Peptide Targeted Quantification By High Resolution Mass Spectrometry A Paradigm Shift? Zhiqi Hao Thermo Fisher Scientific San Jose, CA

Transcription

1 Peptide Targeted Quantification By High Resolution Mass Spectrometry A Paradigm Shift? Zhiqi Hao Thermo Fisher Scientific San Jose, CA

2 Proteomics is Turning Quantitative Hmmm.. Which ones are my targets? Discovery is qualitative & quantitative simultaneously. Profiling thousands of proteins (> 1s proteins). The ultimate goal is to look for targets Labeled or label-free PTM analysis Protein complexes Protein-protein & protein-ligand interactions Pathway analysis 2

3 Quantitative Proteomics Aims at Targets Post discovery Measures behavior of targets in different conditions (1s-1s proteins) But, I meant this protein Relative or absolute quantification Biomarker discovery Verification Validation Quantification Pathway analysis Cell signaling analysis 3

4 Outline 1. Overview of Targeted Peptide Quantification Selected reaction monitoring (SRM) and high resolution, accurate mass (HR/AM) 2. Introducing the Thermo Scientific Q Exactive Benchtop Mass Spectrometer Instrument feature improvements to ensure high performance for targeted quantification Resolution Scan speed Parallel filling / injection Spectrum multiplexing 3. Targeted Quantification using Q Exactive TM MS Methods and Applications MS1 level, precursor ion quantification using multiplexed SIM (msxsim) MS2 level, precursor fragment ion quantification using targeted HCD Example: profiling of kinase inhibition study 4. Summary and Conclusion Differentiating SRM-based quantification and HR/AM for targeted quantification 4

5 Overview of Targeted Peptide Quantification 1. Overview of Targeted Peptide Quantification Selected reaction monitoring (SRM) and high resolution, accurate mass (HR/AM) 2. Introducing the Thermo Scientific Q Exactive Benchtop Mass Spectrometer Instrument feature improvements to ensure high performance for targeted quantification Resolution Scan speed Parallel filling / injection Spectrum multiplexing 3. Targeted Quantification using Q Exactive TM MS Methods and Applications MS1 level, precursor ion quantification using multiplexed SIM (msxsim) MS2 level, precursor fragment ion quantification using targeted HCD Example: profiling of kinase inhibition study 4. Summary and Conclusion Differentiating SRM-based quantification and HR/AM for targeted quantification 5

6 Challenges in Targeted Protein Quantification Precisely quantify tens or hundreds of proteins in complex biological matrix: Pathway analysis Study formation of protein complexes Conduct protein-protein interaction studies Biomarker verification and quantification study Analytical parameters to achieve accurate and reproducible peptide quantification: Selectivity Sensitivity Dynamic range Throughput Reproducibility Repeatability 6

7 No Single Implementation Fits All Commonly applied performance profile Discovery Quan Selectivity Sensitivity Data Density Dynamic Range Reproducibility Repeatability Selectivity Sensitivity Data Density Dynamic Range Reproducibility Repeatability Targeted Quan 7

8 SRM - Traditional Triple Quadrupole Targeted Approach Q1 Q2 Q3 Precursor Ion Pre set CID Product Ion Pre set A proteotypic peptide is selected as being representative of a targeted protein a peptide which is unique for the protein of interest. Q1 is set to transmit only the parent m/z of the proteotypic peptide Q2 is a collision cell. Q3 is set to transmit only a selected product (fragment) ion. Since the Q1 m/z is set to the mass of the intact peptide and Q3 will be set to the m/z of a peptide fragment mass, and only these masses will be transmitted by the quadrupole mass filters, when signal is seen, it means that both the peptide precursor and fragment masses are present. Quantification is based on the intensity of the MS fragment ion. 8

9 SRM-Based Quantification Advantages and Limitations High sensitivity Advantages Low limit of detection (LOD) Low limit of quantification (LOQ) Limitations Comprise scale for sensitivity Longer dwell time to gain sensitivity, lowering throughput High selectivity Multiple transitions to increase selectivity Broad dynamic range 4 orders of quantification linearity routinely achieved High throughput Once method developed, experiment can be done repeatedly for large number of samples Slow method development, too costly for target verification Choice of peptide surrogate Choice of charge state Choice of particular transition Chromatography optimization to eliminate interferences Not suitable to certain samples Large peptides Glycopeptides 9

10 HR/AM - Emerging Orbitrap Approach HR/AM = High Resolution and Accurate Mass Quantification in an Orbitrap TM mass analyzer takes advantage of its resolving power and mass accuracy. Selectivity is ensured by high resolution and accurate mass. Quadrupole mass filter reduces noise, resulting in improved detection limits. Quantification is either MS1 level using SIM scan or MS2 level using HCD. Extracted ion chromatograms (XIC) of specific ions that are used for quantification are generated from SIM scan MS1 or full scan MS2. These types of experiments are targeted only in terms of data processing, and not in the sense of data acquisition, like in an SRM experiment where only the data pertaining to a specific ion is collected. 1

11 HR/AM Targeted Quantification What It Offers Sensitive, accurate, broad dynamic range. High selectivity relies on HR/AM. Minimal method development. No need to select transitions. Suitable to any peptide identified that is unique to the protein. Qual-Quan on the same platform. No switch of instruments from discovery to quantification. Comprehensive information from full MS1 or full MS2 for reinterrogation. Provides information for precursor ions and information for all the transitions. 11

12 Introducing the Q Exactive Hybrid Mass Spectrometer 1. Overview of Targeted Peptide Quantification Selected reaction monitoring (SRM) and high resolution, accurate mass (HR/AM) 2. Introducing the Thermo Scientific Q Exactive Benchtop Mass Spectrometer Instrument feature improvements to ensure high performance for targeted quantification Resolution Scan speed Parallel filling / injection Spectrum multiplexing 3. Targeted Quantification using Q Exactive MS Methods and Applications MS1 level, precursor ion quantification using multiplexed SIM (msxsim) MS2 level, precursor fragment ion quantification using targeted HCD Example: profiling of kinase inhibition study 4. Summary and Conclusion Differentiating SRM-based quantification and HR/AM for targeted quantification 12

13 Q Exactive MS - Performance and Capabilities C-TRAP Parallel Filling and Detection & pagc Spectrum multiplexing HCD Cell MS/MS Orbitrap Mass Analyzer Resolution 14K Mass accuracy better than 2 ppm Quadrupole Mass Filter Quadrupole m/z 5-4 Mass Filter.4 1 m/z amu 5-4 wide prec.4-1 selection amu wide Precursor Improved selection S/N Low detection limit Ion Source Improved sensitivity 13

14 Low Detection Limit for Targeted Quantification Quadrupole reduces noise improved detection limit using SIM scan XIC for m/z (2+) DIKCSNILLNNSGQIK Not detected using Full MS Detected and quantified using SIM MS 14

15 Accurate Quantification and Confirmation from Multiple Isotopes Wide SIM scan allows the use of multiple isotopes for quantification. Selectivity is then ensured by high resolution and accurate mass Relative Abundance # m/z amu IT=15 ms NL=1e8.4 amu, only monoisotopic peak Relative Abundance # amu IT=3.6 ms NL=8.5e7 3 amu, isotopic peak has correct intensity 1 m/z Relative Abundance Relative Abundance # m/z # m/z amu IT=2.7 ms NL=8.6e7 7 amu IT=2.4 ms NL=8.9e7 5 amu, 2 isotopes at the correct intensity 7 amu, 3 isotopes at the correct intensity 15

16 Improved Resolution / Scan Speed in Q Exactive MS Advanced signal processing provides improved resolution / scan speed Resolution Resolution at m/z 2 Resolution at m/z 4 Max. scan speed (Hz) Exactive Q-Exactive Scan Speed [Hz] 16

17 High Resolution Ensures Accurate Quantification Higher resolution provides better selectivity and more-accurate quantification R: 35K R: 7K R: 14K ppm m/z 17

18 Fast Scan Enables Large Scale Targeted Quantification Idle time of instrument is minimized True (practical) 12 Hz HR/AM MS or MS/MS Parallel ion injection/fragmentation and orbitrap detection Predictive AGC 256ms 64ms 18

19 Clear Definition of UHPLC Peaks Relative Abundance Relative Abundance 1 RT: 1.89 RT: Time (min) RT: K 7K Time (min) Peak width (FWHM) =.9 sec Peak width (base) = ~2.8 sec Scans/peak = 35 Scan speed = 12.5 Hz Peak width (FWHM) = 1 sec Peak width (base) = ~3 sec Scans/peak = 11 Scan speed = 3.6 Hz Relative Abundance RT: K Time (min) Peak width (FWHM) =.9 sec Peak width (base) = ~3 sec Scans/peak = 5 Scan speed = 1.6 Hz 19

20 True High Throughput Enabled by Spectrum Multiplexing Principle of 4-plex SIM A G C Orbitrap FTMS acquisition scan 1 A G C Orbitrap FTMS acquisition scan 2 Collecting ions for scan 2 Collecting ions for scan 3 This means, multiplex 4 SIM scan of 12Hz (at 17.5K resolution) can monitor 48 precursors in one second! Relative Abundance Simultaneous monitoring of up to 1 precursors per scan m/z ASMS 211 poster MP13 J.-P.Hauschild et al Multiple C-Trap Fills as a Tool for Massive Parallelization of Orbitrap Mass Spectrometry- a new Concept for Targeted Mass Analysis

21 Targeted Quantification using Q Exactive MS 1. Overview of Targeted Peptide Quantification Selected reaction monitoring (SRM) and high resolution, accurate mass (HR/AM) 2. Introducing the Thermo Scientific Q Exactive Benchtop Mass Spectrometer Instrument features to ensure high performance for targeted quantification Resolution Scan speed Parallel filling / injection Spectrum multiplexing 3. Targeted Quantification using Q Exactive MS Methods and Applications MS1 level, precursor ion quantification using multiplexed SIM (msxsim) MS2 level, precursor fragment ion quantification using targeted HCD Example: profiling of kinase inhibition study 4. Summary and Conclusion Differentiating SRM-based quantification and HR/AM for targeted quantification 21

22 Peptide Quan in Complex Mixture Using Multiplex SIM In multiplexing mode, step 1 and 2 are repeated multiple times to accumulate multiple peptides 1. Selection of targeted precursor ion in the quadrupole 2. Accumulation in C-trap In multiplexing mode, step 1 and 2 are repeated to accumulate multiple precursors before orbitrap measurement. 3. Transfer of ions to orbitrap for detection. In multiplexing mode, multiple precursors isolated separately are measured together. 22

23 HR/AM Quantification - Sensitive and Accurate RT: SM: 5G ppm XICs of target peptide VNQIGTLSESIK (+2, ) Blank serum digest.4 fmol/ul.123 fmol/ul.37 fmol/ul 1.11 fmol/ul 3.33 fmol/ul 1 fmol/ul NL: 8.58E3 NL: 2.46E4 NL: 6.82E4 NL: 2.7E5 NL: 7.55E5 NL: 2.29E6 NL: 5.96E6 MS1 level sensitivity - Quantification is based on precursor ion information Selectivity relies on high resolving power (14K) and accurate mass (<5 ppm). XICs of target peptides can be defined at extremely narrow mass widths (2.5 ppm for example), to generate remarkably clean chromatograms, even from complex matrices. Targeted only in terms of data processing, where extracted ion chromatograms of specific ions are generated from the SIM scan data Time (min) 23

24 Low Attomole Detection Limit, Large Linear Dynamic Range Heavy labeled standard peptides in yeast background A. Sensitivity and Linear dynamic range of peptide targets in 1ng yeast digest Peak area 4E+9 3E+9 2E+9 1E+9 E+ Log1(Peak Area) S/N: >4 1amole 1fmole 1fmole Log 1 (Sample Amount) Low amole detection CV < 1% at 5amole 4 orders of linearity R 2 : Sample amount (fmole) SSAAPPPPPR* GISNEGQNASIK* DIPVPKPK* B. Sensitivity and Linear dynamic range of peptide targets in 1ng yeast digest Peak area 6E+8 5E+8 4E+8 3E+8 2E+8 1E+8 E+ Log1(Peak Area) S/N: amole 1fmole 1fmole Log 1 (Sample Amount) 1 amole-1fmole detection 3-4 orders of linearity R 2 : Sample amount (fmole) NGFILDGFPR* GLILVGGYGTR* 1 ng yeast background 1 ng yeast background 24

25 Quantification and Confirmation Based on Multiple Isotopes Quantification of peptide VNQIGTLSESIK Automatic data processing with Thermo Scientific Pinpoint Software version 1.2 The ion of interest is defined as the co-elution of multiple isotopes. Multiple XIC s for each isotope are used to define the ion The overlay of multiple XIC s for each isotope are used to define an ion. Dilution curve generated on integrated peaks of multiple isotopes Quantification can then be performed on the sum area of all isotopes, or any single or group of isotopes, to maximize S/N. The elution peak are integrated and dilution curves are generated. 25

26 Quantification Statistics Generated by Pinpoint Software Statistics for the dilution line Pinpoint TM software provides statistics for the dilution line. Data is calculated for sample groups as well as individual injections. LODs and LOQs can be identified using these values for any given peptide. Peptide pairs can easily be assigned within Pinpoint software to generate H/L ion ratios and perform absolute quantification. 26

27 Profiling Staurosporine Kinase Inhibition in A549 Cells Multiplex SIM (msxsim) Targeted Quantification Workflow. µm XICs IC5 Curves.1 µm.1 µm.3 µm 1.µM 3. µm 1. µm The cell lysate was incubated with different levels of kinase inhibitor, Kinase- enriched, tryptic digested and analyzed using targeted msxsim. Data was processed and statistics calculated by Pinpoint 1.2. IC5 curve for each kinase was generated based on the relative quantification results. *Thermo Scientific Pierce ActivX Probes and Enzyme Enrichment Kits 27

28 Examples of Targeted Kinase Peptides Peak definition and selectivity Relative Abundance DTVTSELAAVkIVK ( Q12851 GCK) NISHLDLkPQNILLSSLEKPHLK (Q6PHR2 ULK3) Time (min) DLkPSNIFLVDTK (P19525 PKR) Resolution> 7K is required 28

29 Targeted msxsim and Western Blot Results Agree 1 % Kinase Inhibition Staurosporine, um CDK5 AurA RSK2(1) Erk1/2 Available result from Western blot for two of the kinases, Erk1/2 and Rsk2, agrees very well with the targeted msxsim result. Staurosporine has nearly no inhibition to Erk1/2. At.1 μm, the inhibition to Rsk2 was at least 7%. Rsk2 Erk1/ μm Staurosporine 29

30 SRM-Like, MS2 Level HR/AM Targeted Quan Using HCD 1. Selection of targeted precursor ion in the quadrupole. 2. Fragmentation in collision cell. 3. Accumulation of fragment ions. 4. Transfer of fragment ions to Orbitrap mass analyzer for detection. 3

31 SRM-Like, and You Gain More Quantification is based on fragment ion information. XIC of specific product ion is used to define target peptide - MS2 level sensitivity. Additional level of selectivity besides resolving power and mass accuracy. Full MS2 spectrum contains information for all fragment ions (all transitions), no need to select particular transitions. 31

32 Highly Selective, Fast and Sensitive 1 amole of heavy peptides in 25 ng human CSF digest At 7 K, more than 8 data points acquired across LC elution peaks RT: Relative Abundance GPGEDFR Relative Abundance VAHTVAYLGK Relative Abundance YFQGYAR Time (min) 32

33 Low Attomole LOD, 4 Orders of Linear Dynamic Range Heavy-labeled peptides of eicosanoid pathway enzymes in 25 ng CSF digest. 1E E Peak Area y = x R² =.9998 VAHTVAYLGK Peak area y = 382.1x R² =.9999 YFQGYAR Sample amount (amole) Sample amount (amole) Protein Peptide LOQ (amole) LOD (amole) PTGDS GPGEDFR 25 8 PTGS2 QFQYQNR 25 8 PTGS1 LVLTVR 1 3 HPGDS STLPFGK 25 8 PTGES VAHTVAYLGK 3 1 PTGIS FLNPDGSEK 5 17 TBXA1 SVADSVLFLR 1 33 ALOX15 YTLEINVR ALOX12 LWEIIAR LTCS4 YFQGYAR

34 Targeted Quantification Using Q Exactive MS HR/AM Targeted Quantification Instrument method Targeted SIM Targeted MS/MS Quantification LC Peak Area of Precursor LC Peak Area of MS/MS Fragments Selectivity HR/AM MS/MS Pattern, HR/AM Confirmation AM, RT, Isotope Pattern MS/MS Pattern, AM 34

35 Simplified HR/AM Discovery to Targeted Quantification Workflow HR/AM identification Complex protein digest Discovery Method HR/AM Targeted quan method Fewer compromises on selection of peptides No selection of specific transitions. Discovery to targeted quan without switching Instrument Target quan method Development Using Pinpoint Targeted quan data processing List of peptides to target with time schedule 35

36 Summary and Conclusion 1. Overview of Targeted Peptide Quantification Selected reaction monitoring (SRM) and high resolution, accurate mass (HR/AM) 2. Introducing the Thermo Scientific Q Exactive Benchtop Mass Spectrometer Instrument features to ensure high performance for targeted quantification Resolution Scan speed Parallel filling / injection Spectrum multiplexing 3. Targeted Quantification using Q Exactive MS Methods and Applications MS1 level, precursor ion quantification using multiplexed SIM (msxsim) MS2 level, precursor fragment ion quantification using targeted HCD Example: profiling of kinase inhibition study 4. Summary and Conclusion Differentiating SRM-based quantification and HR/AM for targeted quantification 36

37 Targeted Quantitative Proteomics is Facing More Choices SRM Targeted Quan? HR/AM Targeted Quan 37

38 Quote "Triple quadrupoles are unbeatable when you are looking at single transitions with SRM [assays]. Many applications can be done with conventional SRM and there is no point to competing with the triple quadrupole in this area. But when you have multiple transitions for each precursor the more transitions, the more benefit the Q Exactive instrument can provide. - Dr. Alexander Makarov, inventor of the Orbitrap mass analyzer 38

39 SRM-Based Targeted Quantification SRM-based targeted quantification assays are sensitive and specific, but require sophisticated and time-consuming assay development with highly reproducible nano LC separations. Triple-quad based quantification assays are unmatched when one or two transitions are sufficient for specific quantification of analytes. SRM assays provide a very large dynamic range of quantification and can be economically applied to very large number of samples. These distinct advantages of SRM-based assays provide a perfect compliment to a high-resolution targeted quantification approach. 39

40 The Paradigm Shift HR/AM Targeted Quantification The HR/AM quantification approach combines accurate mass and the high resolution capabilities of the Q Exactive mass spectrometer to achieve high-sensitivity, high-precision quantification of peptides. The HR/AM peptide quantification workflow is a paradigm shift as it provides easy transition from qualitative experiments, such as identification or screening to robust early quantification. This new approach also provides a robust method for the accurate quantification of previously hard to quantify proteins, e.g. specific glyco protein isoforms. The workflow provides a perfect compliment to the existing SRMbased workflow on a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer. 4

41 Acknowledgements Andreas Hühmer Yi Zhang Reiko Kiyonami Rosa Viner David Horn Scott Peterman Vlad Zabrouskov Amy Zumwalt Markus Kellmann Catharina Crone Yue Xuan Thomas Moehring Amol Prakash 41