Nat Rev Mol Cell Biol (2006) 7, Mass spectrometry and protein analysis. (2006). Domon and Aebersold Science 312,

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1 Nat Rev Mol Cell Biol (2006) 7, Mass spectrometry and protein analysis. (2006). Domon and Aebersold Science 312,

2 Affinity Capture / Enrichment Key to success in PTM ID Purified protein or 2D gel protein spot Protein Mix or (sub)- Proteomes Peptide Mix +/- Modifications LC-separation Choice of 1D vs 2D; time + gradient offline MALDI MS + (auto MS/MS) +/- targeted MALDI MS/MS Affinity Capture / Enrichment online LC-ESI-MS/MS More non-redundant peptide MS/MS, of better quality increase coverage and/or additional protein hits Specific Scan functions for online datadependent acquisition PTM ID Additional data interrogation, considering modifications

3 SCX Protein IP IMAC Enrichment Strategies - IMAC ± methyl esterification - Titanium dioxide, TiO2 - SCX at low ph - Not specific for ptyr, higher abundance of pser/thr Global versus targeted MS approaches Cataloging versus relative quantification

4 Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. (2005). Rush et al, Nat Biotechnol 23, Cell Signaling Technology Inc. - describe a simple strategy that uses a phosphotyrosinespecific antibody to immunoprecipitate phosphotyrosine peptides directly from digested cellular protein extracts and standard LC-MS/MS methods to identify large numbers of tyrosine phosphorylation sites. - contradict the prevailing view that phosphospecific antibodies are not generally suitable for purifying phosphopeptide An enriched look at tyrosine phosphorylation. (2005). Conrads, T. P., and Veenstra, T. D. Nat Biotechnol 23, The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology) from ascites fluid was coupled noncovalently to protein G agarose (Roche) at 4 mg/ml beads

5 Phosphorylation reduces peptide solution charge state and alters SCX elution at low ph. As much as 68% of an in silico tryptic digest of the human NCBI protein database generates peptides with a predicted solution charge state of (+)2 at ph 2.7, and fewer than 3% with a solution charge state of less than (+)2. At ph 2.7, a theoretical tryptic peptide without histidine residues carries a net solution charge of (+)2 imparted by the amino terminus and the basic group of the carboxylterminal arginine or lysine. Note that the carboxyl terminus and carboxyl groups of glutamate and aspartate residues are protonated at ph 2.7. If singly phosphorylated, the solution charge state of this peptide is reduced to (+)1. Phosphorylation of a similar peptide containing a single histidine residue reduces its solution charge from (+)3 to (+)2, preventing its enrichment in early eluting SCX fractions

6 All phosphopeptides with net solution charges of less than (+)1 were the result of multiple phosphorylation events and/or phosphorylation of carboxyl termini. These peptides had little to no retention on the SCX column, owing to disproportionate charge distribution along the lengths of the peptides. Ballif et al, (2004) Mol Cell Proteomics. 3,

7 Miniaturized immobilized metal affinity chromatography (IMAC) columns High affinity of phosphate groups towards metal-chelated stationary phase, esp Fe 3+ and Ga 3+ Specificity is variable because of affinity for acidic groups (aspartic and glutamic acids) and to electron donors (His) Recovery and enrichment dependent on the particular peptide, type of metal ion, column material, elution procedure Esterification of acidic residues before IMAC enrichment helps to attain much higher specificity Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae Scott B. Ficarro et al. Nature Biotechnology 20, (2002)

8 report a highly selective enrichment procedure for phosphorylated peptides based on TiO 2 microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO 2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO 2.

9 Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. (2007). Jensen, S. S., and Larsen, M. R. Rapid Commun Mass Spectrom 21, IMAC suffers from several disadvantages when combined with reagents frequently used in cell biological experiments, e.g., EDTA, certain detergents, alkaline metal salts and other low molecular mass contaminants. Therefore, largescale phosphoproteomics employing multidimensional chromatography, e.g., ion-exchange chromatography, where the peptides are eluted using an increasing salt concentration, use reversed-phase chromatographic purification steps prior to IMAC enrichment TiO 2 is highly compatible with low ph solutions, detergents, salts and other low molecular weight contaminants tested in this study, whereas IMAC tends to favor multiply phosphorylated peptides when samples are loaded in such solutions.

10 Direct MS mapping of tryptic peptide * * * Enrichment of phosphopeptides with IMAC-Ni 2+ column * N H HO H C CH2 O P O O C OH O Base -elimination - 98 u N H O C C O CH2 Dehydroalanine M.W phosphoserine M.W alkaline phosphatase -80 Da asterisks* indicate the phosphorylated peptides

11 Trypsin Lys-C A potential problem could be in the fact that the -elimination/michael addition creates R and S enantiomers of the aminoethylcysteine derivative and only the R-enantiomer is efficiently hydrolyzed by trypsin.

12 pg pg Nature Biotech (2001) vol 19 New Tools for Quantitative Phosphoproteome Analysis Conrads et al (2002) BBRC 290,

13 Toward the Phosphoproteome Natalie G. Ahn & Katheryn A. Resing Nature Biotechnology 19, (2001) The method of Zhou et al. begins with a proteolytic digest that has been reduced and alkylated to eliminate reactivity from cysteine residues. Following N-terminal and C-terminal protection, phosphoramidate adducts at phosphorylated residues are formed by carbodiimide condensation with cystamine. The free sulfhydryl groups produced from this step are covalently captured onto glass beads coupled to iodoacetic acid. Elution with trifluoroacetic acid then regenerates phosphopeptides for analysis by mass spectrometry. Trial experiments showed that 80% of peptides recovered from yeast extracts were phosphorylated. In contrast, Oda et al. start with a protein mixture in which cysteine reactivity is removed by oxidation with performic acid. Base hydrolysis is used to induce - elimination of phosphate from phosphoserine and phosphothreonine, followed by addition of ethanedithiol to the alkene. The resulting free sulfhydryls are coupled to biotin, allowing purification of phosphoproteins by avidin affinity chromatography. Following elution of phosphoproteins and proteolysis, enrichment of phosphopeptides is carried out by a second round of avidin purification.

14 Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. (2005). Tao, W. A., Wollscheid, B., O'Brien, R., Eng, J. K., Li, X. J., Bodenmiller, B., Watts, J. D., Hood, L., and Aebersold, R. Nat Methods 2, Protect carboxylate groups, introduce stable isotope using Carbodiimide + imidazole; Phosphate react with excess amines on the dendrimer to form phosphoramidate bonds use a commonly-used derivatization of phosphate to phosphoramidate groups, we covalently coupled phosphorylated peptides to a synthetic polyamine (dendrimer) in a single step. The covalently immobilized phosphopeptides were then readily recovered via acid hydrolysis

15 assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.

16 Identification of Phosphopeptides in Complex Mixtures MS scan only is not enough Automated IDA using only MS data does NOT find all phosphopeptides Peptides P0 4 -peptide signal Low abundance of phosphopeptides makes detection in MS mode difficult

17 PTM Discovery Workflow PTM Discovery Workflow Phosphorylation analysis by Precursor Ion Scan-driven LC/MS/MS Precursor Ion Scan (-) + / - polarity switch Enhanced Resolution (+) + / - polarity switch Survey Precursor Ion Scan Detect PTM Enhanced Resolution Scan Get Charge, MW Charge state determination Acquire MSMS Spectra (+) Dynamic Exclusion Product Ion spectra of all ions passing selection criteria Get Sequence

18 Automated Identification and Quantification of Protein Phosphorylation Sites by LC/MS on a Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometer Mol Cell Proteomics (2006) 5: (A) automated LC/MS/MS analysis using a precursor ion scan of m/z 79 (B) The precursor mass detected at 22.1 min by the precursor ion scanning of m/z 79 in the LC separation (C) automatically selected for a positive ion enhanced resolution scan (D) a positive ion enhanced product ion scan

19 MIDAS Workflow for the Discovery of Phosphopeptides H 3 PO 4 (m/z 98) loss is a phosphopeptide-specific Protein sequence P P P P P P Predicted Peptide sequence P P P MS/MS eg M 2+ = loss of P M 2+ = Create MRM list to be monitored select parent P MS Detection & Quantitation CID select product Confirmation & Sequencing P Using the sequence of the protein to be characterized, the phosphopeptides modification sites can be predicted in silico MIDAS workflow is used to detect the phosphopeptide if it is present and then obtain the MS/MS to confirm the sequence and location of phosphorylation P

20 Multiple Reaction Monitoring as a Method for Identifying Protein Posttranslational Modifications J Biomolecular Techniques, (2005) 16:83-90

21 Phospho-Scan: : standard strategy MS Auto-MS/MS Loss of 98, 49 m/z? Yes! No! Ion trap: Combined CID & ETD PTM Discovery Mode MS Auto-MS/MS (CID) Fragment among top N of MS/MS spectrum? Yes! No! Loss of 98 m/z? Yes No Auto-MS 3 of (M+H) + -98, (M+2H) fragments Auto-ETD of (M+3H) 3+

22 [M+3H] 3+ + C 16 H 10. [M+3H] C 16 H 10. Proc Natl Acad Sci USA. 2004; 101(26): ETD results from the gas-phase reaction of multiply protonated peptide molecules with radical anions of polyaromatic hydrocarbons such as fluoranthene After electron transfer, the charge-reduced peptide ion dissociates through the same mechanisms believed responsible in ECD Single-scan ETD MS/MS spectrum resulting from a 50- msec reaction of the triply charged phosphopeptide, with anthracene anions Development of an ECD-like dissociation method for use with a low-cost, widely accessible mass spectrometer such as the QLT anions with sufficiently low electron affinities could function as suitably massive, one-electron donors. Electron transfer to protonated peptides should be exothermic by ev, trigger release of a hydrogen radical, and initiate fragmentation via the same nonergodic pathways accessed in ECD

23 Comparison of single-scan (500- to 600-msec) CAD and ETD mass spectra recorded during data-dependent analyses (nhplc- ESI-MS/MS) of phosphopeptides generated in a tryptic digest of human nuclear proteins.

24 A total of 84,000 ETD and CID tandem MS (MS/MS) spectra from 130 liquid chromatography (LC)-MS/MS runs using three different proteolytic enzymes (Lys-C, trypsin, and Glu-C) allowed us to identify 1,435 unique phosphorylation sites from proteins encoded by 500 genes, of which 1,141 (80%) had not been described previously. Using identical samples for ETD and CID analysis, we found that ETD was superior to CID both in the number of phosphopeptides identified as well as amino acid sequence coverage per phosphopeptide.

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26 Identification of a phosphorylation site is only the first step. Two further aspects have to be addressed to understand the regulatory significance; i.e., (i) how the degree of phosphorylation of a particular site changes over time; and (ii) what the degree of phosphorylation is. Relative quantification SILAC, itraq etc Phosphopeptide spcific derivatization Absolute phosphorylation stoichiometry

27 Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. (2005). Ballif, B. A., Roux, P. P., Gerber, S. A., MacKeigan, J. P., Blenis, J., and Gygi, S. P. Proc Natl Acad Sci U S A 102, Establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein LC-MS/MS was performed by using a 7-T LTQ FT hybrid mass spectrometer 10 LTQ MS/MS spectra were acquired per cycle in a data-dependent fashion from a preceding Fourier transform MS scan (400-1,800 m/z at 100,000 resolution with dynamic exclusion). Raw data were searched with no enzyme specificity against a database containing the sequence for the protein under study by using SEQUEST set to a mass tolerance of 1.1 Da. Fully tryptic phosphopeptides with mass accuracies of <10 ppm were manually validated to assign phosphorylation sites.

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29 P P P + P P P AQUA 10 fmole 1.17 fmole AQUA internal standard peptides SRM Absolute quantification against AQUA std Isotope-labeled Leu 0.59 fmole % phosphorylation = 0.59/( ) = 33.5% Absolute amount of phospho- versus nonphosphopeptide AQUA 10 fmole Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. (2003). Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., and Gygi, S. P. Proc Natl Acad Sci U S A 100,

30 Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. (2005). Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M., and Jensen, O. N. Mol Cell Proteomics 4, SILAC - SCX- IMAC - LTQ-FTMS : Data dependent Neutral Loss MS 3 The mass spectrometer was operated in the data-dependent mode to automatically switch between MS, MS/MS, and neutral loss-dependent MS 3 acquisition. Survey full scan MS spectra (from m/z 300 to 1575) were acquired by FTICR with resolution r = 25,000 at m/z 400 (after accumulation to a target value of 5,000,000 in the linear ion trap). The three most intense ions were sequentially isolated for accurate mass measurements by a FTICR "selected ion monitoring (SIM) scan," which consisted of a 15-Da mass range, r = 50,000, and a target accumulation value of 80,000. These were then simultaneously fragmented in the linear ion trap using collisioninduced dissociation at a target value of 10,000.The data-dependent neutral loss algorithm in the Xcalibur software was enabled for each MS/MS spectra. Datadependent settings were chosen to trigger a MS 3 scan when a neutral loss of 98, 49, or 32.7 Da was detected among the 10 most intense fragment ions. Former target ions selected for MS/MS were dynamically excluded for 30 s. Total cycle time was approximately 3 s.

31 LTQ-FT neutral loss-dependent MS 3 scan cycle a MS 1 full scan is acquired in the FTICR cell for a high dynamic range survey of the total mass range. The three most abundant ions are, in turn, isolated and analyzed. To achieve sub-ppm mass accuracy we acquired a SIM scan in the FTICR of the each peptide ion, which at the same time in the LTQ is fragmented and analyzed (MS 2 ). If a neutral loss precursor ion is detected among the 10 most intense fragment peaks during the MS 2 event, a MS 3 spectrum is automatically generated of this ion.

32 Light MS 2 T F V ps E T A D D I E K y 10 y 9 y 7-49 MS 3 Heavy y 7 y 10 -P y 10 Light Heavy MS MS

33 Phosphoproteomics finds its timing The three populations of cells are stimulated with EGF for different periods of time and extracted. The lysates are mixed in equal proportions, and the tyrosine phosphorylated proteins are affinitypurified with anti-phosphotyrosine antibodies. The proteins are digested with trypsin and identified by mass spectrometry. Because the isotopic labels are distinguishable within the mass spectra, the relative amounts of protein isolated from each time point can be determined. Repeating the experiment with different time points allows a detailed temporal profile to be produced for each protein Nat Biotechnol Sep;22(9): Cells are cultured for several divisions in the presence of one of three isotopically labeled forms of arginine so that all argininecontaining proteins become labeled

34 More than 10,000 phosphopeptides were detected in 116 LC MS analyses. Achieved greater than 99% certainty of phosphopeptide identification through the sub-ppm accuracy of peptide mass measurements, double fragmentation of peptides losing a phospho-group (MS 2 and MS 3 ). Identified 6,600 phosphorylation sites on a total of 2,244 proteins 13 fractions

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36 Multisite protein modification and intramolecular signaling. (2005). Yang, X. J. Oncogene 24,

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39 E1 adenylates the Ubl C-terminal carboxyl group, forming a high-energy Ubl-AMP intermediate which is attacked and covalently bound by the catalytic cysteine of the E1, creating a thioester linkage and releasing AMP transthiolation transthiolation

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43 By combining the tools of affinity purification and MS, substrates, associated proteins, and, in many cases, conjugation sites have been determined. Similar strategies have also been employed to identify other components of these pathways. For example, proteasomalassociated proteins were identified by tagging proteasomal components at the genetic level, purifying the tagged complexes, and identifying associated polypeptides. A powerful adaptation of this approach was seen with the development of tagged, activity-based reagents such as DUB-targeting probes characterized by subsequent MS

44 Against a proteome Wash, elute digest Against specific protein of interest

45 identified 4,210 peptides corresponding to 1,075 candidate ubiquitinconjugated proteins from the 6xHis-ubiquitin sample after subtracting the 48 proteins found in the control experiment. Performing the search a second time with a mass increase of Da as a variable modification to lysine residues resulted in the identification of 110 ubiquitination sites from 72 different proteins A proteomics approach to understanding protein ubiquitination. (2003). Peng et al., and Gygi, S. P. Nat Biotechnol 21, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo

46 A proteomic strategy for gaining insights into protein sumoylation in yeast. (2005). Denison, C., Rudner, A. D., Gerber, S. A., Bakalarski, C. E., Moazed, D., and Gygi, S. P. Mol Cell Proteomics 4, Isolation of SUMO conjugates by a double-affinity purification strategy S. cerevisiae strain expressing (His) 6 -FLAG-SUMO denaturing Ni-NTA chromatography Heavy chain anti-flag antibody purification Yeast are known to contain a number of histidine-rich proteins that bind avidly to Ni- NTA beads; silver staining pattern of proteins isolated by Ni-NTA chromatography from both a (His)8-SUMO strain and a nontagged strain were virtually indistinguishable Light chain 159 proteins unique to the SUMO preparation that were identified by two or more peptides

47 Sumoylation sites identified by MS/MS A total of 6 sites identified Approximately 86% of the proteins identified by two or more peptides and 71% of the one-peptide hits contained at least one SUMO consensus motif.

48 Distinct and overlapping sets of SUMO- 1 and SUMO-2 target proteins revealed by quantitative proteomics. (2006). Vertegaal, A. C., Andersen, J. S., Ogg, S. C., Hay, R. T., Mann, M., and Lamond, A. I. Mol Cell Proteomics 5, His tag = small, compatible with denaturing buffer Denaturing buffer Nuclear lysates Substantial number of proteins are preferentially conjugated to SUMO-1 or SUMO-2/3

49 10 slices 1656 Argcontaining peptides 324 different proteins Arg6 Arg10 14 proteins lack consensus site 53 different proteins 2 Arg-peptides, enriched >1.5 x Search for presence of SUMO motif

50 Glyco-proteomics - Concerted proteomic analysis of glycosylated and deglycosylated sub-proteomes, and glycomics analysis of the released glycans ± PNGase F /endo F SCX + RP 2D nanolc (glyco)-peptides nonretained Targeted Glycoproteome Lectin/mAb columns Proteome Total proteome extracts from Cell, tissues, fluids etc Glycome Glycomics Released total glycan pools MALDI-MS/MS nanoesi-ms/ms Proteomic Map Enriched subsets of Glycopeptides MALDI-MS Mapping + MALDI-MS/MS Sequencing What glycans? On which glycoproteins? Precursor Ion Discovery mode analysis Specific online LC-MS Detection of Glycopeptides Glycopeptides MS/MS sequencing

51 Total proteome extracts from Cell, tissues, fluids etc Glycome Glycomics Released total glycan pools MALDI-MS Mapping + MALDI-MS/MS Sequencing What glycans? Glycomics is an essential element in any glycoproteomic workflow

52 Glyco-proteomics - Concerted proteomic analysis of glycosylated and deglycosylated sub-proteomes, and glycomics analysis of the released glycans (glyco)-peptides Targeted Glycoproteome Proteome Total proteome extracts from Cell, tissues, fluids etc In gel digest ± 18 O label PNGase F periodate Ox Hydrazide capture De-N-glycosylated-Peptides nanolc-ms/ms Shotgun (glyco)proteomics Glycan Analysis Protein ID Glycosylation Sites In gel relase of N- and O-glycans In gel digest of deglycosylated proteins

53 Mol Cell Proteomics Feb;4(2): selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples Nat Biotechnol Jun;21(6):660-6 Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.

54 Glyco-proteomics Lectin Affinity specific capture by metabolic labelling Targeted Glycoproteome (glyco)-peptides Proteome Total proteome extracts from Cell, tissues, fluids etc Isolate and purify single glycoprotein of interest periodate Ox Hydrazide capture PNGase F ± 18 O label In gel digest Glycan Analysis In gel release of N- and O-glycans Lectin Affinity De-N-glycosylated-Peptides Hydrophilic interaction Size exclusion Protein ID Glycosylation Sites Enriched subsets of Glycopeptides In gel digest of deglycosylated proteins Reverse / Normal phase online LC-MS/MS Detection and Sequencing