MALDI TOF protein modification

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Mass Spectrometry for Protein Quantification

and Identification of Posttranslational

Modifications

Joseph A. Loo Joseph A. Loo

Department of Biological Chemistry

David Geffen School of Medicine

Department of Chemistry and Biochemistry

University of California

Los Angeles, CA USA

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Proteomics and posttranslational modifications

Patterson and Aebersold, Nature Genetics (supp.), 33, 311 (2003)

protein-ligand protein-ligand interactions interactions protein-ligand protein-ligand interactions interactions protein protein complexes complexes (machines) (machines) protein protein complexes complexes (machines) (machines) protein families protein families

(activity or structural)

protein families

(activity or structural)

post-translational post-translational modified proteins modified proteins post-translational post-translational modified proteins modified proteins Eukaryotic cell. Examples of protein

properties are shown, including the interaction of proteins

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Proteomic Analysis of Post-translational

Modifications



Post-translational modifications (PTMs)



Covalent processing events that change the properties

of a protein

 proteolytic cleavage

 addition of a modifying group to one or more amino

acids



Determine its activity state, localization, turnover,

interactions with other proteins



Mass spectrometry and other biophysical methods can

be used to determine and localize potential PTMs

 However, PTMs are still challenging aspects of

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Complexity of the Proteome

 Protein processing and modification comprise an important third

dimension of information, beyond those of DNA sequence and protein sequence.

 _{Complexity of the human proteome is far beyond the more than}

30,000 human genes.

 _{The thousands of component proteins of a cell and their}

post-translational modifications may change with the cell cycle,

environmental conditions, developmental stage, and metabolic state.

 Proteomic approaches that advance beyond identifying proteins to _{Proteomic approaches that advance beyond identifying proteins to} elucidating their post-translational modifications are needed.

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 _{Use MS to determine} PTM of isolated

protein

 _{Enzymatic or}

chemical degradation of modified protein  _{HPLC separation of}

peptides

 _{MALDI and/or ESI} used to identify PTM  _{MS/MS used to}

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Proteomic analysis of PTMs

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Glycoprotein Gel Stain

CandyCane glycoprotein molecular weight standards containing alternating glycosylated and nonglycosylated

proteins were electrophoresed through a 13% polyacrylamide gel. After separation, the gel was stained with SYPRO Ruby protein gel stain to detect all eight marker proteins (left). Subsequently, the gel was stained by the standard periodic acid–Schiff base (PAS) method in the Pro-Q Fuchsia

Glycoprotein Gel Stain Kit to detect the glycoproteins alpha₂ -macroglobulin, glucose oxidase, alpha₁-glycoprotein and avidin.

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Nitro-Tyrosine Modification

 _{Oxidative modification of amino acid side chains include methionine} oxidation to the corresponding sulfone, nitrosation or

S-nitrosoglutationylation of cysteine residues, and tyrosine modification to yield o,o’-dityrosine, 3-nitrotyrosine and 3-chlorotyrosine.

 _{Nitric oxide (NO) synthases provide the biological precursor for} nitrating agents that perform this modification in vivo. NO can form nitrating agents in a number of ways including reacting with

superoxide to make peroxynitrite (HOONO) and through enzymatic oxidation of nitrite to generate NO·2

 _{Tyrosine nitration is a well-established protein modification that} occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity.

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Nitro-Tyrosine Modification

“Proteomic method identifies proteins nitrated in vivo during inflammatory challenge,” K. S. Aulak, M. Miyagi, L. Yan, K. A. West, D. Massillon, J. W. Crabb, and D. J. Stuehr, Proc. Natl. Acad. Sci. USA 2001; 98: 12056-12061.

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116 98 55 37 30 20 kDa

3.5 4.5 5.1 5.5 6.0 7.0 8.4 9.53.5 4.5 5.1 5.5 6.0 7.0 8.4 9.5

MAPK phosphatase 2 MAPK phosphatase 2

E2 G1

enolase enolase casein kinase II

casein kinase II

HSP70 HSP70

Naf-1 Naf-1

Diesel Exhaust Particle-Induced Nitro-Tyrosine Modifications

RAW 264.7 macrophage exposed to DEP (Xiao, Loo, and Nel - UCLA)

Sypro Ruby

anti-nitro-tyrosine

trans. factor AP-2ß trans. factor AP-2ß MnSOD

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Phosphorylation

 _{Analysis of the entire complement of phosphorylated proteins in cells:} “phosphoproteome”

 _{Qualitative and quantitative information regarding protein phosphorylation} important

 _{Important in many cellular processes}

 _{signal transduction, gene regulation, cell cycle, apoptosis}

 _{Most common sites of phosphorylation: Ser, Thr, Tyr}

 _{MS can be used to detect and map}

locations for phosphorylation

 _{MW increase from addition of}

phosphate group

 _{treatment with phosphatase allows}

determination of number of phosphate groups

 _{digestion and tandem MS allows for}

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MS/MS and Phosphorylation

 _{Detection of phosphopeptides in complex mixtures can be}

facilitated by neutral loss and precurson ion scanning using

tandem mass spectrometers

 _{Allow selective visualization of peptides containing}

phosphorylated residues

 _{Most commonly performed with triple quadrupole mass}

spectrometers

precursor ion transmission

collision cell (chamber)

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MS/MS and Phosphorylation

 Precursor ion scan

 Q1 is set to allow all the components of the mixture to enter the

collision cell and undergo CAD

 Q3 is fixed at a specific mass value, so that only analytes which

fragment to give a fragment ion of this specific mass will be detected

 _{Phospho-peptide fragments by CAD to give an ion at m/z 79 (PO}₃₎

 Set Q3 to m/z 79: only species which fragment to give a fragment

ion of 79 reach the detector and hence indicating phosphorylation

Q1 Q2

collision cell

Q3

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MS/MS and Phosphorylation



Neutral loss scan

 _{Q1 and Q3 are scanned synchronously but with a specific}

m/z offset

 The entire mixture is allowed to enter the collision cell, but

only those species which fragment to yield a fragment with the same mass as the offset will be observed at the

detector

 pSer and pThr peptides readily lose phosphoric acid during

CAD (98 Da)

 For 2+ ion set offset at 49

 Any species which loses 49 from a doubly charged ion

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Enrichment strategies to analyze

phosphoproteins/peptides



Phosphospecific antibodies

 Anti-pY quite successful

 Anti-pS and anti-pT not as successful, but may be used _(M.

Grønborg, T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O. Ohara, M. Mann, O. N. Jensen, and A. Pandey, “Approach for Identification of

Serine/Threonine-phosphorylated Proteins by Enrichment with Phospho-specific Antibodies.” Mol. Cell. Proteomics 2002, 1:517–527.



Immobilized metal affinity chromatography (IMAC)

 Negatively charged phosphate groups bind to postively charged

metal ions (e.g., Fe3+, Ga3+) immobilized to a chromatographic

support

 Limitation: non-specific binding to acidic side chains (D, E)

 Derivatize all peptides by methyl esterification to reduce

non-specific binding by carboxylate groups.

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Direct MS of phosphopeptides

bound to IMAC beads



Raska et al., Anal.

Chem. 2002, 74, 3429



IMAC beads placed

directly on MALDI target



Matrix solution spotted

onto target



MALDI-MS of peptides

bound to IMAC bead



MALDI-MS/MS (*) to

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

MALDI-MS spectrum

obtained from peptide

bound to IMAC beads

applied directly to

MALDI target



MALDI-MS/MS (Q-TOF)

to locate

phosphorylation site



Sample enrichment with

minimal sample

handling

contains phosphorylated

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Enrichment strategies to analyze



Chemical derivatization



Introduce affinity tag to enrich for

phosphorylated molecules

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phosphoproteins/peptides

 _{Oda et al., Nature Biotech. 2001, 19, 379 for analysis of pS and pT}  _{Remove Cys-reactivity by oxidation with performic acid}

 _{Base hydrolysis induce ß-elimination of phosphate from pS/pT}  Addition of ethanedithiol allows coupling to biotin

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 _{Zhou et al., Nature Biotech. 2001, 19, 375}

 _{Reduce and alkylate Cys-residues to eliminate their}

reactivity

 _{Protect amino groups with t-butyl-dicarbonate (tBoc)}

 Phosphoramidate adducts at

phosphorylated residues are formed by carbodiimide condensation with

cystamine

 _{Free sulfhydryls are covalently}

captured onto glass beads coupled to iodoacetic acid

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Chemical derivatization to

enrich for phosphoproteins



Developed because

other methods based on

affinity/adsorption (e.g.,

IMAC) displayed some

non-specific binding



Chemical derivatization

methods may be overly

complex to be used

routinely



Sensitivity may not be

sufficient for some

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Phosphoprotein Stain

PeppermintStick phosphoprotein molecular weight standards

separated on a 13% SDS

polyacrylamide gel. The markers contain (from largest to smallest) beta-galactosidase, bovine serum albumin (BSA), ovalbumin, beta-casein, avidin and lysozyme. Ovalbumin and beta-casein are

phosphorylated. The gel was stained with Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red). The digital images were pseudocolored.

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Phosphoprotein Stain

Visualization of total protein and phosphoproteins in a 2-D gel

Proteins from a Jurkat T-cell lymphoma line cell lysate were

separated by 2-D gel electrophoresis and stained with Pro-Q Diamond

phosphoprotein gel stain (blueblue)

followed by SYPRO Ruby protein gel

stain (redred). After each dye staining,

the gel was imaged and the resulting composite image was digitally

pseudocolored and overlaid.

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RAW 264.7 exposed to DEP

Global Analysis of Protein Phosphorylation

Pro-Q Diamond

Sypro Ruby

Xiao, Loo, and Nel - UCLA

IEF

9.5 3.54.5 5.1 5.5 6.0 7.0 8.4

5 3 4 1 2 6 7 20 30 37 98 55 9.5

3.54.5 5.1 5.5 6.0 7.0 8.4

30 37 98 55 20 8 9 10 11 12 13 14 TNF

TNF_ convertase convertase

MAGUK p55

PDI

Protein phosphatase 2A

JNK-1

p38 MAPK alpha

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A A B m/z R e l. A b un d. Q Q

H EE

Mass spectrometry is inherently not a

quantitative technique. The intensity of a peptide ion signal does not

accurately reflect the amount of peptide in the sample.

equimolar mixture equimolar mixture

of 2 peptides of 2 peptides

516.725 516.828

m/z

(M+2H)2+_{: [}12_C]-ion

[Val5]-Angiotensin II

1031.5188 (monoisotopic)

Lys-des-Arg9-Bradykinin

1031.5552 (monoisotopic)



 = 0.036= 0.036

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A A B m/z R e l. A bu n d. Q Q

H EE

Two peptides of identical chemical structure that differ in mass because they differ in isotopic composition are expected to generate identical specific signals in a mass spectrometer.

Mass Spectrometry and Quantitative

Measurements

Q

H EE

13_C 13_C

13_C

A

A B

2_D 2_D

Methods coupling mass spectrometry and stable isotope tagging Methods coupling mass spectrometry and stable isotope tagging have been developed for quantitative proteomics.

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ICAT: Isotope-Coded Affinity Tag

 Alkylating group covalently attaches the reagent to reduces Cys-residues.

 A polyether mass-encoded linker contains 8 hydrogens (d0) or 8 deuteriums (d8) that represents the isotope dilution.

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ICAT: Isotope-Coded Affinity Tag

 The Cys-residues in sample 1 is labeled with d0-ICAT and sample 2 is labeled with d8-ICAT.

 The combined samples are digested, and the biotinylated ICAT-labeled peptides are enriched by avidin affinity chromatography and analyzed by LC-MS/MS.

 Each Cys-peptide appears as a pair of signals differing by the mass differential encoded in the tag. The ratio of the signal intensities indicates the abundance ratio of the protein from which the peptide originates.

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Stable Isotope Amino Acid or

15

N-

in vivo

Labeling

 Metabolic stable isotope coding

of proteomes

 _{An equivalent number of cells}

from 2 distinct cultures are grown on media supplemented with

either normal amino acids or 14

N-minimal media, or stable isotope

amino acids (2D/13C/15N) or 15

N-enriched media.

 _{These mass tags are}

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Enzymatic Stable Isotope Coding of

Proteomes

 _{Enzymatic digestion in the presence of}18

O-water incorporates 18O at the carboxy-terminus

of peptides

 _{Proteins from 2 different samples are}

enzymatically digested in normal water or H218O.

R

R₃₃ RR₄₄

NH₂-CH-CO-NH-CH-COOH

...NH-CH-CO-NH-CH-CO-...NH-CH-CO-NH-CH-CO-1818_O_O_H_H

R

R₁₁ RR₂₂

...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH

R

R₁₁ RR₂₂ RR₃₃ RR₄₄

Trypsin /H

Trypsin /H₂₂1818O_O

(Arg, Lys) (Arg, Lys)

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Identification of Low Abundance Proteins



The identification of low abundance

proteins in the presence of high

abundance proteins is problematic

(e.g., “needle in a haystack”)



Pre-fractionation of complex protein

mixtures can alleviate some difficulties

 gel electrophoresis, chromatography,

etc



Removal of known high abundance

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Identification of Low Abundance Proteins

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Additional Readings

 _{R. Aebersold and M. Mann, Mass spectrometry-based proteomics,}

Nature (2003), 422, 198-207.

 _{M. B. Goshe and R. D. Smith, “Stable isotope-coded proteomic mass}

spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 101-109.

 W. A. Tao and R. Aebersold, “Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 110-118.

 _{S. D. Patterson and R. Aebersold, “Proteomics: the first decade and}

beyond.” Nature Genetics 2003; 33 (suppl.): 311-323.

 _{M. Mann and O. N. Jensen, “Proteomic analysis of post-translational}

modification.” Nature Biotech. 2003; 21: 255-261.

 _{D. T. McLachlin and B. T. Chait, “Analysis of phosphorylated proteins}

and peptides by MS.” Curr. Opin. Chem. Biol. 2001; 5: 591-602.

 _{S. Gygi et al., “Quantitative analysis of complex protein mixtures using}

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Proteomics in Practice: A Laboratory Manual of Proteome Analysis

Reiner Westermeier, Tom Naven Wiley-VCH, 2002

PART II: COURSE MANUAL

Step 1: Sample Preparation Step 2: Isoelectric Focusing

Step 3: SDS Polyacrylamide Gel Electrophoresis Step 4: Staining of the Gels

Step 5: Scanning of Gels and Image Analysis Step 6: 2D DIGE

Step 7: Spot Excision

Step 8: Sample Destaining Step 9: In-gel Digestion

Step 10: Microscale Purification

Step 11: Chemical Derivatisation of the Peptide Digest Step 12: MS Analysis

Step 13: Calibration of the MALDI-ToF MS Step 14: Preparing for a Database Search Step 15: PMF Database Search Unsuccessful

PART I: PROTEOMICS TECHNOLOGY Introduction Expression Proteomics Two-dimensional Electrophoresis Spot Handling Mass Spectrometry

Protein Identification by Database Searching

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Proteins and Proteomics: A Laboratory Manual Richard J. Simpson

Cold Spring Harbor Laboratory (2002)

Chapter 1. Introduction to Proteomics

Chapter 2. One–dimensional Polyacrylamide Gel Electrophoresis Chapter 3. Preparing Cellular and Subcellular Extracts

Chapter 4. Preparative Two–dimensional Gel Electrophoresis with Immobilized pH Gradients

Chapter 5. Reversed–phase High–performance Liquid Chromatography Chapter 6. Amino– and Carboxy– terminal Sequence Analysis

Chapter 7. Peptide Mapping and Sequence Analysis of Gel–resolved Proteins Chapter 8. The Use of Mass Spectrometry in Proteomics

Chapter 9. Proteomic Methods for Phosphorylation Site Mapping Chapter 10. Characterization of Protein Complexes