Quantitative Proteomics. Quantitative Proteomics

Transcription

1 Quantitative Proteomics Liwen Zhang Mass Spectrometry and Proteomics Facility The Ohio State University Summer Workshop 216 Quantitative Proteomics Quantitation in proteomics has become a popular area in recent proteomics research with the development of quantitation techniques such as DIGE, SILAC, ICAT, itraq and Label Free. Difference Gel Electrophoresis Gel based using cy-dye chemistry. Isobaric tag for relative and absolute quantitation itraq is a non-gel based technique used to identify and quantify proteins/peptides from different sources in one single experiment by using isotope coded covalent tags that will label the N-terminus and side chain amines of peptides from protein digestions. Stable isotope labeling by amino acids in cell culture SILAC is a non-gel based approach for in vivo incorporation of a label into proteins for MS quantitative proteomics. It relies on metabolic incorporation of a given 'light' or 'heavy' form of the amino acid into the proteins. Label Free Quantitation It has been observed the chromatographic peak areas and number of spectra/peptides observed for a protein in a LC/MS/MS run is correlated with the concentration of that particular protein.

2 DIGE (Difference Gel Electrophoresis) DIGE (Difference Gel Electrophoresis) Spot Volume = [spot 1 on treated]/[spot 1 on standard]

3 DIGE (Difference Gel Electrophoresis),, MASCOT Pros and Cons to DIGE high sensitivity linearity of the dyes utilized straightforward significant reduction of experiment error High reproducibility µg of total protein is required for each biological replicate Requires high resolution 2D gels Not ideal for membrane proteins Not ideal for serum type samples Some protein spots identify more than one protein or do not have enough protein to identify the spot Labor Intensive

4 itraq TM (Applied Biosystems) Overview of itraq Reagents Methodology Isobaric tag for relative and absolute quantitation Cell Culture + Tissue Trypsin digestion Labeling Lys and N terminus Up to 8 samples Fractionation offers more identification

5 Chemistry of TMT Reagents Figure Provided Courtesy of Thermo Fisher Scientifics Workflow of TMT Reagents Figure Provided Courtesy of Thermo Fisher Scientifics

6 Dry Eye Studies: 4 Groups: Normal, Moderate Dry Eye, Mild Dry Eye, Mixed Dry Eyes y2 y3 Group IS Normal Mod Mild Mix 1 IS 1 (126) 24 (127) 241 (128) 243 (129) 269 (1) 2 IS 2 (126) 27 (131) 242 (127) 2 (128) 266 (129) 3 IS 3 (126) 28 (1) 26 (131) 2 (127) 237 (128) 4 IS 4 (126) 263 (129) 246 (1) 238 (131) 214 (127) IS (126) 21 (128) 268 (129) 271 (1) 236 (131) 6 IS 6 (126) 216 (127) 267 (128) 262 (1) 23 (131) Ratio 126:127:128:129:1:131=1:.:.9:.2:.: Relative Ayundance m/z y y y y y9 Protein Name : Human Cystatin-S Peptide Sequence:EQTFGGVNYFFDVEVGR Observed m/z= Theoretical m/z= y y y11 y y14 y1 y Val (129+99) 11. Glu-Val Asp Phe Phe Tyr Asn 98.8 Val Gly Gly Phe.87 Thr Gln b b2 b b b b b b b b b b12 b13 b Gln.94 Thr Phe 6.92 Gly 7.2 Gly Val Asn Tyr Phe Phe 11. Asp Val Glu 1 1 m/z 1 1 itraq MASCOT Results

7 itraq Results DE/NDE (Ratio (# of Detection, p value)) Protein Name Mild/Normal Mod/Normal Mix/Normal Function Aldehyde dehydrogenase, dimeric NADP-preferring OS=Homo sapiens (2.41, 6,.) (1.69, 6,.471) oxidoreductase activity Apolipoprotein A-I OS=Homo sapiens (1.7, 6,.362) transporter activity; protein binding; enzyme regulator activity; lipid binding Cystatin-S OS=Homo sapiens (.1, 6,.14) (.43, 6,.) (., 6,.283) enzyme regulator activity Deleted in malignant brain tumors 1 protein OS=Homo sapiens (., 6,.472) signal transducer activity; protein binding; bacterial cell surface binding Ezrin OS=Homo sapiens (1.79, 6,.1) protein binding; binding Hemopexin OS=Homo sapiens (2.1, 6,.384) (2.11, 6,.486) binding; transporter activity; ion binding Haptoglobin OS=Homo sapiens (1.97, 6,.474) catalytic activity; protein binding; peptidase activity Ig alpha-1 chain C region OS=Homo sapiens (.9, 6,.113) antigen binding; protein binding Ig gamma-1 chain C region OS=Homo sapiens (1.94, 6,.266) antigen binding; protein binding Ig gamma-2 chain C region OS=Homo sapiens (1.87, 6,.369) antigen binding Ig gamma-3 chain C region OS=Homo sapiens (2.8, 6,.8) antigen binding Extracellular glycoprotein lacritin OS=Homo sapiens (.28, 6,.9) (.23, 6,.3) (.48, 6,.428) protein binding; extracellular matrix binding Putative lipocalin 1-like protein 1 OS=Homo sapiens (.43, 6,.26) (.39, 6,.2) (., 6,.119) binding; transporter activity Lipocalin-1 OS=Homo sapiens (.33, 6,.) (.32, 6,.3) (.43, 6,.188) binding; transporter activity; protein binding; enzyme regulator activity Lysozyme C OS=Homo sapiens (.33, 6,.19) (.28, 6,.31) catalytic activity; hydrolase activity; protein binding Polymeric immunoglobulin receptor OS=Homo sapiens (.1, 6,.2) (.43, 6,.16) protein binding Prolactin-inducible protein OS=Homo sapiens (.36, 6,.4) (.38, 6,.8) (.8, 6,.9) protein binding Proline-rich protein 1 OS=Homo sapiens (.44, 6,.23) (.39, 6,.6) enzyme regulator activity Proline-rich protein 4 OS=Homo sapiens (.41, 6,.42) (.34, 6,.81) Secretoglobin family 1D member 1 OS=Homo sapiens (.28, 6,.32) (.21, 6,.6) (.41, 6,.169) binding Mammaglobin-B OS=Homo sapiens (.34, 6,.76) (.26, 6,.9) (.43, 6,.21) steroid binding; binding; hormone binding Serotransferrin OS=Homo sapiens (2.2, 6,.429) protein binding; ion binding Lactotransferrin OS=Homo sapiens (.33, 6,.19) (.34, 6,.72) peptidase activity; ion binding; pattern bindingcarbohydrate binding; hydrolase activity; protein binding Vitamin D-binding protein OS=Homo sapiens (1.6, 6,.449) (1.87, 6,.26) transporter activity; steroid binding; vitamin binding;: protein binding Zinc-alpha-2-glycoprotein OS=Homo sapiens (., 6,.47) (.36, 6,.29) (.9, 6,.2) lipid binding; carboxylic acid binding; hydrolase activity; transporter activity Zymogen granule protein 16 homolog B OS=Homo sapiens (.44, 6,.383) carbohydrate binding Pros and Cons to itraq Requires One less total protein than DIGE ( µg or less) experiment can determine fold change, protein ID and Post translational modification Mass Spectrometry requirements are rigorous High resolution Advanced LC chromatography Long instrument time PQD/HCD needed on IT instrument Up to 8- groups can be compared at the same time Produces enormous amount of data Samples can come from both tissue or cell culture Requiring detailed bioinformatic analysis Expensive

8 SILAC (Stable isotope labeling by amino acids in cell culture) Regular Media (Lys Light) Lys-depleted Media (Lys Heavy) Heavy Isotopic Labeled Amino Acids: No Drug Combination/ Extraction Drug Treatment L-Lysine: 13 C 6, (+6Da), 13 C 6 / 1 N 2 (+8Da), 13 C 6 / 1 N 2 /D9 (+17Da), 1 N 2 /D9 (+11Da), D4 (+4Da) L- Arginine: 13 C 6 (+6Da), 13 C 6 / 1 N 4 (+Da), 13 C 6 / 1 N 4, D7 (+17Da), 1 N 4 /D7 (+11Da) L-Tyrosine: 13 C 9 (+9Da) Fractionation/IP Digestion/ Peptide Identification 1D or 2D Change = Optimization of Heavy Amino Acids Incorporation S G R G K GGK 8 GL GK 12 GG AK 16 RHRK 2 V L R D N I Q G I T K 31 P A I R R L ARRGGVK 44 R I S G L I Y E E T R G V L K 9 V F L E N V I R D A V T Y T E H A K 77 R K 79 T V T A M D V V Y A L K 91 R Q G R T L Y G F G G 1133 M =4*11= Mass

9 17881_13Days # RT: AV: NL: 6.3E T: FTMS + c ESI Full ms [.-2.] m/z RelativeAbundance K* = 13 C 6 / 1 N 2 labeled Lys R* = 13 C 6 / 1 N 4 labeled Arg Cell Culture Labeled for 4 days Cell Culture Labeled for 7 days 17872_1_7 # RT: AV: 13 NL:.19E4 T: FTMS + p ESI Full ms [.-2.] K* = 13 C 6 / 1 N 2 labeled Lys R* = 13 C 6 / 1 N 4 labeled Arg 6 RelativeAbundance M =+8= 18Da 672. Ratio=Heavy/Light=1: m/z DNIQGITKPAIR DNIQGITK*PAIR* 17872_1_7 #12 RT: AV: 1 NL: 1.1E3 T: ITMS + c ESI d Full ms @cid3. [1.-13.] 18Da Da MSMS of DNIQGITK*PAIR* MSMS of DNIQGITKPAIR RelativeAbundance m/z

10 Accession Description Ratio: Heavy/Light Sample 1 Sample 2 P327 Keratin, type I cytoskeletal 9 OS=Homo sapiens P22314 Ubiquitin like modifier activating enzyme 1 OS=Homo sapiens P2326 Adenosylhomocysteinase OS=Homo sapiens.2.11 P1364 Keratin, type I cytoskeletal OS=Homo sapiens P297 Heterogeneous nuclear ribonucleoprotein F OS=Homo sapiens.2.24 O4317 D 3 phosphoglycerate dehydrogenase OS=Homo sapiens P31943 Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens P23771 Trans acting T cell specific transcription factor GATA 3 OS=Homo sapiens O299 Chloride intracellular channel protein 1 OS=Homo sapiens P7741 Adenine phosphoribosyltransferase OS=Homo sapiens Q143 Heterogeneous nuclear ribonucleoprotein D OS=Homo sapiens.39.3 Q9BQE3 Tubulin alpha 1C chain OS=Homo sapiens.3.4 P13639 Elongation factor 2 OS=Homo sapiens P31949 Protein S A11 OS=Homo sapiens P87 Heat shock kda protein 1A/1B OS=Homo sapiens P1186 C 1 tetrahydrofolate synthase, cytoplasmic OS=Homo sapiens P23246 Splicing factor, proline and glutamine rich OS=Homo sapiens P31942 Heterogeneous nuclear ribonucleoprotein H3 OS=Homo sapiens P846 Myotrophin OS=Homo sapiens P kda glucose regulated protein OS=Homo sapiens P8 Phosphoglycerate kinase 1 OS=Homo sapiens..74 P61978 Heterogeneous nuclear ribonucleoprotein K OS=Homo sapiens P62826 GTP binding nuclear protein Ran OS=Homo sapiens P Profilin 2 OS=Homo sapiens O6 Heterogeneous nuclear ribonucleoprotein Q Q99623 Prohibitin 2 OS=Homo sapiens Q68 Peroxiredoxin 1 OS=Homo sapiens P62244 S ribosomal protein S1a OS=Homo sapiens P73 Annexin A2 OS=Homo sapiens P272 Proteasome subunit beta type 6 OS=Homo sapiens P21333 Filamin A OS=Homo sapiens Pros and Cons to itraq One experiment can determine fold change, protein ID and Post translational modification Up to several groups can be compared at the same time Mixing at the beginning, allow more sample preparation steps Expensive Special data analysis platform Samples come from cell cultures Time consuming significant reduction of experiment error High reproducibility

11 Label-free quantification doesn t use a stable isotope containing compound to chemically label the protein. It can determine the relative amount of proteins in two or more biological samples by comparing peptide peak areas or spectral counting. GLLEDLGYDVVVK CASP4_MOUSE Caspase-4 OS=Mus musculus # b Seq. y # G L L E D L G Y D V V V K

12 143_WT_LEG #14664 RT: 89.2 AV: 1 NL: 1.39E3 T: ITMS + c ESI d Full ms @cid3. [1.-2.] _WT_LEG #143 RT: AV: 1 NL: 1.9E3 8 T: ITMS + c ESI d Full ms @cid3. [1.-16.] _WT_LEG #1439 RT: AV: 1 NL: 3.3E T: ITMS + c ESI d Full ms @cid3. [21.-2.] 143_WT_LEG #146 RT: 87.3 AV: 1 NL: 7.47E T: ITMS + c ESI d Full ms @cid3. [2.-2.] _WT_LEG #14467 RT: 88.2 AV: 1 NL: 4.3E3 T: ITMS + c ESI d Full ms @cid3. [ ] m/z m/z m/z m/z m/z Data Dependent Acquisition and Dynamic Exclusion Data Dependent Top Method 143_WT_LEG # RT: AV: 19 NL: 3.22E T: FTMS + c ESI Full ms [.-2.] RelativeAbundance Relative Abundance m/z 1 Relative Abundance Relative Abundance Relative Abundance 4 Relative Abundance MSMS MS Cycle:.6S LC Peak Width >12S Dynamic Exclusion Make MSMS for the lower abundant ions possible!! Proteomics Work Flow Digestion LC/MS MS/MS Peak Area MASCOT ProteomeDiscoverer Spectral Counting MASCOT/Scaffold ProteomeDiscoverer Relative Concentration (empai) MASCOT

13 Label-free quantification by Spectra Counting Condition A Condition B Trypsin Digestion Analysis Label Free Quantitation by Spectral Counting Relative protein quantification is achieved by comparing the number of identified MS/MS spectra from the same protein in each of multiple LC/MS/MS Hongbin Liu, Rovshan G. Sadygov and John R. Yates, III, A Model for Random Sampling and Estimation of Relative Protein Abundance in Shotgun Proteomics Anal. Chem. 24, 76,

14 Label Free Quantitation by Spectral Counting # of reports = # of Treatment Conditions X # of Bio-replicates Scaffold Spectral Counting Results-Scaffold Conditions Bio-Replicates # of Spectra P-Value

15 Spectral Counting Results-Scaffold Spectral Counting Results-Scaffold

16 Spectral Counting Results Label Free Quantitation by Precursor Ion Peak Area Fundamentally, MS1 based measurements are more accurate and precise than spectral counting with a better linear dynamic range. This arises due to a number of weaknesses of spectral counting: No direct measurement of peptide ion properties The response in terms of spectra per peptide ion is not constant across different features. The linear dynamic range of the method can be limited by saturation effects. Dynamic exclusion methods, designed to improve DDA coverage, can also affect the response. There is a stochastic aspect to DDA sampling, hampering reproducibility; DDA sampling is also biased towards more abundant species, for this reason.

17 Label Free Quantitation by Precursor Ion Peak Area

18 Label Free Quantitation by Precursor Ion Peak Area Description Peak Area # of Spectra Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Histone H4 OS=Homo sapiens GN=HIST1H4A PE=1 SV=2 [H4_HUMAN].99E+ 4.24E+.46E Histone H2B type 1 K OS=Homo sapiens GN=HIST1H2BK PE=1 SV=3 [H2B1K_HUMAN] 3.E+ 7.82E+9 1.6E Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 [ACTB_HUMAN] 1.64E+ 1.12E+ 1.47E Beta actin like protein 2 OS=Homo sapiens GN=ACTBL2 PE=1 SV=2 [ACTBL_HUMAN] 1.E+ 4.74E E Actin, alpha cardiac muscle 1 OS=Homo sapiens GN=ACTC1 PE=1 SV=1 [ACTC_HUMAN] 1.34E+ 9.2E E Actin, alpha skeletal muscle OS=Homo sapiens GN=ACTA1 PE=1 SV=1 [ACTS_HUMAN] 1.34E+ 9.2E E Histone H2A.Z OS=Homo sapiens GN=H2AFZ PE=1 SV=2 [H2AZ_HUMAN] 1.18E+ 2.7E+ 3.2E Histone H2A type 1 D OS=Homo sapiens GN=HIST1H2AD PE=1 SV=2 [H2A1D_HUMAN] 1.13E+ 2.73E+ 3.16E Histone H2A type 2 A OS=Homo sapiens GN=HIST2H2AA3 PE=1 SV=3 [H2A2A_HUMAN] 1.13E+ 2.73E+ 3.16E >XX RS27A_HUMAN 1.9E+.32E+9 1.2E Heterogeneous nuclear ribonucleoproteins A2/B1 OS=Homo sapiens GN=HNRNPA2B1 PE=1 SV=2 [ROA2_HUMAN] 9.76E E E Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 [VIME_HUMAN] 8.2E+9.64E E Complement component 1 Q subcomponent binding protein, mitochondrial OS=Homo sapiens GN=C1QBP PE=1 SV=1 [C1QBP_HUMAN] 6.16E+9 3.2E+9.7E >XX TRYP_PIG.32E+9 1.6E+ 6.16E kda heat shock protein, mitochondrial OS=Homo sapiens GN=HSPD1 PE=1 SV=2 [CH_HUMAN].2E+9 3.2E E ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATPB PE=1 SV=3 [ATPB_HUMAN].8E+9 3.2E E D SDS PAGE Fractionation Workflow Same Volume MASCOT Search 9 11 >1 Proteins identified Global Digestion: 92 Proteins SCX : 981 Proteins 1D SDS : >1 Proteins

19 Time (min) Time (min) Multi-dimensional Separation ph. ph 2.6 F 1 $/fraction on the orbitrap XL $7/fraction on the orbitrap Fusion RT: F2 Relative Abundance Time (min) Fractionation (F) Time (min) Gradient separation F1 Slide courtesy of Nilini S. Ranbaduge 37 Human HEK293 Cell 2D 4ug Loading; 3 replicates Sequest HT (D2) 183 Sequest HT (D2) 2938 Sequest HT (E2) Sequest HT (E2) Sequest HT (F2) Sequest HT (F2) 277 Total Label Description 337 Sequest HT (D2) Identified by: Sequest HT (D2) 3488 Sequest HT (E2) Identified by: Sequest HT (E2) 3497 Sequest HT (F2) Identified by: Sequest HT (F2) 3127 Sequest HT (D2)/(E2) Identified by: Sequest HT (D2)/ (E2) 3113 Sequest HT (E2)/(F2) Identified by: Sequest HT (E2)/ (F2) 322 Sequest HT (D2)/(F2) Identified by: Sequest HT (D2)/(F2) 297 Sequest HT (D2)/(E2)/(F2) Identified by: Sequest HT (D2)/(E2)/(F2) Total Label Description 2 Sequest HT (D2) Identified by: Sequest HT (D2) 2473 Sequest HT (E2) Identified by: Sequest HT (E2) Sequest HT (F2) Identified by: Sequest HT (F2) Sequest HT (D2)/(E2) Identified by: Sequest HT (D2)/ (E2) Sequest HT (E2)/(F2) Identified by: Sequest HT (E2)/ (F2) Sequest HT (D2)/(F2) Identified by: Sequest HT (D2)/(F2) 1492 Sequest HT (D2)/(E2)/(F2) Identified by: Sequest HT (D2)/(E2)/(F2) 3398

20 Relative Quantitation of PTM Using Peak Area (Based on Retention Time and SIC) RT: SM: 7B RT: AA: GELLEAIKR123 NL: 1.7E8 Base Peak m/z= F: FTMS + c ESI Full ms [.-2.] MS ICIS 138_TRY Relative Abundance 2 RT: AA: 3238 RT: RT: 41.4 RT: AA: AA: AA: RT: AA: RT: AA: RT: AA: RT: 48.1 AA: NL: 2.39E6 Base Peak m/z= F: FTMS + c ESI Full ms [.-2.] MS ICIS 138_TRY 11GELLEAIK(Ac)R123 2 RT: 31.3 RT: RT: 3.79 RT: 4.2 RT: AA: AA: AA: 462 AA: 7 AA: Time (min) Sequence m/z Mass Peak Area RT Total Peak Percentage Theoretical Observed Error (min) Area (%) 11GELLEAIKR GELLEAIK(Ace)R E+9.

21 Relative Quantitation Using empai empai = PAI -1 Protein content (mol %) = empai/ (empai) x Ishihama Y 1, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M. Exponentially modified protein abundance index (empai) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics. 2 Sep;4(9): Protein name Conc empa Protein Content Rank. Fmol I Conc. empai Conc. empa I Elongation factor 1-a a enolase Heat shock protein HSP -a Vimentin protein S ribosomal protein S Pyruvate kinase, M2 isozyme S ribosomal protein S GTP-binding nuclear protein RAN ADP, ATP carrier protein, fibroblast isoform Peripherin Stress- protein, mitochondrial precursor Fructose-bisphosphate aldolase A IgE-binding protein Calreticulin precursor S ribosomal protein L S ribosomal protein L Peroxiredoxin Voltage-dependent anion-selective channel protein T-complex protein 1, e subunit ATP synthase oligomycin sensitivity conferral protein Phosphate carrier protein, mitochondrial precursor T-complex protein 1, a subunit B Nucleolar RNA helicase II