New Approaches to Quantitative Proteomics Analysis

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1 New Approaches to Quantitative Proteomics Analysis Chris Hodgkins, Market Development Manager, SCIEX ANZ 2 nd November, 2017

2 Who is SCIEX? Founded by Dr. Barry French & others: University of Toronto Introduced API III, the first commercial dedicated LC- MS/MS system Released QTRAP LC-MS/MS which combines triple quad and linear ion trap for the first time Launched the 5500 series which was the most advanced systems of the time Launched the highest number of new products in the history of the company in a single year Launched first Medical Device and IVD solution Added Beckman CE to the portfolio Launched the first benchtop, hi resolution accurate mass solution for biologics characterization X500B QTOF Delivers the first commercial triple quadrupole Launched API MS/MS instrument 4000 LC-MS/MS systems becomes the de facto standard for bioanalysis Introduced API 5000 which was the most sensitive triple quadrupole system Became a Danaher Corporation operating company and acquired the Eksigent LC business Introduced the X-Series, the first accurate mass system for, routine testing X500R QTOF

3 SCIEX Precision Medicine Toolkit SWATH Acquisition on TripleTOF Systems

4 Traditional Proteomics Quantitation Approaches Discovery + Quantitation

5 Data Dependent Acquisition (DDA) Aim: to maximise collection of MS/MS spectra on unique precursor ions (peptides) Survey Scan (MS) Dependent Acquisition Criteria (n precursors) Dependent Scan 1 Dependent Scan 2 Dependent Scan 3 Dependent Scan 4 Dependent Scan n (MS/MS) (MS/MS) (MS/MS) (MS/MS) (MS/MS) Select n precursors Within m/z range? Above? intensity With charge state?

6 Precursor m/z Information Dependent Acquisition (IDA) Time

7 Precursor m/z Information Dependent Acquisition (IDA) Time

8 Protein Sequence Database Searching Converting Spectra to Protein IDs Peptide Sequence: LLSDG LL.. G D S L L L L

9 ProteinPilot - The Paragon Algorithm A Different Strategy for Better Peptide ID (1) Convert raw MS data to peak lists (2) Select peptide hypotheses Paragon Algorithm database search (3) Score peptide hypotheses (4) Infer protein IDs from peptide IDs Pro Group Algorithm

10 TripleTOF 6600 Protein ID Results The continuing quest for depth of coverage 15 high ph fractions of a digested Hela cell lysate Microflow LC and IDA on a TripleTOF 6600 system ProteinPilot Software 5.0 for a combined database search of all fractions 7164 proteins and over peptides identified at a 1% global FDR.

11 Protein Quantitation from Discovery Data Numerous approaches in two basic categories; Labeled Using MS data - (SILAC, Dimethyl) Using MS/MS data (itraq, TMT ) Label-free Using MS data - area under the curve (chromatographic peak integrals) Using MS/MS data eg. spectral counting

12 Targeted Quantitation MRM and MRM HR

13 Targeted Quantitation (MRM/SRM) Mass Analyzer Collision Cell Mass Analyzer Detector Select Peptide Fragment peptide Select Fragment Detect Fragment Highest sensitivity for detecting components in a complex mixture Largest linear dynamic range for quantitation Requires triple quadrupole MS capability robust, stable, cost-effective Well accepted as the MS technique for targeted quantitation

14 Scheduled MRM Algorithm Improving MRM Method Efficiency by Maximizing Analyte Utilization Each MRM monitored only across its expected elution time concurrent MRMs Maintain time spent acquiring MRM transition and number of MRMs effective duty cycle for every peptide (total time spent monitoring concurrent MRMs) Maintain analytical precision

15 MRM HR /Parallel Reaction Monitoring (PRM) Select Peptide Fragment peptide Detect All Fragments TOF MS/MS Spectrum Q1 Q2 TOF High resolution TOF Analyzer for detection of fragment ions Time, min High sensitivity MS/MS for fast data acquisition (up to 100 MS/MS per sec)

16 Targeted Protein Quantitation

17 Precursor m/z Scheduled MRM/MRM HR Time

18 Precursor m/z Scheduled MRM/MRM HR Time

19 MS/MS ALL with SWATH Acquistion

20 What is a SWATH?

21 MS/MS quantitation techniques 1 amu 1 amu Transition Triple quadrupole MRM/SRM TripleTOF / amu m/z MRM HR 0.01 amu 0.01 amu 0.01 amu m/z X amu TripleTOF /6600 SWATH Acquisition

22 MS/MS All with SWATH Acquisition Data-Independent Acquisition SWATH MS High Res./High Mass Acc. Full MS2 Spectra Q1 Filter [25Da] CID TOF Analyzer

23 SWATH Data Extraction y8 y7 y6 Time, min y8 y7 XIC y6

24 Intensity Intensity Variable Window SWATH Acquisition SWATH 2.0 Proteins digested cell line Adjusts Q1 selection window to maintain a roughly constant ion current in each window Narrower window in m/z dense regions Optimal cycle time maintained by adjusting accumulation time and # of windows Reduce number of precursors in dense windows for increased specificity SWATH Da x 75 msec SWATH VW x 25 msec Small molecules crashed plasma Time, min Time, min

25 Precursor m/z Time

26 Key Attribute of SWATH Acquisition Data Completeness for large cohort analysis Conventional Strategies DDA SWATH Strategy DIA Gold Standard Strategy MRM

27 Key Attribute of SWATH Acquisition Data Completeness in very large sample cohorts Samples Proteins Experimental Protein Measurements >1,000,000

28 Reproducibility Challenge: The SWATH Cross-Lab Study

29 Study Design and Implementation 30 SIS peptides spiked into 1 µg HEK293 whole cell lysate background. 3-fold dilution series with 5 levels starting from 5 different top concentrations. 6 orders of linear dynamic range -12 amol to 10 pmol 2 hour gradient, 30cm column (3μm) TripleTOF 5600 Systems with nanolc 64 variable width SWATH windows

30 Consistent detection of proteins across labs accumulated proteins detected 31,880 peptide ions/ ~4,400 proteins (1% protein FDR / 2% assay FDR)

31 Reproducibility (HEK293 proteome) Calculated from 80% complete matrix -- 4,064 proteins

32 Reproducibility (30 x SIL peptides) - Label free quantification - Simple median normalization - 11 different instruments/labs - Mean CV < 25%

33 Average Peak Area Linearity of Spiked Dynamic Range Peptides MRM Style Data Processing MultiQuant Software 30 peptides in HEK293 matrix across 6 orders magnitude concentration Peak area from SWATH data plotted per peptide for each concentration Processed per MRM curves Good linearity of quantitation across ~ 4.5 orders LDR Inset average area per concentration across all sites - normalized Fig 4c - Normalized Averaged Response Curve 1.E E E E E E+04 1.E Concentration (fmol on column)

34 Conclusions For proteomics to become an integral part of Precision Medicine, it needed a quantitative method that could provide accurate, reproducible data on thousands of proteins across thousands of samples. Traditional approaches using Data-Dependent Acquisition are not suitable due to missing values created by stochastic sampling MRM/PRM provides reproducibility, accuracy and precision but at a cost of depth of coverage SWATH (and other DIA approaches) combines the strengths of DDA and targeted approaches into a single method. SWATH forms the centre-piece of SCIEX s Precision Medicine toolkit.

35 Thank You