New Approaches to Quantitative Proteomics Analysis

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

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 1973 1981 1989 2001 2002 2005 2008 2010 2012 2013 2014 2015 2017 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

SCIEX Precision Medicine Toolkit SWATH Acquisition on TripleTOF Systems

Traditional Proteomics Quantitation Approaches Discovery + Quantitation

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?

Precursor m/z Information Dependent Acquisition (IDA) Time

Precursor m/z Information Dependent Acquisition (IDA) Time

Protein Sequence Database Searching Converting Spectra to Protein IDs 594.6297 Peptide Sequence: LLSDG LL.. G D S L L L L

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

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 106 849 peptides identified at a 1% global FDR.

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

Targeted Quantitation MRM and MRM HR

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

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

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)

Targeted Protein Quantitation

Precursor m/z Scheduled MRM/MRM HR Time

Precursor m/z Scheduled MRM/MRM HR Time

MS/MS ALL with SWATH Acquistion

What is a SWATH? http://dictionary.reference.com

MS/MS quantitation techniques 1 amu 1 amu Transition Triple quadrupole MRM/SRM TripleTOF 5600 + /6600 1 amu m/z MRM HR 0.01 amu 0.01 amu 0.01 amu m/z X amu TripleTOF 5600 + /6600 SWATH Acquisition

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

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

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 20000 SWATH 1.0 25 Da x 75 msec SWATH 2.0 100 VW x 25 msec Small molecules crashed plasma 10000 31.09 10000 31.11 30.70 0 30.0 31.0 32.0 Time, min 0 30.0 31.0 32.0 Time, min

Precursor m/z Time

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

Key Attribute of SWATH Acquisition Data Completeness in very large sample cohorts Samples 1.50.100.150.200 250 300 350 400 450 500 550.600 650 700 750.800 850.900.950. Proteins Experimental Protein Measurements >1,000,000

Reproducibility Challenge: The SWATH Cross-Lab Study

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

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

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

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

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+09 1 2 1.E+08 3 4 1.E+07 5 6 7 1.E+06 8 9 1.E+05 10 11 1.E+04 1.E+03 0.1 1 10 100 1000 10000 Concentration (fmol on column)

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.

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