Mass Spectrometry and Proteomics Xudong Yao

Mass Spectrometry and Proteomics Xudong Yao Dept of Chemistry University of Connecticut Storrs, CT April 19, 2005

Proteomics and -omics Roles of mass spectrometry Comparative proteomics Gel or non-gel Label or non-label Chemical proteomics

Analytical Challenges in Post-genome Research Sample complexity Peak capacity Multi-dimensional separation Collective analysis Not traditional, one-by-one analysis Sensitive, specific and quantitative and the answers is Data treatment, analysis and achieving Hardware Software Researchers of multi-disciplinary training

Protein, Proteome and Systems Biology Replication DNA RNA Protein Transcription Translation Genome Transcriptome Proteome Proteomics Analytical Definition of Proteomics Identity, Quantity and Function of All the Proteins in a Mixture Mass Spectrometry

Objectives of Proteomics Identity Quantity Time Proteome Interaction Geometrical Functional Time-Dependence

Roles of Mass Spectrometry

Mass Spectrometry Ion Source (ESI, MALDI) Mass Analyzer Vacuum Ion Detector Data System Sample Introduction Intensity Mass Spectrum m/z

Mass Analyzer Separate ions by mass/charge Common types Quadrupole mass filter (Q), Time-of-Flight (TOF), Ion Trap (IT), Fourier Transform Ion Cyclotron Resonance (FTICR) Tandem mass spectrometry Spatial, such as Q-q-TOF, TOF-TOF, Q-q-Q Temporal, such as IT, FTICR Spatial and Temporal, such as IT-FTICR, Q-q-FTICR, IT-TOF

Mass Spectrometry for Proteins and Peptides UNKNOWN PEPTIDE Structural elucidation Quantitative analysis Sensitive and automatic mixture analysis Sequencing proteins Identification of posttranslational modifications High-order structures and dynamics of proteins and protein dynamics Mass Spectrometer M +S ++A +++N ++++D +++++C ++++++H +++++++E ++++++++M MSANDCHEM Protein/EST Database Protein Identification

Comparative Proteomics Gel or Non-gel Label or Non-label

Comparative Proteomics Relative Changes in proteins including concentration and composition Proteome 1 Proteome 2 Stable Isotope Coding Constant Ratio Separation Relative Quantitation Abnormal Ratios Sampling/Cell Culture Fractionation/Separation Mass Spectrometry Bioinformatics Identification of Proteins

GEL: 2-Dimensional Electrophoresis (2-DE) A Pandey; M Mann, Nat. Biotechnol. 2000 Difficulties with: extreme pi proteins, low abundance, proteins, hydrophobic proteins Inefficient in-gel digestion of proteins for MS analysis Label extensive

NON-GEL: Shotgun Analysis of Protein Complex Easier separation Easier automation Problematic proteins Small, large, hydrophobic, low abundance Easier sample preparation for MS analysis Computational capacity Quantitative capability Link et al. Nat. Biotechnol. 1999

Label: MS-Based Relative Quantitation Large differences in concentration Direct ESI/MALDI MS Small differences in protein concentration Stable isotope dilution: inherent choice Intensity m/z

Introduction of Stable Isotopes Criteria for isotope internal standards Ideally behaving the same before mass analysis Metabolic labeling during biosynthesis/bioprocess Post-biosynthesis/bioprocess labeling: chemical and enzymatic Functional groups on side chains: -SH, -OPO 3 H 3 Termini: N-terminal, C-terminal Active/Binding sites: Chemical Proteomics H 2 N (A 1 A 2 A 3 A n ) i COOH Coding Module

Isotope-Coded Affinity Tag (ICAT) Unique Chemistry for -SH Affinity Tag Isotope-Coded Linker Gygi et al. Nat Biotechnol. 1999

Tryptic Incorporation of Two 18 O Atoms into Peptide C-Terminus H C O C H N E O + H * 2 O H C C * OH + H 2 N * R R

Adenovirus as Model System Minimal real system DNA + Proteins Known genome/proteome Predictable proteolytic peptides Defined architecture Predictable protein expression Comparative quantitation of Ad2/Ad5 proteins Dynamic range of 600-fold Shenk T. In Fundamental Virology, 1996 Capsid protein modeling membrane/hydrophobic proteins Mutations modeling post-translational modifications

3 Ad2 Virion 1 Ad5 Virion H 2 18 O Lys-C Trypsin H 2 16 O Combined Isotope-Coded Peptides Theoretical MW of Ad Tryptic Peptides MALDI-FTICR MS Most of peptides Ad2:Ad5 = 3:1 Protein Identification and Quantitation

MALDI-FTICR Mass Spectrum of Combined Digests Relative Intensity 1597.78, Protein V TSTEVQTDPWFR 2 18 O 2 18 O 1603.83, Protein VII TTVDDAIDAVVEEAR 1628.96, Protein V VLRPGTTVVFTPGER 2 18 O

Controversies and Challenges in Proteolytic Labeling Reported controversies in tryptic 18 O labeling One 18 O incorporation for K-terminated peptides Low efficient incorporation of two 18 O for short peptides Two 18 O in each new peptides Capabilities of endoproteases for 18 O labeling Two 18 O incorporation by trypsin, Lys-C, and Glu-C only One 18 O incorporation by chymotrypsin Challenges for automated, large-scale application Amount and cost of H 2 18 O

Decoupling Proteolytic 18 O Labeling from Protein Digestion Proteins in solution prior to digestion Peptide labeling in small volume of H 2 18 O Separate optimization of digestion and labeling Automatic, high-throughput, large-scale applications

Dissection of Proteolytic Incorporation of Two 18 O Amide Bond Cleavage ½* O H C C R ½* OH + HO E H C R O C H N O + HO H C C H N. HO E R E H 2 *O H C R ½* O C ½* OH. HO E NH 2 H 2 *O H 2 *O H C * O C * OH + HO E H C H 2 O * O C * OH. HO E R R Carboxyl Oxygen Exchange Yao, Afonso, Fenselau. J. Proteom. Res. 2003, 2, 147.

Molecular Basis for Cleavage and Exchange Cleavage S 5 S 4 S 3 S 1 S 2 S 3 S 2 S 1 P 5 P 4 P 3 P P CONH 2 1 P 1 P 2 P 3 Native Peptide Substrate Exchange S 5 S 4 S 3 S 2 S 1 S 1 S 2 S 3 P 5 P 4 P 3 P 2 P 1 COOH Truncated Peptide Substrate Protease catalyzes exchange TWO 18 O INCORPORATION

16 O-to- 18 O Exchange Studied by MALDI-FTICR MS YGGFMR( 16 O 2 ) YGGFMR( 18 O 2 ) YGGFMR( 16 O 18 O) Relative Intensity m/z Time 0.5 min 730.34 732.34 734.34 12.0 min

Determination of Reaction Initial Rates [ R] = [ R o ] I total ( I o Io, I2, I4, Mo, M2, M 4 )

Kinetics Comparison in R- and K-Peptides YGGFMR YGGFMK K cat (min -1 ) 3500±500 2800±300 K M (µm) 1300±300 4400±700 k cat /K M (µm -1 min -1 ) 2.6±0.9 0.64±0.14

Simultaneous Mass Spectrometric Determination of Kinetics for Trypsin-Catalyzed 16 O-to- 18 O Exchange Exchange Rate R R Complete Exchange for Mixture

Chymotrypsin Catalyzes Two 18 O Incorporation Incremental mass of 4 Da from the theoretical First observation of two 18 O incorporation into chymotryptic peptides

Enzymatic 18 O Labeling Universal two 18 O labeling of proteolytic peptides by protease-catalyzed exchange Both K- and R-terminated peptides Chymotrypsin and pepsin for two 18 O labeling, in addition to trypsin, Lys-C, Glu-C, Both short and long peptides 4 Da mass increase at the C-terminus of proteolytic peptides to be differentiated in mass spectrometry

Mass Spectrometry of Peptide- 16 O 2 / 18 O 2 Pairs H 2 N (A 1 A 2 A 3 A n ) i Separation Module COOH Coding Module 18 O-labeling enabled mass spectrometric quantitation Effect of peak resolution on quantitation Analysis on different mass analyzer configurations More than relative quantitation from differential oxygen labeling

Relative Quantitation Using Paired Isotope Clusters Relative Intensity 75 50 I 0 M 100 0 100 I 4 I 2 Observed isotopic distribution of 1:1 mixture of 18 O and 16 O samples 75 50 Theoretical natural isotopic distribution M 2 25 25 M 4 0 0 Mass/Charge Ratio = I I 4 o + 1 M M 2 o I I 2 o + M M 2 o 2 M M 2 o M M 4 o I I 5 1

Correlation of ESI Quantitation with Peptide UV Quantitation MS Ratio of [ 18 O]/[ 16 O] 10 8 6 4 2 Peptide FVNQHLCGSHLVE slope: 0.94±0.03 r 2 : 0.997 0 0 2 4 6 8 10 UV Ratio of [ 18 O]/[ 16 O] Reynolds, Yao, Fenselau. J. Proteom. Res. 2003, 1, 27.

BSA Peptide Sequence Labeling Consistency Sequence Position Unlabeled (I 0 ) Elution Time (minutes) a Labeled (I 4 ) Elution Time Ratio 1 b I 4 /I 0 (minutes) a ACFAVE 589-594 46.48 46.48 0.95 0.87 KKFWGKYLYE 155-164 52.17 52.09 0.84 0.85 TYVPKAFDE 519-527 52.93 52.93 0.99 0.96 DKDVCKNYQE 335-344 58.43 58.43 0.99 0.97 DKGACLLPKIE 196-206 57.36 57.36 0.88 0.87 KQIKKQTALVE 544-554 67.70 67.70 0.90 0.88 LLYYANKYNGVFQE 177-190 75.32 75.32 0.99 1.07 YAVSVLLRLAKE 364-375 77.56 77.48 0.91 0.91 AKDAFLGSFLYE 345-356 88.78 88.78 0.86 0.81 DYLSLILNRLCVLHE 474-488 109.11 109.03 0.85 0.96 Average 0.92 0.92 Standard Deviation 0.06 0.08 c Reynolds, Yao, Fenselau. J. Proteom. Res. 2003, 1, 27.

Correlation between MS and Western-Blot Quantification of Thioredoxin in Doxorubicin-Treated HeLa Cells Mr SDS-PAGE Analysis of Whole Cell-Extracts: Dox Control Dox Control data01_fraction7inverserepeat #1862 RT: 62.36 AV: 1 SM: 15B NL: 3.42E6 T: + NSI d Z ms [ 665.00-675.00] 668.8 100 95 90 85 80 ESI-IT-MS Analysis of Anion-X Fraction 7: (Labeling: Dox Treated 16 O, Control 18 O) Dox 16 O 2 16 O 2 : 18 O 2 5:1 data01_fraction7inverserepeat #1863 RT: 62.40 AV: 1 NL: 7.07E6 T: + c NSI d Full ms2 669.63@35.00 [ 170.00-1350.00] 100 95 90 85 80 582.8 583.5 760.4 889.3 XC= 4.19 Relative Abundance 75 70 65 60 55 50 45 40 669.2 669.7 Control 18 O 2 Relative Abundance 75 70 65 60 55 50 45 40 576.3 689.3 890.4 35 35 1018.4 11 kda 30 25 20 15 670.2 670.7 671.2 30 25 20 15 461.2 574.4 1165.4 1017.4 690.4 319.2 500.6 630.3 762.2 275.7 744.1 405.2 448.0 559.1 876.3 1001.3 1019.4 Coomassie -Stain Western-Blot (Anti-Thioredoxin Ab) 10 5 0 666 667 668 669 670 671 672 673 674 675 m/z Zoom Scan: Relative Quantification [M+2H] +2 =668.8 [M+2H + 18 O 2 ] +2 =670.7 10 5 0 MS 2 Scan: Identification 200 300 400 500 600 700 800 900 1000 1100 1200 1300 [M+H] + =1337.42 K.TAFQEALDAAGDK.L m/z Thioredoxin Courtesy of Dan Clark of Stratagene

Effect of Peak Resolution on 18 O/ 16 O Ratio (I) 1597.78, Protein V TSTEVQTDPWFR 18O 2 18O 2 1603.83, Protein VII TTVDDAIDAVVEEAR MALDI-FTICR 1628.96, Protein V VLRPGTTVVFTPGER 18O 2 Protein II VII IX VIII Terminal % wt 60 14 3 0.3 0.1 Relative Intensity 2.9±0.3 3.7±0.4 3.0±0.5 3.3±0.5 2.6 ±0.3 Yao, Freas, Ramirez, Demirev, Fenselau. Anal. Chem. 2001, 73, 2836.

Effect of Peak Resolution on 18 O/ 16 O Ratio (II) APC2_Human: 0.91 MALDI-TOF APC3_Human: 0.94 A1AH_Human: 0.94 894.3 898.3 1922.7 1926.8 2112.9 2116.9 m/z m/z m/z APC2_Human: 1.2 2+ 447.70 449.68 ESI-IT APC3_Human: 0.87 2+ 961.83 963.84 A1AH_Human: 1.3 3+ 706.23 705.00 m/z m/z m/z Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

Inverse 18 O-labeling Wang, Ma, Quinn, Fu. Anal. Chem 2001, 73, 3742.

Protein Sequence Ions Generated by Tandem Mass Spectrometry Mass Analyzer 1 Fragmenting the Selection Ion Mass Analyzer 2 x 4 y 4 z 4 x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 H O H O H O H O H H H H H H 2 N C C N C C N C C N C C N C O C OH A 1 A 2 A 3 A 4 A 5 a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 a 4 b 4 c 4

Quantitation Based on MS/MS Spectrum (y-ions) y 4 y 3 y 2 y 1 H O H O H O H O H H H H H H 2 N C C N C C N C C N C C N C O C OH MTVEPGLEPEVR A 1 100 b 1 A 2 b 2 A 3 b 3 A 4 b 4 A 5 Relative Intensity 100 16 O 2 18 O 2 Relative Intensity 80 60 40 0 678 680 682 684 m/z 20 0 350 400 450 500 550 600 650 700 750 m/z Yao, Freas, Ramirez, Demirev, Fenselau. Anal. Chem. 2001, 73, 2836.

Effect of Isolation Window Width on Quantitation Using 16 O/ 18 O y-ion Pairs on IT MS Isolation of I o = 689.93 Isolation of I 4 = 691.93 MH 2 2+ 689.85 691.83 MH 2 2+ 689.87 691.82 MS MS 686 688 690 692 694 m/z 686 688 690 692 694 m/z y 10 + 984.60 988.49 MS/MS y 10 + 984.62 988.60 MS/MS 983 985 987 989 991 m/z 983 985 987 989 991 m/z Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

16 O/ 18 O Paired Peptides Facilitate and Validate Peptide Sequencing IT MS/MS Relative Intensity C-terminal mass 575.3 Da Q D A Y V S N-terminal mass 470.1 Da QqTOF MS/MS 400 500 600 700 800 900 1000 1100 1200 m/z

Three 18 O Incorporation Identification of N-Glycosylation Sites on Proteins Relative Intensity 100 75 50 25 Unlabeled 2 18 O-Labeled Unlabeled 3 18 O-Labeled Relative Intensity 100 75 50 25 MS/MS of m/z 1239 + Peptide SSCCYAD*FTE A D F T E 0 468 470 1239 1242 1245 1248 Mass/Charge 0 600 700 800 900 1000 1100 1200 1300 Mass/Charge Glu-C + N-Glycopeptidase F digest of riboflavin binding protein

Four 18 O Incorporation Mapping S-S Linkages in Protein 100 75 4 16 O- Labeled 4 18 O- Labeled Theoretical Distribution Relative Intensity 50 25 1371 1372 1373 1374 1375 Mass/Charge 0 1370 1372 1374 1376 1378 1380 Mass/Charge

Advantages of Modular Design H 2 N (A 1 A 2 A 3 A n ) i Separation Module COOH Coding Module Isotope Coding Universal Important to small proteins Specific Efficient Minimal Structural Modification Chromatographic co-elution Stable during separation Separation Portable to all separation platforms, including affinity separation

Protein Pools of Digitonin Extract of MCF-7 Cells Cancer Cells Digitonin Extraction Digitonin fraction cytosolic soluble cytoskeletal proteins Supernatant 120,000xg 5,000xg Pellet Properties functional proteins soluble proteins Digitonin Extract C4 Fractionation Modified according to Ramsby, Makowski Method Mol. Biol., 112, 1999.

Rel. Int. Rel. Int. LC-MS of Peptides from MCF-7 Digitonin Fraction Base peak chromatography of total peptide mixture Time (min) 4 0 6 0 8 0 1 0 0 1 2 0 40 TOF MS at 78 min Frequency 30 20 10 0 0.5 1.0 1.5 2.0 2.5 3.0 600 650 700 900 950 1000 Yao, Fenselau. ASMS Annual Conference, 2001 m/z MelR/WT Protein Ratio (MelR/WT) = 1.1 I 0 /I 4 Ratio (MelR/WT) = 1.1 ± 0.3 83% (184/223) peptides in 1.1 ± 0.3

Protein Expression Changes in MCF-7 Cells Upon Acquisition of Melphalan Resistance [Most Proteins in a Ratio (MelR/WT) of 1.1] (K)LLPQLTYLDGYDR(E) PHAPI2b /April protein pi = 4.0 MelR/WT = 2.0 (R)GIVTNWDDMEK(I) 602308605F1 NIH_MGC_88 Homo sapiens cdna clone MelR/WT = 2.6 Relative Intensity 784 786 788 m/z 654 656 658 660 Yao, Fenselau. ASMS Annual Conference, 2001

Quantitation of Cytosolic Proteins after C4 Fraction PROTEIN MASS pi WT/MeIR 40S Ribosomal Protein S28 7.9 kda 10.70 0.50 Ubiquitin 8.6 kda 6.56 0.87 Acyl-COA-Binding Protein 10.0 kda 6.11 2.02 Thioredoxin 12.0 kda 4.82 1.13 Prothymosin Alpha 12.2 kda 3.69 2.48 60S Ribosomal Protein L30 13.0 kda 9.65 0.65 Signal Recognition Particle 14.7 kda 10.05 0.56 Profilin 1 15.2 kda 8.47 1.02 Superoxide Dismutase 16.2 kda 5.70 1.09 Stathmin 17.3 kda 5.77 0.11 Cofilin 1 18.7 kda 8.22 1.21 Phosphatidylethanolamine-binding 21.2 kda 7.43 1.47 60S Ribosomal Protein L14 23.4 kda 10.94 0.40 60S Ribosomal Protein L13 24.3 kda 11.65 1.09 Nuclear Ubiquitous Casein 26.3 kda 5.00 0.52 Hepatoma-Derived Growth Factor 26.9 KDa 4.70 1.82 Brown, Fenselau. J. Proteom. Res. 2003, 3, 455.

Analysis of Human Plasma Sample: Example 1 Human Plasma Abundance Protein Depletion Denaturing SEC Large Proteins Fraction X Small Proteins RP Tryptic Digestion 1x Peptide Pool 1 1x Peptide Pool 2 Enzymatic 16 O-Labeling 2x MG 16 O-Peptides Enzymatic 18 O-Labeling 1x MG 18 O-Peptides Combine Differentially-Labeled Peptides SCX MALDI-MS/MS µ-rp-hplc ESI-MS/MS Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

LC-MALDI & LC-ESI MS Analysis of Differentially 18 O/ 16 O-Labeled Peptides Present in Human Plasma LC-MALDI-MS (TOF) 1708.5 1712.5 LC-nanoESI-MS (QTOF) 748.43 570.29 571.63 748.3 752.47 752.3 LC-ESI-MS (IT) 748.42 570.32 571.65 752.42 Rel. Int. m/z Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

Analysis of Human Plasma Sample: Example 2 Qian et. al., Smith MCP 2005, March 7

Assembling Separation Module and Coding Module H 2 N (A 1 A 2 A 3 A n ) i Separation Module COOH Coding Module Protein H 2 18/16 O Glu-C 16/18 O 2 16/18 O 2 16/18 O 2 16/18 O 2 16/18 O 2 O I O NH O O NH O NH S NH FVNQHLCGSHLVE 16 O-16 O 2 O-18 O 2 O CH2 16/18 O 16/18 OH Relative Intensity Biotin Biotin S Mass/Charge

16 O/ 18 O-Labeling and Affinity Enrichment to Quantitate Proteins Liu, Qian, Strittmatter, Camp, Anderson, Thrall, Smith. Anal. Chem. 2004, 76, 5345.

16 O/ 18 O-Labeling and Affinity Enrichment to Quantitate Protein Phosphorylation Bonenfant, Schmelzle, Jacinto, Crespo, Mini, Hall, Jenoe. PNAS 2003, 100, 880.

Introduction to Chemical Proteomics

Profiling Enzyme Activity Cravatt et al.