions resulting inDTT becoming a six-member ring structure. 2-sequential thiol-disulfide exchange reactions resulting inDTT becoming a six-member ring structure. 2-mercaptoethanol (ME), another disulfide reducing agent, iscommonly used in lieu of DTT. Furthermore, it should benoted that while both DTT and ME sufficiently reducedisulfide bridges in proteins, there is a propensity for themto reform. Thus the protein sample is commonly treatedwith 2-Iodoacetamide, an alkylating sulfhydryl reagent,which binds covalently to free sulfides and preventsdisulfide bridges from reforming.

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Reduction of a disulfide bond by two thiol-disulfide exchange reactions involving DTT

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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, andhydrophobic)

• 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

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IEF and IPG (immobilized pH Gradient)

Strip of paper Made by covalentlyintegrating acrylamide andvariable pH ampholytes.Separation on basis of pI, not MWAvailable in different pH ranges

3-103-104-85-7

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

pH 3 4 5 6 7 8 9 10

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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.applied.• Resolution is determined by the slope of the pH gradient and the field

strength.

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Immobilized pH gradient gel strips

Many can be run in parallel for greater 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)

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The Second Dimension …Running the Gel

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SDS GelIPG strip-pressed down into the SDS-PAGE gel

Negative electrodepH 3 4 5 6 7 8 9 10

Positive electrodeSimilar mw but different pI

Similar pI but different mw

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Different IPG pH ranges yield Different Results

pH 4 pH 5

pH 4 pH 9

pH 5 pH 7

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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 reagentCopper 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

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2D Gel Results

• 401 spots (peptides) identified• 279 gene products

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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

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Trypsin Digestion of Gel spot

Cut out spot

Extracting a Gel Spot

Run Mass spec

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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 liverTumorCy5--Tumor

Both

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Conclusions• 2D gel electrophoresis is a popular method for

protein display, separation, visualization, andquantitation

• A good precursor to MS, but not required• 2D gels provide pI, MW data, and• 2D gels provide pI, MW data, and

photodocumentation• Web tools are now available that permit partial

analysis and comparison of 2D gels usingsoftware and simulators

• 2D gels are fun to run62

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Improved Sample Throughput- automated spot cuttingImproved Sample Throughput

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Why alternative approaches?Drawbacks:

– The frequent under-representation of membrane(glyco)proteins in common 2D-GE due to a low solubilizingpower of the non-ionic and zwitterionic detergents used.

– Many of them are only weakly stained by conventionaldyes due to the high carbohydrate content.

• Therefore, alternative approaches have to be appliedfor isolation, purification and/or characterization of

• Therefore, alternative approaches have to be appliedfor isolation, purification and/or characterization ofmembrane (glyco)proteins:– Blue native electrophoresis (BN-PAGE):

Coomassie Brilliant Blue dye provides the necessarycharges to the protein complexes for the electrophoreticseparation.

– 2D benzyldimethyl-n-hexadecylammoniumchloride/SDS-PAGE

– SDS-PAGE with nano-HPLC65

• A sensitive detection and characterization of theglycosylation pattern of electroblotted proteins may beachieved by carbohydrate-specific lectins.

• Using a panel of lectins with different specificities,glycoproteins can be probed for defined oligosaccharideepitopes.

Why alternative approaches?

epitopes.• Additional information on the type of glycans attached can

be obtained by combining gel electrophoretic separationand/or lectin probing with treatment by specific exo- andendo-glycosidases as well as respective amidases.

• Using this approach, an initial assessment of theglycosylation properties of a given glycoprotein may beachieved.

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Robust Analytical Methods forProtein Characterization

• Chemical derivatization:• The ionization state of amino acids changes

with pH

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Names, Abbreviations, and Properties of The Twenty Amino Acids

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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 -2.4pK of alpha-NH2 groups: 9.0 -10.8pK 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 distinguishedCysteine and Tryptophan are problematic in some methods

Amino-Terminal Sequencing by Edman Degradation:Amino-Terminal Sequencing by Edman Degradation:• Very sensitive• Standard method-still best approach to NH2-terminus

Very steep learning curve to do it wellBlocked proteins cannot be analyzedMixtures are challengingPeptides with long repeats are problematicPTMs (Post Translational Modifications)are often missed but can be dealt withOften not competitive with MS for internal sequence 70

Polyacrylamide Gel ElectrophoresisA crude measure of molecular weight and purity

• Analytical or preparative separations• Coupled with Blotting-sensitive & selective detectionIsoelectric Focusing

Analytical or preparative separations

Robust Analytical Methods for Protein Characterization

Used for mapping disease markers (e.g. Chronic granulomatous diseases)Variety of pH gradientsAutomated, high throughput instruments

Two Dimensional IEF –PAGE• Orthogonal separations-large separation space• Detection of small changes in complex samples

Separation of post-translationally modified proteinsDynamic Range problems due to sample loading capacity,,

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Chemical Methods for Protein Characterization

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Chemical Methods for Protein Characterization: Proteolysis

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Edman degradation

• Edman degradation, developed by PehrEdman, is a method of sequencing aminoacids in a peptide. In this method, the amino-terminal residue is labeled and cleaved fromterminal residue is labeled and cleaved fromthe peptide without disrupting the peptidebonds between other amino acid residues.

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Edman degradation: Mechanism

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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.

• This procedure can then be repeated again to identify thenext amino acid.

• Drawback: to this technique is that the peptides beingsequenced in this manner cannot have more than 50 to 60residues (and in practice, under 30).

• The peptide length is limited due to the cyclical derivitizationnot always going to completion.

Edman degradation: Mechanism

not always going to completion.• The derivitization problem can be resolved by cleaving large

peptides into smaller peptides before proceeding with thereaction. It is able to accurately sequence up to 30 aminoacids with modern machines capable of over 99% efficiencyper amino acid.

• Advantage: It only uses 10 - 100 picomoles of peptide for thesequencing process. Edman degradation reaction isautomated 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.)

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

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

Tosyllysine Chloromethyl Ketone HCl

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Tosyllysine Chloromethyl Ketone HCl

Combining Analytical Methods for Protein Characterization

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Methods suitable for the separationand characterization of glycopeptides

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Strategies for the analysis of releasedglycoprotein-glycans

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Mass Spectrometry inProteomicsProteomics

Methods & Theory

Proteomics Tools

• Molecular Biology Tools

• Separation & Display 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 • 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?

• Ionization method

• Mass analyzer– MALDI-TOF

• MW

Create ions Separate ions Detect ions

• Mass spectrum

• Database

86

method– MALDI– Electrospray(Proteins must be charged

and dry)

• MW – Triple Quadrapole

• AA seq– MALDI-QqTOF

• AA seq and MW

– QqTOF

• AA seq and protein modif.

• Database analysis

Generalized Protein Identification by MS

Spot removed from gel

Fragmented using trypsin

Spectrum of fragments generated

Libr

ary

Artificial spectra built

Artificially trypsinated

Database of sequences

(i.e. SwissProt)

MATCH

Libr

ary

Methods forproteinprotein

identification

MS Principles• Analytical method to measure the

molecular or atomic weight of samples• Different compounds can be uniquely

identified by their mass

CH3CH2OH

NOH

HO

-CH2-

-CH2CH-NH2

COOH

HO

HO

Butorphanol L-dopa Ethanol

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 aloneformula 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 • 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

IonizerIonizer

+_

Mass AnalyzerMass Analyzer DetectorDetector

Mass spectrometersLinear Time Of Flight tube

Reflector Time Of Flight tube

detector

ion source

ion source

time of flight

• Time of flight (TOF) (MALDI)– Measures the time required for ions to fly

down the length of a chamber. – Often combined with MALDI (MALDI-TOF)

Detections from multiple laser bursts are averaged. Multiple laser

• Tandem MS- MS/MS-separation and identification of compounds in complex mixtures

reflectordetector

time of flight

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)

m/z ratio:

All proteins are sorted based on a

mass to charge ratio (m/z)

Molecular weight divided by the charge on this protein

Typical Mass Spectrum

aspirin

Relative Relative AbundanceAbundance

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 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

DMM

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 SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumps

InletIon

SourceMassFilter Detector

DataSystem

Sample PlateTargetHPLCGCSolids probe

MALDIESIIonSprayFABLSIMSEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

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– 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 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+

CH OH CH O=H+ + HCH3OH CH2O=H+ + H

CH3OH + CH3 + OH

CHO=H+ + HCH2O=H+

Why wouldn’t Electron Impact be suitable for analyzing proteins?

Why You Can’t Use EI ForAnalyzing 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)

MALDI

cyano-hydroxycinnamic acid

Gold tip needle

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)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 • 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• 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 DesorptionAssisted Laser DesorptionIonizationIonization

337 nm UV laser

MALDI

cyano-hydroxycinnamic acid

MALDIMALDI

• Sample is ionized by bombarding sample with laser light

• Sample is mixed with a UV absorbant matrix • 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 PlateHT Spotting on a MALDI Plate

MALDI IonizationMALDI Ionization

++

+

+

-

--

++

-+

Analyte

Matrix

Laser

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

• Dissociation of matrix, phase +

+

+-

---++

+

++

• 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”)

+

+

+

MALDIMALDI• 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• 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 MALDIPrinciple for MALDI--TOF MASS TOF MASS

pulsedUV or IR laser(3-4 ns)

detector

peptide mixtureembedded in light absorbing chemicals (matrix)

++

+++

++

+ ++

vacuum

strong electric field

Time Of Flight tubecloud ofprotonatedpeptide moleculesaccV

Principle for MALDIPrinciple for MALDI--TOF MASSTOF MASSLinear Time Of Flight tube

Reflector Time Of Flight tube

detector

ion source

time of flight

Reflector Time Of Flight tube

reflector

ion source

detector

time of flight

MALDIMALDI == SELDISELDI

337 nm UV laser

MALDI

cyano-hydroxycinnaminic acid

SELDI

MALDIMALDI//SELDISELDI SpectraSpectra

Normal

Tumor

Mass

Mass Spectrometer Schematic

InletIon

DetectorData

High Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumps

MassFilterInlet

Ion Source Detector

DataSystem

Sample PlateTargetHPLCGCSolids probe

MALDIESIIonSprayFABLSIMSEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

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)• 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 TIP

MCPDETECTOR

PUSHER

TOF

IONSOURCE

HEXAPOLECOLLISIONCELL

HEXAPOLE

HEXAPOLE

QUADRUPOLEREFLECTRONSKIMMER

PUSHER

Mass Spec Equation (TOF)

mz

2Vt2=

L2z

m = mass of ion L = drift tube lengthz = charge of ion t = time of travelV = voltage

L2

Ion Trap Mass Analyzer

• Ion traps are ion trapping devices that make use of a three-dimensional quadrupole field to trap and mass-analyze ionsmass-analyze ions

• invented by Wolfgang Paul (Nobel Prize1989)

• Offer good mass resolving power

FT-ICRFourier-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 1974Marshal 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

(trypsin)

Peptides are separated by hydrophobicity on reverse phase column (2D)

separates peptides by charge

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

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 proteinmasses) 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 FingerprintingPrinciples of Fingerprinting

>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe

>Protein 2

Sequence Mass (M+H) Tryptic Fragments

4842.05 4842.05

acedfhsakdfgeasdfpkivtmeeewendadnfekqwfe

acek>Protein 2acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe

>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfe

4842.05 4842.05

4842.054842.05

acekdfhsadfgeasdfpkivtmeeewenkdadnfeqwfe

acedfhsadfgekasdfpkivtmeeewendakdnfegwfe

Principles of FingerprintingPrinciples of Fingerprinting

>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe

>Protein 2

Sequence Mass (M+H) Mass Spectrum

4842.05

>Protein 2acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe

>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfe

4842.05

4842.05

Predicting Peptide Cleavages

http://ca.expasy.org/tools/peptidecutter/

http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

Protease Cleavage RulesRules

Trypsin XXX[KR]--[!P]XXX

Chymotrypsin XX[FYW]--[!P]XXX

Sometimes Sometimes inhibition occursinhibition occurs

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.• 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 MassesCalculating Peptide Masses• Sum the monoisotopic residue massesMonoisotopicMonoisotopic MassMass:: thethe sumsum ofof thethe exactexact oror accurateaccurate massesmasses

ofof thethe lightestlightest stablestable isotopeisotope ofof thethe atomsatoms inin aa moleculemolecule• Add mass of H2O (18.01056)• Add mass of H+ (1.00785 to get M+H)• If Met is oxidized add 15.99491• 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-122H-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 isotopesisotopes

• Average mass is the abundance weighted mass of all isotopic components

Mass Calculation (Glycine)

NH2—CH2—COOH

R —NH—CH —CO—R

Amino acid

ResidueR1—NH—CH2—CO—R3Residue

Monoisotopic Mass1H = 1.00782512C = 12.0000014N = 14.0030716O = 15.99491

Glycine Amino Acid Mass5xH + 2xC + 2xO + 1xN= 75.032015 amuGlycine Residue Mass3xH + 2xC + 1xO + 1xN=57.021455 amu

Amino Acid Residue Masses

Glycine 57.02147Alanine 71.03712Serine 87.03203Proline 97.05277Valine 99.06842

Aspartic acid 115.02695Glutamine 128.05858Lysine 128.09497Glutamic acid 129.0426Methionine 131.04049

Monoisotopic Mass

Valine 99.06842Threonine 101.04768Cysteine 103.00919Isoleucine 113.08407Leucine 113.08407Asparagine 114.04293

Methionine 131.04049Histidine 137.05891Phenylalanine 147.06842Arginine 156.10112Tyrosine 163.06333Tryptophan 186.07932

Amino Acid Residue Masses

Glycine 57.0520Alanine 71.0788Serine 87.0782Proline 97.1167Valine 99.1326

Aspartic acid 115.0886Glutamine 128.1308Lysine 128.1742Glutamic acid 129.1155Methionine 131.1986

Average Mass

Valine 99.1326Threonine 101.1051Cysteine 103.1448Isoleucine 113.1595Leucine 113.1595Asparagine 114.1039

Methionine 131.1986Histidine 137.1412Phenylalanine 147.1766Arginine 156.1876Tyrosine 163.1760Tryptophan 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 PMFLimitations With PMF

• Requires that the protein of interest already be in a sequence database

• Spurious or missing critical mass peaks always lead to problemslead 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 SpectrometryTandem Mass Spectrometry• Purpose is to fragment ions from parent ion to

provide structural information about a molecule

• Also allows mass separation and AA identificationof 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 & ProteomicsMS & Proteomics

Tandem Mass SpectrometryTandem Mass Spectrometry

• Different MS-MS configurations– Quadrupole-quadrupole (low energy)– Magnetic sector-quadrupole (high)– Magnetic sector-quadrupole (high)– Quadrupole-time-of-flight (low energy)– Time-of-flight-time-of-flight (low energy)

How Tandem MSHow Tandem MSsequencing workssequencing 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

Ser-Glu-Leu-Ile-Arg-Trp

Collision Cell

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

Ser-Glu-Leu

Ser-Glu-Leu-Ile

Etc…

Advantages of Tandem Mass SpecAdvantages 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 capabilityHigh-thoughput capability

Disadvantages of Tandem Mass Spec

Very expensive-Campus

Hardware: $1000

Setup: $300

1 run: $1000

Requires sequence databases for analysis

MSMS--MS & ProteomicsMS & Proteomics

• Provides precise sequence-specific data

• More informative than

• Requires more handling, refinement and sample manipulation

Advantages Disadvantages

• More informative than PMF methods (>90%)

• Can be used for de-novo sequencing (not entirely dependent on databases)

• Can be used to ID post-trans. modifications

• Requires more expensive and complicated equipment

• Requires high level expertise

• Slower, not generally high throughput

Different MSDifferent MS--MS ModesMS Modes

• Product or Daughter Ion Scanning– first analyzer selects ion for further fragmentation– most often used for peptide sequencing

• Precursor or Parent Ion Scanning• 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): aquantitative 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

O

Biotin Affinity tag: Binds tightly to streptavidin-agarose resin

Linker: Heavy version will have

Reactive group: Thiol-reactive group will bind to Cys

S OI

NH

**

* *

O

OON

H

O

O

NHNH

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

The ICAT Reagent

How ICAT works?

Lyse &Label

Affinity isolation on streptavidin beads

QuantificationMS

IdentificationMS/MS

NH2-EACDPLR-COOH

Proteolysis(ie trypsin)

MIX

100

m/z200 400 600

0

100

550 570 5900

m/z

Light

Heavy

NH2-EACDPLR-COOH

ICAT Quantitation

ICATICATAdvantages vs. DisadvantagesAdvantages vs. Disadvantages

• Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%)

• Can be used on complex mixtures of proteins

• Yield and non specificity

• Slight chromatography differences

• Expensive

mixtures of proteins

• Cys-specific label reduces sample complexity

• Peptides can be sequenced directly if tandem MS-MS is used

• Tag fragmentation

• Meaning of relative quantification information

• No presence of cysteine residues or not accessible by ICAT reagent

Mass Spectrometer SchematicMass Spectrometer Schematic

High Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumps

MassFilterInlet

Ion Source Detector

DataSystem

Sample PlateTargetHPLCGCSolids probe

MALDIESIIonSprayFABLSIMSEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

MS DetectorsMS 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 ionelectronic 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 DetectorsMass Detectors

Electron Multiplier (Dynode)

Limitations of ProteomicsLimitations 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-proteins exist in multiple isoforms-no protein equivalent of PCR exists for amplification

of small samples

Shotgun Proteomics:Shotgun Proteomics:MuMultiltiddimensionalimensional PProteinrotein

IIdentificationdentification TTechnologyechnology(MudPIT)(MudPIT)(MudPIT)(MudPIT)

Fractionation &Isolation

2-DE Liquid Chromatography

General Strategy for Proteomics CharacterizationGeneral Strategy for Proteomics Characterization

Peptides

Mass Spectrometry

Database Search

Characterization

• Identification• Post Translational modifications• Quantification

MALDI-TOF MS-(LC)-ESI-MS/MS

Tandem Mass Spectrometer

Digestion

2D Chromatography

Protein Mixture

Peptide Mixture

Overview of Shotgun Proteomics: MudPIT

SEQUEST®

DTASelect & Contrast

SCXRP

PySpzS5609 #2438 RT: 66.03 AV: 1 NL: 8.37E6T: + c d Full ms2 729.75@35.00 [ 190.00-1470.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

545.31

658.36

900.36

1031.40

913.421240.53

782.23896.29

1032.43895.33546.19 771.24

1028.41

721.31

431.15 801.38

1241.39914.34427.27 559.13

1258.56317.17 669.39 1033.60 1312.35651.14408.74 1027.221142.43

915.53432.40 882.07600.24399.24986.50 1123.49217.91 1356.10481.13 869.23 1195.44

MS/MS Spectrum

> 1,000 Proteins Identified

MudPIT

Trypsin+ proteins

IEX-HPLC RP-HPLC

+ proteins

p53

SCX RP

2D Chromatography

Acquiring MS/MS DatasetsAcquiring MS/MS Datasets

MudPIT Cycle load sample wash salt step wash RP gradient re-equilibration

x 3~18

Tandem MS SpectrumPeptide Sequence is Inferred from Fragment ions

MS/MS of Peptide Mixtures

LC

MSMS(MW Profile)

MS/MS(AA Identity)

Summary of MudPITSummary 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.

22--DEDE vsvs MudPITMudPIT

• Widely used, highly commercialized

• High resolving power

• Highly automated process

• Identified proteins with extreme pI values, low abundance and low abundance and those from membranethose from membrane

• Visual presentation

• Limited dynamic range• Only good for highly soluble and

high abundance proteins• Large amount of sample required

• Thousands of proteins can be identified

• Not yet commercialized• Expensive• Computationally intensive• Quantitation

THE END