MS Based Proteomics: Recent Case Studies Using Advanced Instrumentation

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MS Based Proteomics: Recent Case Studies Using Advanced Instrumentation Chris Adams, PH.D. Stanford University Mass Spectrometry http://mass-spec.stanford.edu/

For personal use only. Please do not reuse or reproduce without the author s permission. 2

Instrumentation and Capabilities Proteomics Front End(s)- Eksigent Nano2D LC Mass Spectrometer(s)- LTQ Orbitrap Velos (ETD) LTQ

Instrumental Upgrades: More data of higher quality

Sample Prep & Data Analysis- Sample Prep -In-gel digests -Solution -Enrichments -Labels (itraq, TMT) SP DA Data Analysis -Sequest -Mascot -Scaffold for visualization -Statistics (FDR) -Qualitative ti v. Quantitative

Current Case Studies Global proteome analysis Quantitative Proteomics- One task many alternatives.. PTM mapping

Project: 1 Global l Proteome Professor Virginia i i Walbot, Dr. Dave Skibbe http://www.stanford.edu/~walbot/ Tumorigenic Fungal Peptides Causing Plant Cancer: Defining Expression Timing and Modeling Structural Similarities to Host Proteins Ustilago maydis causes cancer within a few days of infecting maize by stimulating extra cell divisions, endopolyploidization (up to 64N), and enormous cell expansion. Although U. maydis grows on many hosts, only maize forms tumors where the pathogen can complete its lifecycle. Gene deletion demonstrated that secreted fungal proteins many of which are small and encoded by gene families are required for tumors. D. Skibbe, et al. Science 328, 89 (2010)

Project: 1 Global Proteome (cont.) huitlacoche WEET-LA-KO-CHEE

1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 6 / 2 3 / 2 0 1 0 4 : 1 8 : 1 8 P M R T : 0. 0 0 0 0 0-5 9. 9 9 3 2 0 1 0 0 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 2 0. 7 8 3 8 7 2 1. 0 0 6 7 1 2 3. 2 5 5 7 1 2 6. 1 3 8 7 1 2 7. 9 3 3 5 3 3 2. 4 9 0 2 1 3 2. 8 8 6 3 7 3 7. 2 8 6 0 3 4 9. 0 5 2 3 7 3 0 1 9. 0 8 7 0 4 2 5 2 0 1 6. 0 6 2 7 1 1 5 3 0. 4 6 4 5 4 1 1. 7 8 3 3 7 1 0 1 3. 8 2 9 5 4 1 1. 4 7 7 7 1 5 5 0. 1 5 4 0 4 4 2. 5 8 5 5 4 4 6. 7 4 5 3 8 5 4. 8 6 4 3 8 3. 3 5 2 0 4 5. 2 1 3 5 3 1 0. 3 1 2 7 1 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 T i m e ( m i n ) 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 6 / 2 3 / 2 0 1 0 4 : 1 8 : 1 8 P M 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A # 2 8 1 5 R T : 2 6. 0 1 A V : 1 N L : 3. 3 0 E 8 T : F T M S + p N S I F u l l m s [ 4 0 0. 0 0-1 8 0 0. 0 0 ] 6 3 4. 3 2 7 3 1 0 0 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 N L : 2. 0 7 E 9 T I C M S 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 1 2 6 7. 6 4 6 5 8 0 7. 9 2 2 2 7 2 8. 3 9 2 8 4 9 6. 2 5 3 3 z = 3 1 0 9 2. 0 8 5 6 6 6 9. 8 4 5 1 8 8 4. 4 5 3 0 6 1 5. 3 0 6 7 9 6 1. 4 9 9 3 9 9 1. 4 9 9 5 1 1 2 7. 1 0 2 3 1 2 2 9. 6 0 5 0 1 2 7 9. 1 3 8 8 z = 3 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 1 1 5 0 1 2 0 0 1 2 5 0 1 3 0 0 1 3 5 0 m / z 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 6 / 2 3 / 2 0 1 0 4 : 1 8 :1 8 P M 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A # 2 8 1 6 R T : 2 6. 0 2 A V : 1 N L : 7. 3 6 E 5 T : F T M S + p N S I d F u l l m s 2 8 0 7. 9 2 @ h c d 4 5. 0 0 [ 1 0 0. 0 0-1 6 3 0. 0 0 ] 1 0 0 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 6 3 6. 3 3 4 9 5 1 7. 3 3 4 4 6 5 4. 3 4 5 2 7 6 7. 4 2 9 3 9 1 3. 5 3 4 1 7 8 5. 4 3 8 8 5 4 6. 2 1 9 1 6 3 2. 3 6 0 0 7 4 5. 4 4 5 8 5 5 7. 2 9 3 6 7 3 9. 4 3 5 3 8 1 6. 4 8 1 9 z =? 1 0 2 6. 6 2 3 7 6 2 6. 3 5 2 3 8 9 5. 5 1 9 3 z =? z =? 6 7 0. 3 7 7 8 1 1 5 5. 6 6 5 2 8 8 0. 5 1 7 4 1 2 8 4. 6 8 2 3 z =? 1 0 0 7. 5 0 9 9 1 0 4 4. 0 4 6 5 z =? z =? 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 1 1 5 0 1 2 0 0 1 2 5 0 1 3 0 0 Lane B1 B1 B2 Global l Proteome (cont.) Infected Uninfect In-gel tryptic digest 1 2 3 4 5 6 peptides Reversed Phase nanolc Relative Abundance B3 Full Scan Orbitrap B4 Relative Abundance B5 MS/MS top 15 Ion Trap B6 Relative Abundance

Global l Proteome (cont.) 100118_dskibbe_walbot_l1_1D RT:4.6354-57.5517 100 537.82 412.99 647.88541.64 430.72554.30 657.34 628.87 811.43 50 B1 483.79 629.33 621.34 602.05 867.13 401.23 408.22 1087.1616 25 100 0 445.12 445.12620.26 480.33 896.50 892.25 412.98 537.82 50 867.13 B2 771.89 647.88 541.64 811.43 555.76 483.79 430.72 485.74 689.89 0 445.12445.12 402.25 401.23 480.33 896.50 892.50 100 513.31 421.76 489.26 626.87 908.48 50 B3 708.90 717.39 511.27 472.90464.80 897.52 499.91 559.66 638.37 997.20 736.43 480.33 480.33 480.33 615.16 482.24524.56 100 0 421.76 421.76 50 B4 692.89 475.79 527.34 414.21 523.29 639.33 559.66 1017.19 1113.19 100 0 415.21 445.12 524.56 578.77 615.40851.80 480.33 451.16 421.76 B5 50 681.83 470.30 492.25 418.23 544.27 587.83 449.09 441.28 594.351167.07 738.04 1176.93 100 0 615.16 445.12403.23 416.73 1314.61 868.61 480.33 415.21 413.01 453.76 50 B6 460.25 453.26 502.30 632.85 594.02 615.40 449.09 415.74 785.05 720.90 927.65 615.40 0 445.12 487.73 467.74 664.36 480.33 719.44 5 10 15 20 25 30 35 40 45 50 55 Time (min) Relative Abundance Instrument time: 6 bands x 6 lanes = 36 hours

1 2 3 4 5 6 1 peptide ID 1412 proteins 0.8 % Protein FDR 5%PeptideFDR Protein Inference Global l Proteome (cont.) 3500 Numbe er of Protein ns 3000 2500 2000 1500 1000 500 0 86.7 96.1 99.5 99.2 98.5 99.9 Protein Confidence Inverval 38 detected fungal proteins Total 1412

Global l Proteome (cont.) unts pectral Cou No ormalized S 200 150 100 50 1 2 3 1 2 3 Infected Mock

Global l Proteome (cont.) No rmalized Sp pectral Cou unts 120 80 20 1 2 3 1 2 3 Infected Mock

Quantitative i Proteomics Ex. 1 Spectral Counting Spectral count correlates well with protein abundance Fold change can be calculated and statistically evaluated Simple and straightforward implementation Sensitive to protein abundance changes for abundant proteins 2 fold change easily detected with high confidence Limitations The response to increasing protein amount is saturable Noisy data at low spectral counts large difference in spectral count necessary to determine significant change

Quantitative Proteomics Ex. 1 Dr. Jonathan Rothbard, Steinman Lab http://steinmanlab.stanford.edu/index.html Define the serological clients of shspb5 in normal and patient's plasma Small heat shock proteins are a family of intracellular chaperones that are cytoprotective due to their ability to bind partially unfolded proteins and prevent aggregation. Recently, HspB5 (alpha B crystallin) has been shown to be therapeutically effective when administered intraperatoneally in animal models of multiple sclerosis, rheumatoid arthritis, stroke, and ischemia reperfusion injury. In all cases, there is significant immune suppression. To test the hypothesis that the mode of action is the chaperone activity of the protein in plasma, the proteomic content of clients has been defined by mass spectrometry. More specifically, proteins associated with HspB5 in normal and patient's plasma. 15

1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 6 / 2 3 / 2 0 1 0 4 : 1 8 : 1 8 P M R T : 0. 0 0 0 0 0-5 9. 9 9 3 2 0 1 0 0 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 2 0. 7 8 3 8 7 2 1. 0 0 6 7 1 2 3. 2 5 5 7 1 2 6. 1 3 8 7 1 2 7. 9 3 3 5 3 3 2. 4 9 0 2 1 3 2. 8 8 6 3 7 3 7. 2 8 6 0 3 4 9. 0 5 2 3 7 3 0 1 9. 0 8 7 0 4 2 5 2 0 1 6. 0 6 2 7 1 1 5 3 0. 4 6 4 5 4 1 1. 7 8 3 3 7 1 0 1 3. 8 2 9 5 4 1 1. 4 7 7 7 1 5 5 0. 1 5 4 0 4 4 2. 5 8 5 5 4 4 6. 7 4 5 3 8 5 4. 8 6 4 3 8 3. 3 5 2 0 4 5. 2 1 3 5 3 1 0. 3 1 2 7 1 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 T i m e ( m i n ) 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 6 / 2 3 / 2 0 1 0 4 : 1 8 : 1 8 P M 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A # 2 8 1 5 R T : 2 6. 0 1 A V : 1 N L : 3. 3 0 E 8 T : F T M S + p N S I F u l l m s [ 4 0 0. 0 0-1 8 0 0. 0 0 ] 6 3 4. 3 2 7 3 1 0 0 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 N L : 2. 0 7 E 9 T I C M S 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 1 2 6 7. 6 4 6 5 8 0 7. 9 2 2 2 7 2 8. 3 9 2 8 4 9 6. 2 5 3 3 z = 3 1 0 9 2. 0 8 5 6 6 6 9. 8 4 5 1 8 8 4. 4 5 3 0 6 1 5. 3 0 6 7 9 6 1. 4 9 9 3 9 9 1. 4 9 9 5 1 1 2 7. 1 0 2 3 1 2 2 9. 6 0 5 0 1 2 7 9. 1 3 8 8 z = 3 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 1 1 5 0 1 2 0 0 1 2 5 0 1 3 0 0 1 3 5 0 m / z 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A 6 / 2 3 / 2 0 1 0 4 : 1 8 :1 8 P M 1 0 0 6 2 3 _ R e d w o o d B i o S c i _ R a d u k a _ 2 9 A # 2 8 1 6 R T : 2 6. 0 2 A V : 1 N L : 7. 3 6 E 5 T : F T M S + p N S I d F u l l m s 2 8 0 7. 9 2 @ h c d 4 5. 0 0 [ 1 0 0. 0 0-1 6 3 0. 0 0 ] 9 1 3. 5 3 4 1 1 0 0 9 5 9 0 8 5 8 0 7 5 6 3 6. 3 3 4 9 7 0 6 5 6 0 5 5 5 0 7 6 7. 4 2 9 3 4 5 5 1 7. 3 3 4 4 6 5 4. 3 4 5 2 4 0 3 5 3 0 7 8 5. 4 3 8 8 5 4 6. 2 1 9 1 2 5 6 3 2. 3 6 0 0 2 0 7 4 5. 4 4 5 8 1 5 5 5 7. 2 9 3 6 7 3 9. 4 3 5 3 8 1 6. 4 8 1 9 z =? 1 0 2 6. 6 2 3 7 1 0 6 2 6. 3 5 2 3 8 9 5. 5 1 9 3 z =? z =? 6 7 0. 3 7 7 8 1 1 5 5. 6 6 5 2 8 8 0. 5 1 7 4 1 2 8 4. 6 8 2 3 5 z =? 1 0 0 7. 5 0 9 9 1 0 4 4. 0 4 6 5 z =? z =? 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 1 1 5 0 1 2 0 0 1 2 5 0 1 3 0 0 Quantitative Proteomics Ex. 1 MS/ Normal Serum @ 23 C @ 37 C @ 42 C HspB5 IP HspB5 *G120 Solution tryptic digest peptides Reversed Phase nanolc Relative Abundance Full Scan Orbitrap Relative Abundance MS/MS top 8 Ion Trap Relative Abundance 16

Quantitative Proteomics Ex. 1

Quantitative Proteomics Ex. 1 B5_23C B5_37C 3 10 0 154 18 17 44 B5_42C G120_23C G120_37C 4 2 1 194 13 12 17 G120_42C

Quantitative Proteomics Ex. 1 Proteins Identified (Avg. 6 LC MS runs) Proteins ID Spectra Identified (Avg. 6 LC MS runs) Spectra ID G120 42C G120 42C B5 42C B5 42C Sam mple G120 37C Sam mple G120 37C B5 37C B5 37C G120 23C G120 23C B5 23C B5 23C 0 50 100 150 200 Proteins ID 0 500 1000 1500 2000 Spectra ID

Quantitative Proteomics Ex. 1 IPI00745872 (100%), 69,366.9 Da IPI:IPI00745872.2 Tax_Id=9606 Gene_Symbol=ALB Isoform 1 of Serum albumin 45 unique peptides, 64 unique spectra, 81 total spectra, 438/609 amino acids (72% coverage) M K W V T F I S L L F L F S S A Y S R G V F R R D A H K S E V A H R F K D L G E E N F K A L V L I A F A Q Y L Q Q C P F E D H V K L V N E V T E F A K T C V A D E S A E N C D K S L H T L F G D K L C T V A T L R E T Y G E M A D C C A K Q E P E R N E C F L Q H K D D N P N L P R L V R P E V D V M C T A F H D N E E T F L K K Y L Y E I A R R H P Y F Y A P E L L F F A K R Y K A A F T E C C Q A A D K A A C L L P K L D E L R D E G K A S S A K Q R L K C A S L Q K F G E R A F K A W A V A R L S Q R F P K A E F A E V S K L V T D L T K V H T E C C H G D L L E C A D D R A D L A K Y I C E N Q D S I S S K L K E C C E K P L L E K S H C I A E V E N D E M P A D L P S L A A D F V E S K D V C K N Y A E A K D V F L G M F L Y E Y A R R H P D Y S V V L L L R L A K T Y E T T L E K C C A A A D P H E C Y A K V F D E F K P L V E E P Q N L I K Q N C E L F E Q L G E Y K F Q N A L L V R Y T K K V P Q V S T P T L V E V S R N L G K V G S K C C K H P E A K R M P C A E D Y L S V V L N Q L C V L H E K T P V S D R V T K C C T E S L V N R R P C F S A L E V D E T Y V P K E F N A E T F T F H A D I C T L S E K E R Q I K K Q T A L V E L V K H K P K A T K E Q L K A V M D D F A A F V E K C C K A D D K E T C F A E E G K K L V A A S Q A A L G L Non specific binding: Albumin

Quantitative Proteomics Ex. 1 IPI00019591 (100%), 140,943.5 Da IPI:IPI00019591.2 Tax_Id=9606 Gene_Symbol=CFB cdna FLJ55673, highly similar to Complement factor B 27 unique peptides, 32 unique spectra, 33 total spectra, 333/1266 amino acids (26% coverage) M G P L M V L F C L L F L Y P G L A D S A P S C P Q N V N I S G G T F T L S H G W A P G S L L T Y S C P Q G L Y P S P A S R L C K S S G Q W Q T P G A T R S L S K A V C K P G H C P N P G I S L G A V R T G F R F G H G D K V R Y R C S S N L V L T G S S E R E C Q G N G V W S G T E P I C R Q P Y S Y D F P E D V A P A L G T S F S H M L G A T N P T Q K T K D H E N G T G T N T Y A A L N S V Y L M M N N Q M R L L G M E T M A W Q E I R H A I I L L T D G K S N M G G S P K T A V D H I R E I L N I N Q K R N D Y L D I Y A I G V G K L D V D W R E L N E L G S K K D G E R H A F I L Q D T K A L H Q V F E H M L D V S K L T D T I C G V G N M S A N A S D Q E R T P W H V T I K P K S Q E T C R G A L I S D Q W V L T A A H C F R D G N D H S L W R V N V G D P K S Q W G K E F L I E K A V I S P G F D V F A K K N Q G I L E F Y G D D I A L L K L A Q K V K M S T H A R P I C L P C T M E A N L A L R R P Q G S T C R D H E N E L L N K Q S V P A H F V A L N G S K L N I N L K M G V E W T S C A E V V S Q E K T M F P N L T D V R E V V T D Q F L C S G T Q E D E S P C K G V T T T P W S L A R P Q G S C S L E G V E I K G G S F R L L Q E G Q A L E Y V C P S G F Y P Y P V Q T R T C R S T G S W S T L K T Q D Q K T V R K A E C R A I H C P R P H D F E N G E Y W P R S P Y Y N V S D E I S F H C Y D G Y T L R G S A N R T C Q V N G R W S G Q T A I C D N G A G Y C S N P G I P I G T R K V G S Q Y R L E D S V T Y H C S R G L T L R G S Q R R T C Q E G G S W S G T E P S C Q D S F M Y D T P Q E V A E A F L S S L T E T I E G V D A E D G H G P G E Q Q K R K I V L D P S G S M N I Y L V L D G S D S I G A S N F T G A K K C L V N L I E K V A S Y G V K P R Y G L V T Y A T Y P K I W V K V S E A D S S N A D W V T K Q L N E I N Y E D H K L K S G T N T K K A L Q A V Y S M M S W P D D V P P E G W N R T R H V I I L M T D G L H N M G G D P I T V I D E I R D L L Y I G K D R K N P R E D Y L D V Y V F G V G P L V N Q V N I N A L A S K K D N E Q H V F K V K D M E N L E D V F Y Q M I D E S Q S L S L C G M V W E H R K G T D Y H K Q P W Q A K I S V I R P S K G H E S C M G A V V S E Y F V L T A A H C F T V D D K E H S I K V S V G G E K R D L E I E V V L F H P N Y N I N G K K E A G I P E F Y D Y D V A L I K L K N K L K Y G Q T I R P I C L P C T E G T T R A L R L P P T T T C Q Q Q K E E L L P A Q D I K A L F V S E E E K K L T R K E V Y I K N G D K K G S C E R D A Q Y A P G Y D K V K D I S E V V T P R F L C T G G V S P Y A D P N T C R G D S G G P L I V H K R S R F I Q V G V I S W G V V D V C K N Q K R Q K Q V P A H A R D F H I N L F Q V L P W L K E K L Q D E D L G F L Specific binding: Complement factor B

Quantitative Proteomics Continued

Quantitative Proteomics Ex. 2 Isobaric labels

Quantitative i Proteomics Ex. 2 Dr. Wen-Jun Shen, Kraemer Lab http://med.stanford.edu/profiles/fredric_kraemer/ Understanding protein expression in relation to cellular response to fatty acid/hdl treatments Plex

Quantitative i Proteomics Ex. 2 TMT_4Plex_rep_1 TMT_4Plex_rep_2 2 peptides, >95% C.I. 65 323 55 TMT_4Plex_rep_1 TMT_4Plex_rep_2 Proteins 440 1564 374 Peptides

Quantitative Proteomics Ex. 2 Run in duplicate, 2 hour LC MSMS gradients HCD for reporter ion detection 443 proteins identified, >99% Protein, >95% (2)Peptide >70% of protein ID d quantification values reported TMT-126 100% 1,849.06 AMU, +2 H (Parent Error: 79 ppm) 80% tive Intensity Relat 60% 40% TMT-127 20% Minimum Value (5.0%) 0% 110.0 115.0 120.0 125.0 130.0 135.0 140.0 145.0 150.0 m/z

Quantitative i Proteomics Ex. 2

Quantitative i Proteomics Ex. 2

Quantitative i Proteomics Ex. 2

Acknowledgements SUMS Allis Chien Karolina Krasinska Pavel Aronov Maurizio Splendore LudmilaL d Alexandrova Elias Lab Snyder Lab Vincent and Stella Coates Foundation

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