Fundamentals of Protein and Peptide Analyses by Mass Spectrometry. Arthur Moseley Genome Academy April, 2013

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1 Fundamentals of Protein and Peptide Analyses by Mass Spectrometry Arthur Moseley Genome Academy April, 2013

2 Workflows utilizing high-resolution high-mass accuracy LC-MS/MS Qualitative Protein Identification SDS-PAGE Gels Recombinant Expressed Purified Proteins Protein Interaction Networks Immunoprecipitations or Tagged-Protein Expression Systems Gel or Gel-Free protocols Qualitative or quantitative analysis (Label-Free or SILAC) PTM Characterization A C B A Phosphorylation (TiO 2 Enrichments), S-Nitrosylation (RAC enrichments), Acetylation (Antibody Based Enrichment) Qualitative or Quantitative Analysis (Label-Free or various SILAC/covalent labeling strategies) Differential Protein Expression Analysis Global (non-targeted) quantitation of individual protein expression as a function of disease, treatment, time, etc. Vs.

3 Analytical Focus is Most Often Directed to Sample Analysis, but Efficient and Reproducible Sample Prep is Essential Sample (lysate, sub-cellular fraction) Peptide and Protein Quantitation Depletion and/or Selective Enrichment Integration of quantitative and qualitative data (Rosetta Elucidator) Peptide ID quality scoring & translating peptides to proteins (Rosetta Elucidator or Proteome Software s Scaffold) Database search of product ion spectra (Waters Identity E and Matrix Science Mascot) Depleted / Enriched Proteome DIGEST Image Conversion, Image alignment and Quantitative Analysis (Rosetta Elucidator) Automated translation to DB searchable format Waters PLGS, Rosetta Elucidator, Matrix Science Distiller Automated data transfer to NetApp enterprise data storage nanoscale UPLC MS MS E or MS/MS High resolution, Accurate Mass Mass Spectrometry Quantitative Pipeline Qualitative Pipeline High Resolution Accurate Mass Measurements Precursor Ions and Product Ions

4 Differential Protein Expression Analysis Label-Free Quantitation Workflow Condition 1 Condition 2 Image Translation (n=12) Solubilize and Process Protein 1D- or 2D- LC-MS Condition 3 Condition 4 Retention Time Alignment Intensity Normalization Retention Time Master Image Isotope Group Retention Time m/z m/z Intensity m/z Retention Time Retention Time Retention Time

5 Rigorously use Quantitatively Reproducible Analytical Methods Forget not the basics of analytical chemistry Highly reproducible chromatography is required A high sampling rate across the chromatographic peak is required for accurate quantitation Ideally want sampling points across chromatographic profile Highly reproducible chromatography is required for sample-to-sample comparisons High resolution, accurate mass (precursor & products) tandem mass spectrometry technology needed For quantitative selectivity (near isobaric cross-talk) For accurate qualitative identifications 1% FPR at peptide level (Decoy DB; Peptide Prophet) No QCs No Replication No Common Standard = No Quantifiably Reliable Data = No Quantifiably Reliable Data = No Meaningful Comparison across Projects

6 Selected Ion Chromatograms - area/volume are the quantitative measure Peptide Quantity Sum of all features from that peptide Quantitation of a peptide Across four biological groups Peptide CV = 8-12% Protein CV = 3-4% Protein Quantity Sum of all peptides from that protein Analytical Variability of ~ 35,000 peptides Daily QC Sample (pool of QC plasma sample) Analytical + Biological Variability of ~35,000 peptides Patient Samples ~ 40% Isotope Groups CV < 10% ~ 70% Isotope Groups CV < 20% ~ 90% Isotope Groups CV < 25% ~ 2 % Isotope Groups CV < 25% 25% CV Plasma Peptides

7 Incorporation of Qualitative Identifications and Protein Quantitation Export Peak List Mascot Identity E m/z Forward/Reverse Database Retention Time Import Search Results PeptideTeller algorithm to yield peptide level 1% FDR Intensity Retention Time Peptide Quantity Sum of all features from that peptide Protein Quantity Sum of all peptides from that protein Intensity Injection

8 Differential Protein Expression Analysis Increasing the Depth of Coverage in a Discovery Experiment 1,000s-10,000s Number of Analytes Plasma, Tissue, Proximal Fluids, etc. Number of Samples 100-1,000 1,000s 10s Want to Need to Current Solutions * Improve Qualitative Depth of Coverage * Improve Confidence in Spectral Assignments * Maintain Quantitative Precision * Incorporate into Label- Free Workflow * Keep Analysis Time and Cost Low * Ion-Mobility Enabled Data- Independent Acquisitions * Multidimensional LC Separations

9 Ion-Mobility Enabled Data-Independent Acquisitions PLGS Software Ion mobility Waters.com

10 Differential Protein Expression Analysis Label-Free Quantitation Workflow Multidimensional LC Condition 1 Condition 1 Condition 2 Condition 2 Image Translation Fraction 1 (n=3) (n=8) Fxn 3 Fxn 2 Fxn 1 Condition 3 Condition 3 Condition 4 Condition 4 Retention Time Alignment m/z Intensity Normalization Retention Time m/z Peptide Quantity Sum of all features from that peptide (all fractions) Intensity Retention Time Retention Time Retention Time Protein Quantity Sum of all peptides from that protein Injection

11 RP/RP Multidimensional Liquid Chromatography First Dimension Reverse Phase Separation, ph um x 5 cm column 5um XBridge C 18 particles Second Dimension Reverse Phase Separation, ph um x 15 cm column 1.7um BEH130 C 18 particles Key Advantages High orthogonality High resolution Excellent reproducibility Low sample requirements (~3 ug for 5-fraction, ~5 ug for 10-fraction) Waters.com

12 NanoAcquity UPLC System with 2D Technology 5 um Xbridge C um x 5 cm A: 20 mm Ammonium Formate, ph 10 B: Neat ACN A: 0.1% F.A. B: ACN/0.1% F.A. 1.7 um BEH130 C18 75 um x 15 cm %B 2nd Dimension % 20.4% 10.8% 14.0% 16.7% Minutes %B 1st Dimension

13 Increased Coverage from 2D RP/RP Fractionation Experimental Design Triplicate Analysis 1 ug Ecoli Lysate Investigator supplied pellet Rapigest/sonication in 50 mm ammonium bicarbonate 1D LC Separation 90 min gradient 1.7 um BEH130 C18 75 um x 25 cm UPLC column 2D LC Separation 5-fraction and 10-fraction Synapt G2 HDMS Resolution Mode IM-DIA 0.6s LE Scan, 0.6s HE Scan HE Scan: 27V 50V CE Ramp Spectra Processed/Searched in PLGS (v2.5) (100% FDR) Import spectra to Scaffold (v3.6) (Proteome Software) Peptide Prophet Algorithm (Keller, A et. al. Achem. 2002, 74.) Fxn 5 Fxn 4 Fxn 3 Fxn 2 Fxn 1

14 Equal Distribution of Peptides Across Fractions Identified Peptides 5-Fraction LC/LC Average Identifications Average Cumulative Identifications Cumulative Identifications st Dimension Fraction Identified Peptides 10-Fraction LC/LC Average Identifications Average Cumulative Identifications Cumulative Identifications st Dimension Fraction

15 Protein Identifications Increased Depth of Coverage 2D LC/LC <1.0% Peptide Level False Discovery Rate (Scaffold) Peptide Identifications fold fold fold fold Identifications Identifications min 1D 5fxn LC/LC 10fxn LC/LC 90-min 1D 5fxn LC/LC 10fxn LC/LC Protein Overlap 90 min 1D Protein Overlap 5fxn 2D Protein Overlap 10fxn 2D 568/1449 1D 1016/ fxn 78% 3/3 74% 3/3` 73% 3/3 1422/ fxn 88% 2/3 86% 2/3 85% 2/3

16 Assessing the Analytical Reproducibility in a Multidimensional LC/LC-MS Experiment Biological Rep n= 17 n= M Pre-Treatment Needle Biopsy Tumor Samples Post-Treatment Needle Biopsy Tumor Samples Solubilize Reduce/alkylate/trypsin digest 5-Fraction 2D UPLC/UPLC Synapt G2 Resolution Mode IM-DIA Molecular Weight Rosetta Elucidator Label-Free Quantitation Peptide Teller Annotation (1% FDR)

17 Assessing Analytical Reproducibility Incorporating Quality Controls QC 1: yeast ADH spiked into each sample at a constant fmol quantity per ug of lysate QC 2: create a pooled sample from equal portions of all samples and run (same analytical method) periodically throughout the study Day 1 Injection 1 Individual biological (randomized) replicates with spiked yeast ADH run in singlicate Day 9 Injection 42 QC pool run periodically throughout study

18 Global Protein Expression Profiles Principle Component Analysis Protein Level (n=1278) 36/40 samples (4 outliers removed) Z-score normalized Agglomerative Cluster Protein Level (n=1278) 36/40 samples (4 outliers removed) Z-score normalized Intensity Proteins (n=1278)

19 Coefficient of Variation Distributions 7 QC pool injections over 9 days of data acquisition Peptides (n=6024) Protein (n=1278) % % Frequency % 60% 40% 20% Cumulative % Frequency % 60% 40% 20% Cumulative % 0 0% 0 0% More Coefficient of Variation % (bins of 5%) More Coefficient of Variation % (bins of 5%) Average CV: 16.2% Median CV: 12.0% 80% of the signals have CVs < 22.0% 50% of the signals have CVs < 12.0% Average CV: 11.9% Median CV: 8.8% 80% of the signals have CVs < 16.4% 50% of the signals have CVs < 8.8%

20 Analytical Variation Across Dynamic Range Peptide Level (n=6024) Individual peptide Average for that order of magnitude Protein Level (n=1278) Individual protein Average for that order of magnitude %CV Peptide Intensity %CV Protein Intensity E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 Log10 Summed Peptide Intensity 0 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 Log10 Summed Protein Intensity

21 Mass Spectrometer Tandem Mass Spectrometry (MS/MS) for Peptide Identification Tandem MS permits selection and isolation of specific ions for subsequent analysis. Tandem instruments have multiple mass analyzers. Tandem Mass Spectrometer Synonyms - Tandem mass spectrometer - MS/MS

22 Fundamentals of MS/MS for Peptide ID 1. Precursor ions are selected and isolated 2. Collision-Induced-Dissociation results in fragmentation 3. Product Ions are characterized with the second mass analyzer Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER ION SOURCE PRECURSOR ION SELECTION NEUTRAL GAS COLLISIONS PRODUCT ION DETECTION DETECTOR

23 Quadrupole/Time-of-Flight Tandem Mass Spectrometer PUSHER TOF DETECTOR ZSPRAY TM Ion Source SAMPLING CONE SKIMMER RF HEXAPOLE RF HEXAPOLE QUADRUPOLE IN NARROW BANDPASS MODE HEXAPOLE GAS COLLISION CELL Q - TOF MS/MS MODE REFLECTRON

24 Common Peptide Product Ion Types - fortunately, peptide fragment in predictable locations along the peptide backbone z n-1 z n-2 z 2 z 1 y n-1 y n-2 y 1 x n-1 +2H x n-2 +2H x 1 +2H NH 2 _ CH _ CO _ NH _ CH _ CO _ NH _ CH _ CO... NH _ CH _ CO _ NH _ CH _ CO 2 H R 1 R 2 R 3 R n-1 R n a 1 a 2 a n-1 b 1 +2H b 2 +2H +2H b n-1 +2H c1 c 2 c n-2 c n-1 K. Biemann in Methods in Enzymology, 1990, 193, p

25 MS/MS Spectrumof Doubly Charged Glu-Fibrinopeptide: y-series ions Glu fib 50 ul of 0.06 um qt07123 AccMass 75 (Cen,2, 80.00, Ar,5000.0,0.00) 100 % : TOF MSMS ES+ R A S F F G E E N D N V (185.91) ymax y y b y y y y y y y y y **Remember, y-ions read C->N direction **Last y ion (y14) = peptide M+H M/z

26 Peptide Molecular Weight - Mass accuracy very important - Experimental MW compared with peptide MWs from in-silico digestion of proteome database - Generate list of MW matching (+/- mass error) Peptide Fragment MW - Mass accuracy very important - Experimental fragment MW compared with MWs from in-silico fragmentation of peptides from list Overview of Peptide Identification

27 Search Engines for Interpreting Peptide MS/MS Spectra Mascot MS-Tag (Protein Prospector) Omssa PepFrag (Prowl) pfind Phenyx Sonar (Knexus) X!Tandem (The GPM) XProteo Not on the Internet Identity E, Sequest, Paragon, Myrimatch, SpectrumMill, greylag

28 Search Parameters database taxonomy enzyme missed cleavages fixed modifications variable modifications protein MW protein pi estimated mass measurement error NOTE outstanding Help page with a lot of useful information If you only visit one web site for Peptide DB searching, this is the one

29 Interpretation of Peptide DB Searches Scaffold (Proteome Software) is a free viewer which accepts results from most MS/MS DB search engines Allows detailed evaluation of qualitative results from a sample Allows detailed comparison of qualitative results across experiments Provide Gene Ontology information on the identified proteins Free Download at

30 Instructions for Scaffold Download From Select Downloads Download Scaffold 4 Insure you select version appropriate for your computer You do not need to request an evaluation key as you will be using this as a viewer only (no cost) Selecting the Tour button allows you to view Flash presentations on use of this software for proteomic analyses Taking the Tours is Highly Recommended

31 Downloading Protein Databases for use in Scaffold - UniProt Example Visit Select Downloads from top left of web page From the UniProtKB select UniProtKB/Swiss-Prot FASTA This downloads a WinZip file, from which you extract the uniprot_sprot.fasta file

32 Scaffold Overview of Two Cell Lines, Two treatments Each - high resolution, accurate mass LC/MS/MS dataset - all fields sorted by clicking on header

33 Scaffold Overview of Four Phenotypes of a Cell Line Note each sample was analyzed in triplicate previous slide showed a composite result

34 Details of Identification of Actin -

35 Correlation of Peptide IDs Across Experiments

36 Details of the Identification of a Peptide in a Sample

37 Details of the Identification of a Peptide in a Sample

38 Scaffold View for Sequence Homology Challenges

39 Automated Generation of Venn Diagram Overlaps between Phenotypes in Scaffold Protein Overlap State 1 State State 3 Peptide Overlap State 1 State State 3

40 Scaffold Supplemental Information - Gene Ontology Biological Function and Compartment Information from NCBI

41 The Most important Step (?) Sample Preparation Goals of sample preparation Concentrate dilute samples Remove salts / buffer exchange Remove detergents Fractionate proteins MW Isoelectric Point Isolate sub-proteomes Depletion Enrichment Reduce, alkylate, digest Ultimately to reproducibly produce proteolytic peptides for LC/MS analysis

42 Cell/Tissue Lysis Duke Proteomics Core Facility Protocols Cell lines, soft tissues and solubilization of lysates typically accomplished using probe sonication Mass Spectrometry Appropriate Protocol for Cell Lysis using Probe Sonication Duke.pdf Hard tissues require more vigorous methods such as a Tissue Tearor in conjunction with TRIzol reagent Tissue Homogenization and RNA + Protein Extraction using TRIzol reagent dproteinextraction.pdf

43 Simple Sample Concentration If sample is in compatible/volatile buffers either simple speed-vac or lyophilization (freeze-drying) both work well Speed-vac is used for removal of organic solvents with minimal aqueous content Can be used in conjunction with lyophilization Lyophilization preferred with aqueous solvents Insure sample is well frozen before applying vacuum (!!) Separate cut and pierced cap is used with microcentrifuge tubes to minimize sample loss Lyophilization preferred when possible as resultant product suffers less degradation, is more stable and is more easily reconstituted Lyophilization originally developed during WWII for preserving blood serum for medical treatment of wounded

44 Desalting/Buffer Exchange/Concentration Spin columns with MW filters provide consistent results Use spin filters with small volume reservoir to preclude complete flow through Amicon Ultra 10 kda centrifugal filters work well Always rinse spin filters 2X with final buffer to remove PEGs from membranes Dialysis membranes are typically problematic due to polymeric leaching, Note that the molecular weight cutoff of these spin columns is broad and wide Always select higher/lower MW than you anticipate needing i.e. 10 kda cutoff for collection of peptides from flow-through fraction Note that spin filters lead to some sample loss Alternatives for low amounts samples Gel cleanup run sample only partially into 1D gel In-gel digestion of band Zeba Spin (Thermo) desalting columns Gel filtration columns» 7kDa exclusion volume» ~ 5% loss with 5 ug total protein loading

45 Detergent Cleanup/Sample Concentration ProteoSpin Detergent Removal spin column (Norgen Biotek) Effective for detergent removal ionic, non-ionic and zwitterionic detergents Relatively high sample losses minimal 50 ug total protein Mixed mode chromatography with SPE cartridges or 96 well plate formats Ion exchange and hydrophobic retention mechanisms Oasis MCX (Waters) have proven effective and provide high recovery Load in acid, wash in acid, wash with methanol, elute in base (NH4OH) Remove base via lyophilization or speed-vac Available in SPE cartridge or 96-well plate format Can run wells dry w/o impacting cleanup or recovery Simple precipitation using organic solvents methanol or acetone

46 Spin Filter Based/Aided Sample Preparation - Manza, Stamer, Ham, Codreanu and Leibler, Proteomics, 5, , Wisniewski, Zougman, Nagaraj and Mann, Nature Methods, 6(5), , Leibler and Ham, Nature Methods, 6(11), , 2009

47 Basic Sample Digestion Protocols Duke Proteomics Core Facility Protocols In-gel digestion of 1D Bands and 2D Spots Protocol easily adapted for other proteases Lys-C, Pepsin, Glu-C Add GG for activation of Glu-C; Glu-Glu (0.5 mm) is an dipeptide activator which enhances GluC activity by 20-50% but is not required In-Solution Tryptic Digestion Protocol Perform Bradford assay to determine protein concentration of each solution. Normalize concentrations for samples within a group using 50 mm AmBic. Aim for a protein concentration of between 0.1 and 1 µg/µl at the end of this protocol Notes Any sample manipulation prior to trypsin digestion should be done in a BSC or laminar flow hood Wear nitrile (not latex) gloves Wear a lab coat and make sure there is no gap between your coat sleeve and the gloves (lab tape works well)

48 Sample Enrichment and Depletion Due to LC/MS sample loading and dynamic range limitations, the coverage of the proteins of interest is almost always improved by selective enrichments and/or depletions Depletion/fractionation methods MW cutoff spin filters 1D Gels (!) Immunodepletion Enrichment methods Brute force MW and isoelectric point Selective Immunodepletion Resin-assisted capture

49 Sample Depletion of Abundant Plasma Proteins using Immunodepletion Analyses of biofluids confounded by high amounts of plasma-derived proteins Fourteen high abundance plasma proteins account for 94% of total protein in plasma albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2- macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein Al, apolipoprotein All, complement C3, and transthyretin These proteins found in a variety of biofluids Including plasma, serum, BALF, synovial fluid, cerebrospinal fluid Immunodepletion using immobilized antibodies is the preferred approach Liu, et al, MCP, 2006 Whiteaker, et al, JPR, 2007 detailed comparison of techniques Tu, et al, JPR 2010 Spin-column and LC column formats available from multiple vendors Agilent MARS is the standard approach in many labs Expensive Quality Control must be applied Column conditioning is absolutely require to minimize non-specific protein removal

50 Reproducibility of Sample Depletion Multiple QC metrics required to insure reproducibility Including depletion of reference plasma pool sample each daily (5% of samples) MARS 14 Depletion Unbound Bound Chromatogram AUC QC Bound vs Unbound Fractions DAD1 A, Sig=280,16 Ref=360,100 (042009\ D) DAD1 A, Sig=280,16 Ref=360,100 (042009\ D) mau Area: Area: min Bradford Assay QC Protein levels pre and post depletion 1D SDS-PAGE QC JMP Box Plot Analysis of band volumes

51 Selective Enrichment/Fractionation - based on isoelectric point Fractionation OFFGEL Fractionation (Agilent) Can be performed at protein or peptide level In-solution fraction collection (12 or 24 fractions) Reference 50 fraction SCX 24 fraction pi

52 Selective Enrichment - based on immunoprecipitation of tagged protein constructs Tagging of target proteins via fusion constructs (N-terminal or C-terminal) precludes challenges in generating antibody that specifically targets the protein of interest Various tags are in widespread use for many uses, including mapping of PTMs and studies of protein complexes Some concerns exist regarding biological relevance because the tag itself may obscure native interactions or introduce non-native interactions Protein Affinity Purification Products are widely available One comprehensive source with reference guides available at Pierce Co-immunoprecipitation (co-ip) Troubleshooting Guide

53 Recommended Resins for Enrichment of Proteins with Commonly Incorporated Tags, and their Binding Partners Duke Proteomics Core Facility Protocols Acknowledgements for Recommended Resins - Dr. Josh Anderson, Dr. Matt Foster, Dr. Lukasz Kozubowski, Dr. Erik Soderblom, Dr. Will Thompson, all from Duke University School of Medicine

54 Recommended Resins for Enrichment of Proteins with Commonly Incorporated Tags, and their Binding Partners Duke Proteomics Core Facility Protocols Acknowledgements for Recommended Resins - Dr. Josh Anderson - Dr. Matt Foster - Dr. Lukasz Kozubowski - Dr. Erik Soderblom - Dr. Will Thompson - all from Duke University School of Medicine

55 Selective Enrichment - based on immunoprecipitation using antibodies Use of selective antibodies has proven quite useful, but presence of antibody in final solution can present an overwhelming background in LC/MS/MS analysis Immobilization of antibody to resins minimizes this issue but can present additional problems Steric hindrance of native antibody-antigen complex Variety of crosslinking methods available, including Carbohydrate immobilization using hydrazide-activated agarose beads and polyacrylamide resins Crosslinking Primary Antibody to Protein A/G resin using dimethylpimelimidate

56 Enrichment of PTM Sub-Proteomes DIGESTION CHEMICAL ENRICHMENT TiO2 or IMAC ANTIBODY ENRICHMENT (e.g. acetyl-k, UBI-K, SUMO-K) ANTIBODY ENRICHMENT DIGESTION PTM-SWITCH AND RESIN-ASSISTED CAPTURE 1. BLOCK NATIVE AMINO ACID 2.SPECIFICALLY CLEAVE PTM = Free amino acid = Chemically blocked amino acid = Amino acid with posttranslational modification = Free amino acid, formerly with PTM 3. ENRICH a) RAC (direct on-resin) b) BST (derivatize with biotin, enrich on SA resin)

57 Selective Enrichment - based on resin assisted capture Several methods have been developed to enrich for proteins based on their PTM Protocol for Post Capture Sample Processing of SNO RAC and Acyl RAC Proteomics Samples Proteomic analysis of S nitrosylation and denitrosylation by resin assisted capture. Forrester MT, Thompson JW, Foster, MW, Nogueira L, Moseley MA, Stamler JS. Nat Biotechnol Jun; 27(6): Site specific analysis of protein S acylation by resin assisted capture (Acyl RAC). Forrester MT, Hess DT, Thompson JW, Hultman RC, Moseley MA, Stamler JS, Casey PJ. J Lipid Res Epub Nov 2.

58 Phosphorylation Analysis by Mass Spectrometry

59 A. Data Acquisition Workflow Phosphoproteomic Workflow Overview B. Performance of Enrichment Extract and solubilize protein, digest, spike bovine 30 fmol/ug (N) Number of Peptides Reproducibility Across 4 Enrichments Phosphopeptide Metrics Average % CV= 10.3% Median %CV = 7.8% 80% of Phosphopeptides %CV < 12.4% %CV C. Data Analysis (glioblastoma xenograft tumors) TiO 2 Phosphopeptide Enrichment (1) Qualitative only LC-MS/MS (3) Qualitative/Quantitative LC-MS/MS Non- Aligned Aligned

60 A Robust, Single Step Enrichment Protocol TiO2 enrichment conditions with the best combination of specificity, yield, and reproducibility Non-Phosphorylated Peptides Phosphorylated Peptides Summed Non-Phosphorylated Peptide Intensity Summed Phosphorylated Peptide Intensity Soderblom et al, Analytical Chemistry 2011 May 15;83(10):

61 Specificity as Function of [DHB] and Loading Global Phosphorylated Peptide Specificity (%) Singly Phosphorylated Peptide Specificity (%) Multiply Phosphorylated Peptide Specificity (%) Resulting Generic Conditions: Enrich at 2X Peptide/Resin Capacity and mg/ml DHB Soderblom et al, Analytical Chemistry 2011 May 15;83(10):

62 Drastic Effect of Enrichment Conditions on Singlyversus Multiply-Phosphorylated Species SSPKDEEEAEpSPAEDEEEDEVEK SpSPKDEEEAEpSPAEDEEEDEVEK Relative Intensity of a Phosphopeptide from Nuclear Casein Kinase, Quantified in Singly and Doubly-Phosphorylated Forms Soderblom et al, Analytical Chemistry 2011 May 15;83(10):

63 Quantitative Accuracy and Reproducibility of Phosphopeptide Enrichment Workflow TiO2 TiO2 TiO2 X fmol casein/ 1 ug lysate Frequency (3) LC-MS (3)LC-MS (3) LC-MS Analytical Reproducibility Analytical + Enrichment Reproducibility Log10 Intensity 25 fmol/ug 25 fmol 75 fmol TiO2 TiO2 (3) LC-MS (3)LC-MS CV% (Bins of 10%) Median Analytical CV: 6.3% Median Analytical + Enrichment CV: 22.8% Log 10 Intensity 75 fmol /ug Expected Fold-Change: 3.0 Average Measured Fold-Change: 3.1 Soderblom et al, Analytical Chemistry 2011 May 15;83(10):

64 Migration to Automated Ti0 2 Enrichment at a Capillary LC Scale Format Average %CV Median %CV 80% peptides below Spin Column Capillary Column Dr. Brenna M. Richardson, Dr. Erik J. Soderblom, et al., manuscript in preparation.

65 Phosphoproteomics Projects Exemplars Project Total Protein/Sample Unique Samples Unique 1% FDR 1 Unique 5% FDR 1 Average Technical % CV Drug Treated Mouse Lung 600 µg % Mouse Breast Cancer Tumor 2000 µg % Drug Treated Mouse Brain 1000 µg % Cardiovascular Disease Human Heart Tissue 1000 µg % Drug Treated Human Tumor 2000 µg % Treated Human Airway Epithelial Cells 890 µg % Nanoparticle Treated Mouse Lung Tissue 930 µg % Sickle Cell Human Red Blood Cell Ghosts (membrane fraction) Study µg % Infected Epithelial cells 750 µg % Stimulated/Inhibited Mouse Lung Tissue Study µg % Stimulated/Inhibited Mouse Lung Tissue Study µg % Treated Sickle Cell Human Red Blood Cell Ghosts (membrane fraction) Study µg % Cardiovascular Disease Human Heart Tissue 600 µg % Challenged /Treated Mouse Lung Tissue Study µg % G-protein coupled receptor Knockdown Zebrafish Embryo lysate 200 µg % 1. 1% or 5% occurrence of reverse entries from target decoy database mascot searches

66 Enrichment of Phosphoproteomes DIGESTION CHEMICAL ENRICHMENT TiO2 or IMAC or Phospho-motif specific antibody

67 Unique Insights into the Phosphoproteome Obtained by these Four Different Methods Antibody Phospho-Motif Specific Unique Peptides Antibody Phospho-Motif Specific And TiO2 Unique Peptides 649 Proteins 0.3% Protein FDR 3526 Spectra 0.1% Peptide FDR

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