Increasing the Peak Capacity of LC/MS/MS Systems for the Analysis of Complex Biological Samples

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1 Increasing the Peak Capacity of LC/MS/MS Systems for the Analysis of Complex Biological Samples Mark A. McDowall Pharmaceutical Discovery & Life Sciences Waters Corporation, Manchester, UK 2011 Waters Corporation 1

2 Discovery Metabolomics Workflow Experimental Design Comprehensive Data Acquisition Differential Analysis ID Metabolites of Interest Report Generation 2011 Waters Corporation 2

3 Discovery Proteomics Workflow Experimental Design Comprehensive Data Acquisition Differential Analysis ID Proteins of Interest Hi3 Protein Quantification Report Generation 2011 Waters Corporation 3

4 Binocular Vision of Biological Processes PROTEOMICS METABOLOMICS 2011 Waters Corporation 4

5 Limitations in Bottom-Up LC/MS/MS Dynamic range of sample > dynamic range of MS Many peptides in a small analytical space Under-sampling of precursor ions in LC/MS/MS Up to 50% of LC/MS/MS Spectra are chimeric 1 Sensitivity 1. Houel S. et al, J. Proteome Res., 2010, 9 (8), LC / I MS / MS ^ 2011 Waters Corporation 5

6 Dispersive / Parallel Analytical Systems the timeing is right! Non-Dispersive (Filter) Separations SPE Ion Mobility FAIMS / DIMS Mass Spectrometry Quadrupole ITD (Precursor Selection) Magnetic Sector Dispersive Separations HPLC / UPLC Ion Mobility TW-IMS Mass Spectrometry TOF MS FT-ICR OBT HPLC (Seconds) > TW-IMS (mseconds) > TOF-MS (µseconds) 2011 Waters Corporation 6

7 Data-Dependent MS/MS Targets the Most Abundant Peptides Michalski et. al., J. of Prot. Res., 2011, 10, HeLa, 1D SDS, LTQ OrbiTrap Velos, > Waters Corporation 7

Data-Dependent MS/MS co-fragmented peptides frequently observed Most of the time, two peptides or more are fragmented at once Not a problem for abundant peptides In the median case, only 14%

8 Data-Dependent MS/MS co-fragmented peptides frequently observed Most of the time, two peptides or more are fragmented at once Not a problem for abundant peptides In the median case, only 14% of the peptide current in the isolation window is due to the precursor ion. Michalski et. al., J. of Prot. Res., 2011, 10, HeLa, 1D SDS, LTQ OrbiTrap Velos, > Waters Corporation 8

9 Peak Capacity Pc ~ Separation Space Peak Width 2011 Waters Corporation 9

$Orthogonal Separations Uneven Distribution Pc (system) = Pc 1 x Pc 2 x F F = fraction of bins$

10 Peak Capacity multiple dimensions (LC, MS, IMS, etc) IDEAL SITUATION Fully Orthogonal Separations Regular Distribution Pc (system) = Pc 1 x Pc 2 REAL SITUATION Partially Orthogonal Separations Uneven Distribution Pc (system) = Pc 1 x Pc 2 x F F = fraction of bins occupied Giddings JC. J Chromatogr A (1995);703:3 Gilar M. J Sep Sci (2005);28: Waters Corporation 10

11 Increasing Peak Capacity Travelling Wave Ion Mobility 100 Relative Intensity (%) (GRGDS) Å² IMS Resolution (Ω/ΔΩ) = 44 FWHM (SDGRG) Å² 20 0 SYNAPT G2 2.5mBar N Arrival Time (ms) ~ 22 R max = maximum resolution for travelling wave ion mobility (Ca 40FWHM) K = ion mobility coefficient Derived from: Shvartsberg AA. Anal Chem (2008);80: Personal communication: Jason Wildgoose 2011 Waters Corporation 11

12 Increasing Peak Capacity Travelling Wave Ion Mobility N (system) = N LC x N IM x N MS 2 10,000 s (HRMS) > 1000 s (2D-LC) > 100 s (1D-LC) > 10 s (IMS) 2011 Waters Corporation 12

13 IMS Increases Peak Capacity by creating a datacube N m/z N (system) = N LC x x N MS 2 10,000 s (HRMS) > 1000 s (2D-LC) > 100 s (1D-LC) 2011 Waters Corporation 13

14 Data Independent UPLC/MS/MS E increasing peak capacity & MS duty cycle ^ LC / I MS / MS ^ E VERYTHING 2011 Waters Corporation 14

15 Waters TriWave Technology Waters Corporation 15

16 Increasing Peak Capacity UPLC/IMS/MS E deconvoluting chimericy a Co-Eluting Peptides Ionized Precursors Precursors IMS Separated Precursors Transferred to TOF MS 2011 Waters Corporation 16

17 Increasing Peak Capacity UPLC/IMS/MS E deconvoluting chimericy b Co-Eluting Peptides Ionized Precursors Precursors IMS Separated Precursors & Products Time Aligned 2011 Waters Corporation 17

18 Shewanella oneidensis IMS Separation of Co-eluting (UPLC) Peptides 2x PRECURSORS WITHIN +/-1 DA (i.e. THEORETICAL MS/MS WINDOW) MOBILITY SEPARATION Peptide A Peptide B PEPTIDE B AT SAME UPLC RETENTION TIME PEPTIDE A Peptide A Peptide B Drift Time (bins) Drift Time 2011 Waters Corporation 18

19 Impact of Ion Mobility Enhanced Precursor/Product Alignment IMS OFF IMS ON 2011 Waters Corporation 19

20 Increasing Peak Capacity 2D RPxRP UPLC (High ph/low ph) 100 % g 20mM ammonium formate, ph % acetonitrile in 50 min % 1 B in 70 minutes 20 mm NH4OH ph 10 Bovine_Hemoglobin_Digest_Stored_091803_ : Scan Time ES TIC 4.51e9 neutral acidic basic acidic basic TIC 4.37e8 ph Δ pi % ph % Formic acid, ph % acetonitrile in 50 min Gilar M. et al, J. Sep. Sci. 2005, 28, Waters Corporation 20

21 Increasing Peak Capacity 2D RPxRP UPLC (High ph/low ph) Acidic peptides are more effectively retained in RP at acidic ph. Basic peptides are more effectively retained at basic ph. Gilar M. et. al, J. Sep. Sci. 2005, 28, Waters Corporation 21

22 Increasing Peak Capacity Caenorhabditis elegans 1D Chromatography 1 µg C. elegans < 2 hours 2D-3 Fraction 1.5 µg C. elegans < 4 hours 50% ACN 2D-5 Fraction 2.5 µg C. elegans < 6 hours 17.7% ACN 50% ACN 13.1% ACN 20.4% ACN 16.7% ACN 14% ACN 10.8% ACN 2011 Waters Corporation 22

23 Increasing Number of Identified Peptides with Increase in Peak Capacity Peptides MS E MSE HDMS E HDMSE D 2D-3 Fraction 2D-5 Fraction LC Method 2011 Waters Corporation 23

24 Increasing Number of Identified Proteins with Increase in Peak Capacity Proteins MS E MSE HDMS E HDMSE D 2D-3 Fraction 2D-5 Fraction Resolution 20,000 LC Method 5 ppm precursor window 12 ppm product window Proteins had to be in 2/3 replicates 2011 Waters Corporation 24

25 Increased Proteome Coverage of Caenorhabditis elegans 1D UPLC/HDMS E 914 Proteins 475 Proteins 1D UPLC/MS E 503 Proteins 2011 Waters Corporation 25

26 Increased Proteome Coverage of Caenorhabditis elegans 2D(3) UPLC/HDMS E 1415 Proteins 732 Proteins 2D(3) UPLC/MS E 798 Proteins 2011 Waters Corporation 26

27 Increased Proteome Coverage of Caenorhabditis elegans 2D(5) UPLC/HDMS E 1902 Proteins 934 Proteins 2D(5) UPLC/MS E 1017 Proteins 2011 Waters Corporation 27

28 Hi3 Absolute Quantification of Proteins C. elegans Identified in 1D, 2D-3Fraction, and 2D-5Fraction Identified in 2D-3Fraction and 2D-5Fraction Identified in 2D-5Fraction only peptide intensity Protein X i= 1 3 peptide intensity Internalstnd. i= 1 3 [ Internal stnd] 2011 Waters Corporation 28

Qualititative & Quantitative Analysis mouse brain Sample Tryptic protein digests of 4 biological replicates of double knock-out mice and 4 controls Estimated protein concentration 2-6 µg/µl 1D

29 Qualititative & Quantitative Analysis mouse brain Sample Tryptic protein digests of 4 biological replicates of double knock-out mice and 4 controls Estimated protein concentration 2-6 µg/µl 1D UPLC/IMS/MS E conditions 120 min gradient from 1 to 40% acetonitrile (0.1% formic acid) 1 µl injected; calculated amount 1.4 µg/injection UPLC/IMS/MS E (Data Independent Acquisition) o IMS = 40 FWHM o ToF MS = 25,000 FWHM 2011 Waters Corporation 29

30 1D UPLC/IMS/MS E 2597 Proteins 2011 Waters Corporation 30

Technical Reproducibility 134 270 289 1615 Loading 1.

31 Technical Reproducibility Loading 1.4 µg Gradient time 120 min FDR protein 0.71% proteins (> 2/3 technical replicates) 2011 Waters Corporation 31

32 PCA of UPLC/IMS/MS E Data 12 independent UPLC-IMS-MS E experiments. Variance of technical replicates is significantly smaller than biological replicates t[2] J48_001 J48_002 J48_003 J52_001 J52_002 J52_003 J51_003 J51_002 J51_ J49_003 J49_001 J49_ t[1] 2011 Waters Corporation 32

33 Hi3 Quantification of the 1961 Replicating Proteins peptide intensity protein x i= 1 3 peptide intensity internal standard i= 1 3 [ internal standard] Absolute quantification of proteins by LCMS E : a virtue of parallel MS acquisition. Silva et al. MCP 5 (2006) Waters Corporation 33

34 Increasing Sensitivity 2011 Waters Corporation 34

35 Increasing Sensitivity by incorporating a conjoined Ion Guide 2011 Waters Corporation 35

36 Increasing Sensitivity by incorporating a conjoined TW Ion Guide No Electric Field 1 mbar N 2 N 2 Flow 2011 Waters Corporation 36

37 Increasing Sensitivity by incorporating a conjoined TW Ion Guide With Electric Field (25 V between guides) 1 mbar N 2 T-Wave E T-Wave N 2 Flow 2011 Waters Corporation 37

38 Increasing Sensitivity by incorporating a conjoined TW Ion Guide 2011 Waters Corporation 38

39 Synapt G2-S vs. Synapt G2 the effect of the StepWave Ion Guide 2011 Waters Corporation 39

peptides 8000 6000 4000 2000 G2-S G2 0 10 ng 50 ng 400 ng Column

40 UPLC/HDMS E E.coli Peptide Identification Rates Peptides # peptides G2-S G ng 50 ng 400 ng Column Load E. coli (Cytosolic Fraction ) Tryptic Digest UPLC/HDMS E 75 µm column 90 min gradient 2011 Waters Corporation 40

400 300 200 100 0 10 ng 50 ng 400 ng Column Load G2-S G2 E.

41 UPLC/HDMS E E.coli Protein Identification Rates Proteins ng 50 ng 400 ng Column Load G2-S G2 E. coli (Cytosolic Fraction ) Tryptic Digest UPLC/HDMS E 75 µm column 90 min gradient 2011 Waters Corporation 41

42 UPLC/HDMS E E.coli Peptide Precursor Resolution m/z 401 m/z 552 m/z 800 Peptide Resolution >45K FWHM for Precursor Ions Across Wide m/z Range 2011 Waters Corporation 42

43 UPLC/HDMS E E.coli Mass Error Distribution RMS Error: 0.9ppm 2011 Waters Corporation 43

44 Summary Protein digests can be extremely complex. Number of unique precursors >100 ions/sec. LC/MS/MS is a (self limiting) serial process. LC/IMS/MS is a multiplexed alternative. Chimericy is a major limitation in LC/MS/MS. LC/IMS/MS significantly reduces chimericy. StepWave significantly increase sensitivity Waters Corporation 44

45 Acknowledgements Waters Corporation, Manchester UK Tim Riley Jim Langridge Chris Hughes Lee Gethings Jonathan Williams Barry Dyson Keith Richardson Waters Corporation, Milford MA Scott Geromanos Craig Dorschel Martha Stapels Dan Golick Steve Ciavarini Jose De Corral PLGS (UPLC/IMS/MS E )Interest Group Konstantinos Thalassinos University College London, UK Stefan Tenzer Mainz University, Germany Twan America Plant Research International, NL Arthur Moseley & Will Thompson Duke University, USA Andrew Ottens Virginia Commonwealth University, USA Greg Cavey Southwest Michigan Innovation Center, USA Yishai Levin Weizmann Institute of Science, Israel 2011 Waters Corporation 45

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