Fourier-Transform MS for Top Down Proteomics

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

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

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)

Top Down Proteomics: Measuring Intact Proteins

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

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

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

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 = 0.9992 1.0E+07 1.0E+06 Classic y = 96.788x R² = 0.9985 1.0E+04 y = 26.934x R² = 0.9989 1.0E+05 y = 51.111x R² = 0.9999 1.0E+03 1.0E+04 Advanced Active Ion Beam Guide 1.0E+02 1.000 10.000 100.000 1000.000 Injection (fg) 1.0E+03 10 100 1000 10000 100000 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

60 Overview of Orbitrap Fusion 90 Lumos MS for Top Down 30 (b) New Orbitrap Tribrid mass spectrometer: 100 80 60 Orbitrap Fusion Lumos MS Advanced Quadrupole Technology Relative Abundance Relative Abundance Relative Relative Abundance Abundance Relative Abundanc Relative Abundance 100 80 50 70 100 80 40 60 90 70 50 20 40 70 90 50 10 30 60 80 40 20 100 50 70 0 30 10 90 40 60 20 100 0 80 30 50 10 NTKO_H3.1_full_60k #1 RT: 0.00 AV: 1 NL: 2.24E6 T: FTMS + p NSI Full ms [500.00-1500.00] 854.09 z=18 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 70 90 20 40 100 0 60 80 10 30 90 50 70 20 100 0 80 40 60 10 90 70 30 50 100 80 0 60 20 40 70 90 50 10 30 60 80 40 20 0 50 70 30 10 40 60 20 0 30 50 10 848.33 z=? 850 855 860 865 870 m/z 20 400 10 30 200 10 0 853.43 z=18 852.59 z=18 852.43 z=18 851.76 z=18 851.09 z=18 850.31 z=18 854.93 z=18 855.09 z=18 856.43 z=18 857.20 z=18 857.98 z=18 859.65 z=18 ETD Histone HD H3.1 High Transmission at < 1 Th Isolation Windows 861.31 z=18 863.48 z=18 865.49 867.49 z=? z=? Advanced Vacuum Technology 849 850 851 852 853 8 m/z 849 850 851 852 853 8 m/z 849 850 851 852 853 8 m/z 849 850 851 852 853 m/z 849 850 851 852 853 m/z

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

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

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 10-64 Proteoform Score: 730 ProSightPD or (Xtract + ProSight Lite) (Fellers R, et al, Proteomics 15:1235, 2015) ETD-HD 100 90 80 MS2 ETD Spectrum 855.65 z=18 906.04 z=17 NL: 1.47E histone_h3 _810.7_hc FTMS + p 810.7000@ [300.0000 Relative Abundance 70 60 50 40 30 20 10 346.22 z=1 367.57 z=3 465.78 z=2 474.31 z=1 810.56 z=19 543.85 z=2 587.36 z=2 703.42 z=1 655.39 z=2 737.44 z=? 774.46 z=1 961.67 z=16 1025.64 z=15 1094.69 z=? 1184.66 z=? 1273.71 z=1 1397.32 z=? 0 100 905.98 z=17 NL: 5.93E Histone_H _810.7_NO

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

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

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

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

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

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

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. 2015 Aug 13. mcp.m115.053819. [Epub ahead of print] Dr. Luca Fornelli

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

TOP DOWN PROTEOMICS: RECENT PROGRESS

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); 30-60 kda range coming soon... New Metrics for Proteoform Scoring

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

Reducing Top Down to Practice, circa 2012

The Best Top Down Platform, 2015 and beyond

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

GELFrEE Separation of Human Cell Extract 50 37 25 60 Files HCD/ETD/CID DD 18 Files Med-High-HCD 20 15 34 Files 16 HCD DD 28 High-High HCD 10

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

ETD for PTM Localization on Integral Membrane Proteins For further discussion of integral membrane protein fragmentation see Owen Skinner, TP 008 29

The High Mass Problem Large proteins have poor chromatographic properties Top-down LC/MS tends to identify smaller proteins Tran et al., Nature 2011, 480:254 258 30

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

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

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

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

Comparison of RP-4H F2 25cm and RP- RT: 0.00-80.02 6.16 100 6.64 80 60 40 20 0 100 80 60 40 20 0 5.64 4H F2 50cm fraction 6 37.08 5.10 37.49 24.97 38.90 46.13 4.70 31.60 36.50 10.15 24.62 11.38 47.34 53.26 64.86 67.80 14.44 16.64 53.75 2.47 69.14 78.22 13.27 27.16 40.59 39.84 0 10 20 30 40 50 60 70 80 Time (min) 45.53 48.78 6.33 6.62 56.10 5.63 26.89 33.82 54.12 56.48 49.48 RP-4H 25cm 99 gene products 116 proteoforms RP-4H 50cm 101 gene products 136 proteoforms 16.68 25.27 66.48 19.13 57.87 24.73 69.11 70.47 NL: 7.26E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130623_RP- 4H_100umIDx25cm_HeLa _GF06 NL: 5.96E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130624_RP- 4H_100umIDx50cm_HeLa _GF06

RT: 0.00-130.13 100 90 80 70 Comparison of different gradient lengths with the RP-4H 100cm F2 at 0.4µl/min flow rate (fraction 8) 42.78 46.94 62 gene products 371 proteoforms NL: 1.89E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130714_RP- 4H_100umIDx100cm_110mi n_hela_gf08 60 50 40 39.62 49.55 50.63 30 31.86 38.25 20 10 0 100 90 80 70 60 50 40 30 20 10 0 2.26 5.56 17.33 18.13 37.56 28.79 24.32 53.17 55.78 16.27 18.80 58.48 15.13 69.40 70.78 75.18 76.65 59.78 67.41 51.50 52.90 58.62 63.33 57.85 49.88 73.33 79.65 46.93 40.19 80.11 43.57 91.17 26.93 39.96 84.17 35.79 89.55 13.79 15.27 17.91 120.69 9.56 22.43 92.55 99.50 104.79 112.79 8.07 119.70 125.22 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (min) 67.94 85 gene products 581 proteoforms NL: 2.34E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130714_RP- 4H_100umIDx100cm_160mi n_hela_gf08

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)

Comparison of RP-4H F2 25cm and RP- RT: 0.00-80.02 6.16 100 6.64 80 60 40 20 0 100 80 60 40 20 0 5.64 4H F2 50cm fraction 6 37.08 5.10 37.49 24.97 38.90 46.13 4.70 31.60 36.50 10.15 24.62 11.38 47.34 53.26 64.86 67.80 14.44 16.64 53.75 2.47 69.14 78.22 13.27 27.16 40.59 39.84 0 10 20 30 40 50 60 70 80 Time (min) 45.53 48.78 6.33 6.62 56.10 5.63 26.89 33.82 54.12 56.48 49.48 RP-4H 25cm 99 gene products 116 proteoforms RP-4H 50cm 101 gene products 136 proteoforms 16.68 25.27 66.48 19.13 57.87 24.73 69.11 70.47 NL: 7.26E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130623_RP- 4H_100umIDx25cm_HeLa _GF06 NL: 5.96E6 Base Peak F: FTMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130624_RP- 4H_100umIDx50cm_HeLa _GF06

Comparison of RP-4H F2 25cm and RP- RT: 0.00-80.09 100 80 60 40 20 4H F2 50cm fraction 9 22.72 22.49 33.94 32.08 31.30 29.67 34.87 37.42 47.59 RP-4H 25cm 15 gene products 70 proteoforms 38.58 39.58 47.98 21.79 53.07 20.23 53.30 10.84 6.25 54.15 60.02 64.53 69.16 NL: 1.09E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130623_RP- 4H_100umIDx25cm_HeLa _GF09 0 100 80 60 12.75 70.84 78.95 30.56 36.32 34.93 40.48 42.33 51.86 RP-4H 50cm 59 gene products 273 proteoforms NL: 2.24E7 Base Peak F: ITMS + p NSI sid=15.00 Full ms [500.00-2000.00] MS 20130624_RP- 4H_100umIDx50cm_HeLa _GF09 40 20 0 44.95 45.10 53.86 27.68 25.66 54.47 13.74 57.47 6.50 24.79 66.26 70.68 72.94 0 10 20 30 40 50 60 70 80 Time (min)

TOP DOWN PROTEOMICS: FUTURE DIRECTIONS

Recent Progress on Proteoform-Resolved Measurements

Top Down Label-free Quantitation Ntai et al. Anal. Chem. 2014.

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

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 0-1000+) - 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

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. PFR2000374) associated with the proteoform. 4. a C-Score - metric of confidence for the proteoform

Statistical Metrics for classifying a hit in Top Down Proteomics

The C-Score : Improving Proteoform Characterization

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 0-1000+) - 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

Top Down Proteomics: 2 Big Ideas

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

Organ/Tiss ue Cells Organelles Protein Complexes Protein Molecules

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

Acknowledgments 53

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