Fourier-Transform MS for Top Down Proteomics

Transcription

1 Fourier-Transform MS for Top Down Proteomics Proteomics Northwestern University Thermo User Meeting, December 9 th, 2015 Hosted by Thermo Fisher Scientific Odense, Denmark

2 On the Use of Top Down Proteomics in Targeted and Discovery Mode C & E News Webinar, October 26 th, 2015 Sponsored by Thermo Fisher Scientific

3 Executive Summary Orbitrap Fusion Lumos MS Features and Demonstrations Complete Characterization of Human Histone H3 Retuximab and EDC Characterization onthe-fly TD and MD (IdeS) on a Chromatographic Time Scale Top Down Proteomics Updates and Future Direction Proteoform Identification and Characterization Proteoform Quantitation (0-30 kda)

4 Top Down Proteomics: Measuring Intact Proteins

5 The proteoform: Cannonical Sequence (UniProtKB) Endogenous proteolysis mrna splicing Mutations SNPs A distinct molecular form of a protein product arising from a single gene -- The Consortium for Top Down Proteomics Site specific features -govern activity, localization, interactions, half-life Consortium for Top Down Proteomics, Nat. Methods, 2013

6 Overview of Orbitrap Fusion Lumos MS for Top Down (b) New Orbitrap Tribrid mass spectrometer: Orbitrap Fusion Lumos MS ETD HD Advanced Quadrupole Technology Advanced Active Ion Beam Guide Advanced Vacuum Technology Electrodynamic Ion Funnel High Capacity Transfer Tube

7 Overview of Orbitrap Fusion Lumos MS for Top Down (b) New Orbitrap Tribrid mass spectrometer: Orbitrap Fusion Lumos MS Advanced Active Ion Beam Guide High Capacity Transfer Tube Electrodynamic Ion Funnel High Capacity Transfer Tube

8 Area Area Overview of Orbitrap Fusion Lumos MS for Top Down (b) New Orbitrap Tribrid mass spectrometer: Orbitrap Fusion Lumos MS Letter Box Inlet 2-5x Improvement Enhanced Sensitivity In Sensitivity 1.0E+06 Enhanced 1.0E+08 Enhanced 1.0E+05 Classic y = 123.8x R² = E E+06 Classic y = x R² = E+04 y = x R² = E+05 y = x R² = E E+04 Advanced Active Ion Beam Guide 1.0E Injection (fg) 1.0E Injection (fg) Electrodynamic Ion Funnel High flow Alprazolam dilution series 5x improvement in sensitivity 27 Proprietary & Confidential High Capacity Transfer Tube Low flow AEFVEVTK dilution series 2x improvement in sensitivity

9 60 Overview of Orbitrap Fusion 90 Lumos MS for Top Down 30 (b) New Orbitrap Tribrid mass spectrometer: Orbitrap Fusion Lumos MS Advanced Quadrupole Technology Relative Abundance Relative Abundance Relative Relative Abundance Abundance Relative Abundanc Relative Abundance NTKO_H3.1_full_60k #1 RT: 0.00 AV: 1 NL: 2.24E6 T: FTMS + p NSI Full ms [ ] z= z=? m/z z= z= z= z= z= z= z= z= z= z= z= z=18 ETD Histone HD H3.1 High Transmission at < 1 Th Isolation Windows z= z= z=? z=? Advanced Vacuum Technology m/z m/z m/z m/z m/z

10 Overview of Orbitrap Fusion Lumos MS for Top Down (b) New Orbitrap Tribrid mass spectrometer: Orbitrap Fusion Lumos MS ETD HD ETD HD of Intact Monoclonal Antibody Unmatched S:N for ETD Fragment Ions High Capacity Transfer Tube

11 Overview of Orbitrap Fusion Lumos MS for Top Down (b) New Orbitrap Tribrid mass spectrometer: Orbitrap Fusion Lumos MS Isotopically Resolved Light Chain Isotopic Resolution on Larger Molecules with Higher Sensitivity Advanced Quadrupole Technology Advanced Active Ion Beam Guide Advanced Vacuum Technology Electrodynamic Ion Funnel High Capacity Transfer Tube

12 New Tribrid Lumos MS and ProSight Allow Complete Characterization of Human Histone H3 MS1 Spectrum of Histone H3.1 Fragment Ion Map 7 methylations: K9me2K18me1K27me2K36me2 Quadrupole Isolation (0.6 Th) Isolated Precursors p-score: 4 x Proteoform Score: 730 ProSightPD or (Xtract + ProSight Lite) (Fellers R, et al, Proteomics 15:1235, 2015) ETD-HD MS2 ETD Spectrum z= z=17 NL: 1.47E histone_h3 _810.7_hc FTMS + p @ [ Relative Abundance z= z= z= z= z= z= z= z= z= z=? z= z= z= z=? z=? z= z=? z=17 NL: 5.93E Histone_H _810.7_NO

13 Isolating Proteoforms of Histone H3.1 (e.g., with 8 Methylations) Quad Isolate: 0.6 Th

14 Isolating Proteoforms of Histone H3.1 (e.g., with 8 Methylations) Quad Isolate: 0.6 Th

15 Isolating Proteoforms of Histone H3.1 (e.g., with 8 Methylations) Quad Isolate: 0.6 Th

16 Characterizing Histone H3.1 Proteoforms with 7 Methylations: ETD-HD + EThCD

17 Characterizing Histone H3.1 Proteoforms with 7 Methylations: ETD-HD + EThCD

18 Characterizing Histone H3.1 Proteoforms with 7 Methylations: ETD-HD + EThCD C28 Ion Me5 Me4 Me2 Me3 Me6 Me7

19 Dr. Yupeng Zheng Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression. Mol. Cell. Proteomics Aug 13. mcp.m [Epub ahead of print] Dr. Luca Fornelli

20 Improved Top-Down and Middle-Down Characterization of Complex Biopharmaceuticals on a Modified Tribrid Mass Spectrometer Dr. Luca Fornelli Dr. Philip Compton

21 TOP DOWN PROTEOMICS: RECENT PROGRESS

22 Executive Summary on TDPs: Proteoform-Resolved Measurements are Proving Powerful and Valuable Protein Identification and Proteoform Characterization (Can now do thousands in a week ) Proteoform Quantitation now possible (0-30 kda); kda range coming soon... New Metrics for Proteoform Scoring

23 Top Down Proteomics of >1000 Proteins, >3000 Proteoforms Tran, Kelleher, et al., Nature 2011; 480:7376, p. 254

24 Reducing Top Down to Practice, circa 2012

25 The Best Top Down Platform, 2015 and beyond

26 Top Down Proteomics of Mouse Xenografts (PDX) WHIM 2 WHIM 2 WHIM 16 WHIM 16 Ntai, Kelleher, et al., Mol. Cell. Proteom. In Review

27 GELFrEE Separation of Human Cell Extract Files HCD/ETD/CID DD 18 Files Med-High-HCD Files 16 HCD DD 28 High-High HCD 10

28 Analysis of a Single Enriched Mitochondrial GELFrEE Fraction 153 unique protein IDs 218 proteoforms 67 membrane proteins Catherman, A.D. et al., Mol. Cell. Proteomics, 2013, in press. 28

29 ETD for PTM Localization on Integral Membrane Proteins For further discussion of integral membrane protein fragmentation see Owen Skinner, TP

30 The High Mass Problem Large proteins have poor chromatographic properties Top-down LC/MS tends to identify smaller proteins Tran et al., Nature 2011, 480:

31 1. Platform for efficient characterization of biotherapeutics (a) LC-MS/MS Efficient IgG desalting/fragment separation Minimal sample manipulation Denaturing conditions: reverse phase chromatography (H 2 O/ACN) Monolithic columns (Thermo Scientific), 200 µm i.d., 250 mm, 10 µl/min flow rate RP-5H (C4) New! RP-4H (ethylvinylbenzene/divinylbenzene) Kelly Flook Shane Bechler Yury Agroskin 31

32 Intact Protein Separations Yury Agroskin Kelly Flook Neil Kelleher Shane Bechler Kihun Kim Ioanna Ntai

33 Extended mass range for top-down proteomics (these are IT data)

34 Improved intact protein separations ~50-65 kda proteins 47 gene products 94 proteoforms 64 gene products 149 proteoforms

35 Comparison of RP-4H F2 25cm and RP- RT: H F2 50cm fraction Time (min) RP-4H 25cm 99 gene products 116 proteoforms RP-4H 50cm 101 gene products 136 proteoforms NL: 7.26E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx25cm_HeLa _GF06 NL: 5.96E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx50cm_HeLa _GF06

36 RT: Comparison of different gradient lengths with the RP-4H 100cm F2 at 0.4µl/min flow rate (fraction 8) gene products 371 proteoforms NL: 1.89E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx100cm_110mi n_hela_gf Time (min) gene products 581 proteoforms NL: 2.34E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx100cm_160mi n_hela_gf08

37 Column and Operating Parameters Column Flow rate Column temperature Eluents Injection PLRP-S 1000 Å 5μ Analytical (100 μm ID 25 cm) Loading 1.5 μl/min Gradient 1 μl/min 40 C RP-4H Analytical (100 μm ID 50 cm) Loading 1.5 μl/min Gradient 1 μl/min A 95% H 2 O/5% ACN + 0.1% Formic Acid B 5% H 2 O/95% ACN + 0.1% Formic Acid 6 μl (standards), 5 μl (GELFrEE fractions)

38 Comparison of RP-4H F2 25cm and RP- RT: H F2 50cm fraction Time (min) RP-4H 25cm 99 gene products 116 proteoforms RP-4H 50cm 101 gene products 136 proteoforms NL: 7.26E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx25cm_HeLa _GF06 NL: 5.96E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx50cm_HeLa _GF06

39 Comparison of RP-4H F2 25cm and RP- RT: H F2 50cm fraction RP-4H 25cm 15 gene products 70 proteoforms NL: 1.09E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx25cm_HeLa _GF RP-4H 50cm 59 gene products 273 proteoforms NL: 2.24E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [ ] MS _RP- 4H_100umIDx50cm_HeLa _GF Time (min)

40 TOP DOWN PROTEOMICS: FUTURE DIRECTIONS

41 Recent Progress on Proteoform-Resolved Measurements

42 Top Down Label-free Quantitation Ntai et al. Anal. Chem

43 3 Measurement Functions of Top Down Proteomics Protein Identification Characterization of Proteoforms Quantitation of Proteoforms Can Now Keep Score for Each

44 Key Metrics for Top Down Proteomics - Identification: p-value, E-value, or q-value - Proteoform Characterization Score (PCS), or C-Score for short PCS: 730 (scale is ) - Quantitation Confidence (instantaneous q-value, ANOVA)... and... How many Proteoforms are fragmented?... and how well? Swiss-Prot Accession No: P00000 p-value: 4 x 10-64

45 Report 4 things in Top Down Searches of the Future: 1. the UniProt Accession Number associated with the I.D.. 2. E-value (or Q-value) - some metric of confidence for the I.D. 3. The Proteoform repository identifier : (e.g. PFR ) associated with the proteoform. 4. a C-Score - metric of confidence for the proteoform

46 Statistical Metrics for classifying a hit in Top Down Proteomics

47 The C-Score : Improving Proteoform Characterization

48 Metrics of All 3 should be very high on the Orbitrap Fusion Lumos MS - Identification: p-value, E-value, or q-value - Proteoform Characterization Score (PCS), or C-Score for short PCS: 730 (scale is ) - Quantitation Confidence (instantaneous q-value, ANOVA)... and... How many Proteoforms are fragmented?... and how well? Swiss-Prot Accession No: P00000 p-value: 4 x 10-64

49 Top Down Proteomics: 2 Big Ideas

50 The Proteoform Hypothesis (It s About Tighter Correlations to Phenotype) 2 proteoforms 1 proteoform 3 peptides 2 peptides 1 peptide Proteoforms in Translational Research Need for N and Strategic Alignment with Population and Genomic-Based Medicine

51 Organ/Tiss ue Cells Organelles Protein Complexes Protein Molecules

52 Conclusions Orbitrap Fusion Lumos MS is suitable for both top-down MS of intact 150 kda abs and middle-down MS of ~25 kda ab fragments on the LC time scale ETD HD improved substantially the S/N ratio of MS/MS spectra (~3-fold improvement) Lumos MS technology allowed the characterization of EDC through: Mass determination of both non-isotopically and isotopically resolved EDC fragments Localization of conjugation sites by ETD HD 52

53 Acknowledgments 53

54 Sponsors and Supporters of the Proteomics Center at Northwestern