6 x 5 Ways to Ensure Your LC-MS/MS is Healthy

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1 6 x 5 Ways to Ensure Your LC-MS/MS is Healthy (Also known as - Tracking Performance with the 6 x 5 LC-MS/MS Peptide Reference Mixture) Mike Rosenblatt, Ph.D. Group Leader Mass Spec Reagents 215.

2 We monitor our fitness So why not track the fitness of our instruments

3 Reference Materials for Mass Spectrometry Instrument Performance QC Sensitivity, Dynamic Range, Separation, etc. Evaluation of Sample Prep Methods Method Development: Proteolysis, Enrichment, Fractionation, etc. Instrument Method Development Optimization of Instrument Parameters for Discovery Experiments Optimization for Quantitative Experiments (SILAC, itraq, SRM)

4 A Peptide Mixture for LC-MS/MS Methods Development and QC Mass Spec (MS) instruments are utilized in research, drug discovery, disease monitoring, drug testing, environmental and food safety, etc. These instruments need to be monitored before precious samples get sacrificed. No reagent exists to determine all LC and MS parameters, in particular sensitivity and dynamic range in a single experimental run. What if we could introduce a single reagent to report all key instrument performance parameters in a single run? And what if we provide (free) software to make it even easier?

5 Designing an Idealized Peptide Mixture 215.

6 Peptide Selection Process Large collection of Peptides from Human Serum List reduced to 8282 Unique Peptides Removed P,M,W,C,N,Q, N-terminal E/D Kept peptides between 7 and 15 AA long Rejected insoluble and synthetically difficult Synthesized 96 crude peptides

7 Retention Time (mins) Retention Time Analysis of 96 Crude Peptides Bin 6 Bin 5 Bin 4 Bin 3 Bin 2 Bin Peptide Identifier

8 Peptide Selection Process Large collection of Peptides from Human Serum List was reduce to 8282 Unique Peptides Removed P,M,W,C,N,Q, N-terminal E/D Kept all peptides between 7 and 15 AA long Rejected insoluble and synthetically difficult Synthesized 96 crude peptides Decided on final, six peptides

9 MS Peak Intensity (Total Ion Current) LC Analysis and More. most hydrophilic peptide C 18 LC Gradient (increasing hydrophobicity) most hydrophobic peptide LC Chromatogram Peptide Retention Time (minutes) VTSGSTSTSR LASVSVSR YVYVADVAAK VVGGLVALR LLSLGAGEFK LGFTDLFSK LC Parameters: Retention Time Peak Width Peak Height

10 MS Peak Intensity (Total Ion Current) LC Analysis and More. most hydrophilic peptide C 18 LC Gradient (increasing hydrophobicity) most hydrophobic peptide LC Chromatogram Peptide Retention Time (minutes) LASVSVSR VTSGSTSTSR YVYVADVAAK VVGGLVALR LLSLGAGEFK LGFTDLFSK Producing a set of peptides for LC monitoring is straightforward But how will we measure MS sensitivity and dynamic range?

11 Isotopologues An isotopologue is a molecule that is identical in all properties with the only exception being it s molecular mass. This is achieved through the incorporation of stable isotopes. An example of a stable heavy isotopically labeled amino acid is: Heavy Leucine A peptide isotopologue pair would be two peptides with differing numbers of stable heavy labeled isotopes (i.e. VTSGSTSTSRVersus VTSGSTSTSR)

12 MS Peak Intensity (Total Ion Current) A Closer Look at the 6 5 Mixture Peptide 1 (most hydrophilic) LC Gradient (increasing hydrophobicity) LC Chromatogram Peptide 6 (most hydrophobic) Peptide Retention Time (minutes) Peptide 1 Mass VTSGSTSTSR VTSGSTSTSR VTSGSTSTSR 12.5 VTSGSTSTSR VTSGSTSTSR Each peptide isotopologue contains one or more heavy labeled amino acid We are able to mix these synthetic peptides at different concentrations Each chromatographic peak is a combination of All 5 Isotopologues Ok, but what about Dynamic Range?

13 How can we use the Isotopologues to give us Linear Dynamic Range? Corrected Intensity Peptide Isotopologue Mass Mass difference (Daltons) relative to next isotopologue Relative Concentration VTSGSTSTSR X VTSGSTSTSR X VTSGSTSTSR X VTSGSTSTSR X VTSGSTSTSR X Intensity (log scale).1 fmole 1 fmole 1 fmole 1 fmole 1 fmole LLSLGAGEFK LLSLGAGEFK.1 fmole LLSLGAGEFK LLSLGAGEFK 1 fmole LLSLGAGEFK 1 fmole 1 fmole 1 fmole Lowest Detected amount Linear Dynamic range (Peptide Mass, m/z) (amount on column) Isotopologue peptides are mixed at specific ratios to give a standard curve!

14 Log (Peak Height) Log (intensity) Total Ion Intensity (TIC) A mixture of 6 x 5 = 3 peptides for monitoring LC-MS/MS parameters Hydrophobicity VTSGSTSTSR LASVSVSR YVYVADVAAK VVGGLVALR LLSLGAGEFK LGFTDLFSK 59.3(m/z) 428.2(m/z) 566.8(m/z) 459.8(m/z) 537.3(m/z) 1X [Pep] 535.3(m/z) (A) 54.7 (m/z) 52.2 (m/z) (m/z) (m/z) (m/z) 42.7 (m/z) (m/z) (m/z) (m/z) (m/z) (m/z) (m/z) (m/z) (m/z) 45.3 (m/z) (m/z) (m/z) (m/z) (m/z) (m/z).1x [Pep].1X [Pep].1X [Pep].1X [Pep] 53.3 (m/z) (m/z) (m/z) (m/z) (Time(min) ) (B) (A) (B) (C) (D) (E) (F) (Mass/charge) (Mass/charge) (Mass/charge) (Mass/charge) (Mass/charge) (Mass/charge) (C) (A) (B) (C) (D) (E) (F) Log (mole) Log (mole) Log (mole) Log (fmole) Log (fmole) Log (fmole)

15 Characterization and QC of the Mixture 215.

16 Chromatography RT: VTSGSTSTSR LASVSVSR VVGGLVALR YVYVADVAAK LLSLGAGEFK NL: 1.11E9 Base Peak F: FTMS + p NSI Full ms [ ] MS msb16749_4 Conditions: Buffer A:.1 % Formic Acid Buffer B:.1 % Formic Acid in Acetonitrile Gradient: 4 % B in one hour Column:.75mm 15 cm C 18 column LGFTDLFSK Time (min)

17 Isotope Spacing Peptide MW LLSLGAGEFK LLSLGAGEFK LLSLGAGEFK LLSLGAGEFK LLSLGAGEFK Da 3.5 Da 3.5 Da 5 Da This spectrum, where peptides are mixed 1:1:1:1:1, exemplifies a typical mass spectrum. Because each mass is at least 5 daltons (Z=1), we see no crosstalk between isotopic envelopes

18 In Process Check: AAA correlates with MS intensity Because we perform AAA we are able to normalize all concentrations per isotopologue

19 Log (Peak Height) Log (Peak Height) Establishing Full Dynamic Range, LOD, and LOQ LASVSVSR (Heavy isotope; ) RT: pmole fmol 1 fmol 5 fmol 1 fmol 1 fmol amol 1 amol Time (min) NL: 3.2E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS MSB1583_1pmol82213 NL: 2.14E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_5fmol82213 NL: 4.95E8 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_1fmol82213 NL: 2.32E8 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_5fmol82213 NL: 3.33E7 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_1fmol82213 NL: 3.6E6 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_1fmol82213 NL: 2.15E6 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_5amol82213 NL: 1.84E6 m/z= F: FTMS + p NSI Full ms [3.-16.] MS msb1583_1amol fmol 1 fmol Log (fmole) y = 1.695x R² = Log (fmole) This should be done first to establish the optimal loading condition. In this example.5 1 pmole would be optimal. 1 pmol

20 Relative Abundance Analysis of XIC s (extracted Ion Chromatograms) C:\Users\...\MSB17521_2 5/8/214 6:29:4 AM RT: SM: 7B (m/z ; 5 fmole) (m/z ; 5 fmole) (m/z ; 5 fmole) (m/z ;.5 fmole) NL: 2.65E m/z= (m/z 521.8;.5 fmole) MS MSB17521_ Time (min) NL: 1.2E9 m/z= MS MSB17521_ 2 NL: 1.E8 m/z= MS MSB17521_ 2 NL: 1.6E7 m/z= MS MSB17521_ 2 NL: 1.24E6 m/z= MS MSB17521_

21 Log (intensity) Log (intensity) Log (intensity) Log (intensity) Log (intensiy) Log (intensity) Experimental Determination of Instrument Dynamic Range y = 1.754x R² = amol VTSGSTSTSR LASVSVSR YVYVADVAAK 5 amol 5 fmol Log (fmole) 5 fmol 5 fmol y =.893x R² = Log (fmole) y =.9471x R² = Log (fmole) VVGGLVALR LLSLGAGEFK LGFTDLFSK y = 1.7x R² = y =.841x R² = y =.823x R² = Log (fmole) Log (fmole) Log (fmole) Instrument: Thermo Q-Exactive. A dynamic range of 4 logs (using 5 points) is confirmed as well as a minimum sensitivity of 5amol

22 A Software Tool for Easy Analysis Parameter Category Reported Retention Time LC yes Peak Width LC yes Base Peak Height LC yes Graphical XIC analysis LC yes Lowest Detectable Quantity MS yes Linear Dynamic Range* MS yes LOD and LOQ** MS Not Directly Slope and r 2 of sensitivity curve graphical analysis MS yes Mass Accuracy MS yes Lowest Quantity detected MS yes Performance over time Both yes Multi-Analysis (Instrument) Comparisons Both yes

23 PReMiS Software Tool for Analysis of Peptide Data 215.

24 Software Front End

25 Processing A New Sample

26 Reporting LC and MS Parameters

27 Reporting LC and MS Parameters

28 Reporting on Chromatography of all Peptides

29 Instrument Linearity and Sensitivity

30 One Month of Retention Times

31 Retention Time (Minutes) Multi-Analysis Reporting Feature: Retention Time Comparison Q-Exactive 2 Q-Exactive 1 LTQ Orbitrap Velos

32 Tracking Instrument Sensitivity

33 Documenting Instrument Setup

34 Analysis in Complex Backgrounds 215.

35 Relative Abundance Total Ion Intensity (TIC) Internal Scoring Function Enables Confident Peak Detection (Especially Complex Samples) Score: Peptides coelute + Heaviest Peptides has sufficient intensity + Peptides have good linear Fit + Peptides elute in Correct Order + Heaviest Peptide has correct isotopic pattern Pep 1 Pep 2 Pep 3 Pep 4 Pep 5 P1>P2>P3 etc. MSB16751_4 # RT: AV: 11 NL: 1.12E8 T: FTMS + p NSI Full ms [ ] (Time(min) ) m/z Scoring allows selection of correct peptide, especially in complex backgrounds

36 Finding Peptides in Neat Samples is Easy RT: * Time (min) NL: 1.11E9 Base Peak F: FTMS + p NSI Full ms [ ] MS MSB16749_ NL: 1.49E9 m/z= F: FTMS + p NSI Full ms [ ] MS MSB16749_ NL: 8.53E8 Base Peak m/z= F: FTMS + p NSI Full ms [ ] MS MSB16749_4 * * * NL: 7.43E8 Base Peak m/z= F: FTMS + p NSI Full ms [ ] MS MSB16749_4 NL: 8.61E8 Base Peak m/z= F: FTMS + p NSI Full ms [ ] MS MSB16749_ NL: 8.31E8 Base Peak m/z= F: FTMS + p NSI Full ms [ ] MS MSB16749_4 * NL: 4.65E8 Base Peak m/z= F: FTMS + p NSI Full ms [ ] MS MSB16749_4 * * Software determined RT

37 Identification of Correct RT s is Possible in Complex Mixtures RT: * * NL: 1.2E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS 37.9 Promega_K562_1583_1pmol * NL: 1.44E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS Promega_K562_1583_1pmol * Time (min) NL: 1.6E1 TIC MS Promega_K562_1583_1pmol NL: 2.39E8 m/z= F: FTMS + p NSI Full ms [3.-16.] MS Promega_K562_1583_1pmol NL: 1.69E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS Promega_K562_1583_1pmol NL: 1.33E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS Promega_K562_1583_1pmol * * Human K562 Whole Cell Proteome Digest NL: 1.18E9 m/z= F: FTMS + p NSI Full ms [3.-16.] MS Promega_K562_1583_1pmol * Software determined RT

38 Additional Applications 215.

39 Intensity MS1 vs. Scheduled PRM Quantitation MS 1 PRM PRM PRM PRM PRM II III IV V 9 min Gradient VI I Time (min)

40 PRM log(peak area) MS1 log(peak area) MS1 vs. PRM Linear Dynamic Range Pep II Pep III Pep IV Pep V Pep VI log(fmol) log(fmol) log(fmol) log(fmol) log(fmol) log(fmol) log(fmol) log(fmol) log(fmol) log(fmol)

41 PRM Log(peak area) MS1 Log(peak area) Ion Intensity Improved Sensitivity With PRM 5 fmol 5 fmol 5 fmol 5 amol 5 amol Pep I Pep II Pep III Pep IV Pep V Pep VI Peptide Elution Time Intensity Intensity Time (min) Log[fmol] Time (min)

42 PRM Log(Peak Area Ratio) MS1 Log(Peak Area Ratio) MS1 vs. PRM Dynamic Range Stability Pep IV - VVGGLVALR 6 C.V. = 63.8% (1X/.1X) 4 C.V. = 8.56% (1X/.1X) 2 C.V. = 2.59% (1X/.1X) C.V. = 1.6% (1X/.1X) Injection Number C.V. = 5.25% C.V. = 3.86% C.V. = 3.19% C.V. = 3.79% (1X/.1X) (1X/.1X) (1X/.1X) (1X/.1X) Injection Number

43 Dynamic Range of QqQ Instrument Q1 Isolation Width Q1 Q2 Q3.2 m/z.7 m/z 1.2 m/z Determine optimal Q1 isolation width for increased linear dynamic range

44 1.2 m/z.7 m/z.2 m/z Noise Level as a Function of Q1 Isolation Width 5 attomoles on column Peak Area RSD = 2.38% Peak Area RSD = 6.3% Peak Area RSD = 1.17%

45 Log(Peak Area Ratio) Log(Peak Area) Scheduled SRM Dynamic Range Stability Pep II Pep III Pep IV Pep V Pep VI Log(fmol) Log(fmol) Log(fmol) Log(fmol) Log(fmol) 6 Pep IV - VVGGLVALR C.V. = 7.8% C.V. = 1.89% C.V. = 1.82% C.V. = 1.21% Injection Number (1X/.1X) (1X/.1X) (1X/.1X) (1X/.1X)

46 Summary o o o o o o o 3 peptides: 6 sequences x 5 isomers per sequence. 1-fold abundance gradient from heaviest isomer to lightest. One injection gives up to 4 orders of dynamic range and can track sensitivity down to the attomole level Complementary software provided to report LC and MS parameters as well as tracking historical performance or comparing instruments Software can read.raw (Thermo),.wiff (AB Sciex), and.mzml formats Mixture can be analyzed neat or spiked into complex samples All peptides are analyzed by AAA quantified so as to insure accurate reporting of sensitivity and dynamic range

47 Acknowledgements Promega: Marjeta Urh Ethan Strauss Gary Kobs Shika Gupta NC State: Professor Michael Bereman Joshua Beri

48 Thank You for Your Time and Attention! Questions? If you think of a question later, contact Technical Services: techserv@promega.com 215.