Sample Preparation Automation and QQQ Workflows for Peptide Quantitation in DMPK

Transcription

1 Sample Preparation Automation and QQQ Workflows for Peptide Quantitation in DMPK Alex Zhu & Yanan Yang LCMS Applications Scientist Agilent Technologies

2 Why is Agilent Workflow a Good Choice for DMPK? AssayMAP Automated Sample Prep Ultra High Sensitivity wide dynamic range Quantitation with confirmation Excellent Reproducibility Simple MRM method development Great Robustness 2

3 Agilent Workflow for DMPK Sample Prep. Method Dev. Data Acq. Data Ana QQQ AssayMAP Bravo 6495 QQQ MH Quant Skyline App Library tmrm Skyline All Ions MSMS 3

4 AssayMAP Technology Components Target Customer Automated workflows designed for analytical chemists Microchromatography Cartridges Quantitative binding & elution Simple User Interface Uses customer language - not automation language Affinity (protein) Purification In-Solution Digestion Peptide Cleanup (desalting) Protein Cleanup (desalting) Phosphopeptide Enrichment IMAC Cartridge Customization Fractionation Sample Normalization Liquid handling utilities Protein purification PA-W (protein A) PG-W (protein G) SA-W (streptavidin) Reversed-phase cleanup: C18 (peptide) RP-S (peptide) RP-W (denatured mabs) Peptide Fractionation: SCX RP-S C18 Phosphopeptide enrichment: TiO 2 Positive Displacement Pipetting Fe(III)-NTA Syringes interface directly with cartridges and enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding 4

5 Workflow Library 5

6 Workflows 6

7 Purification Reproducibility and Recovery Using PG-W Cartridges 7

8 Consistent and Robust Affinity purification Denaturing Reduction Alkylation Digestion Across 96 sample replicates EIC overlays of mab peptides from a single row of samples (n = 12)

9 AssayMAP Power Users Daniel Spellman (Merck, West Point, USA) Field: large molecule pharmacokinetics AssayMAP Application: enrichment, digestion, cleanup Dan has made AssayMAP a critical part of a workflow used at Merck. AssayMAP provides significant reductions in both consumable cost and time to results. No more messy mag beads! Jacob Jaffe, The Broad Institute in Cambridge and MIT, USA Field: High-Throughput Proteomics AssayMap Application: mab & phosphopeptide enrichment, digestion, cleanup "Using the combination of extremely consistent, parallelized digestion with automated reverse-phase cleanup via AssayMAP, at a scale appropriate for ultrasensitive proteomics applications, has enabled us to contemplate collaborative studies of previously unheard-of scales and throughput. 9

10 Comparison Standard vs. High Throughput Magnetic Bead AssayMAP Kevin Bateman (Merck-WP, European Bioanalysis Forum, Barcelona Nov

11 6495 QQQ LC/MS - Premium Performance Improved sensitivity (IDL / MDL) Average 3x in S/N for peptides comparing to 6490 Improved precision and excellent accuracy at the lowest levels Proven 6 orders of linear dynamic range Proven robustness in complex matrix biological matrix (plasma) Improved mass range (2250), fast scan speed and MRM acquisition rate 11

12 Enhancing Sensitivity for Higher Flow LC Using More Efficient Ionization of Peptides Heat Sink with Active Cooling 2 x AJS normalized response 0.8 Heated Sheath Gas 0.6 Thermal Gradient Focusing Region 0.4 ESI relative response 0.2 MS Inlet Acquisition Time (min) Agilent JetStream interface: Thermal gradient focusing electrospray Usable with flow rates from 10 µl/min and up Yields 3-5x increase in sensitivity for peptides 12

13 Agilent Jet Stream Off On

14 Enhancing Sensitivity by Increasing Ion Sampling and Transmitting 6 Bore Capillary High Pressure Stage 1 Low Pressure Stage 2 Existing RF Ion Guide Samples more ions Removes neutrals Gives higher ion yield 14

15 6495 QQQ Technologies Continued Development Proven ifunnel Technology Agilent Jet Stream Hexabore Capillary Dual Ion Funnel Increased ion generation Enhanced ion sampling New Enhanced Q1 Ion Optics Improved ion transmission New Tapered Hexapole Collision Cell Effective ion collection and transmission New Detector with High Energy Conversion Dynode Improved ion detection with low noise 15

16 P e a k A r e a ( 1 0 ^ 3 ) Collision Energy Optimization Using Skyline SW Opt CE CE Replicate Skyline SW will export MRM list or acquisition method to run on Agilent QQQ with optimal CE for each transition

17 Agilent QQQ and Skyline Software Automation Tool for Automatic CE optimization Automation tool automatically creates QQQ methods and acquires data for CE optimization, reloads and analyzes the results, exports the final optimized QQQ method (MRM, dmrm, tmrm) and use it to runs any real samples queued up in the worklist 17

18 Agilent QQQ and Skyline Software Automation Tool for Automatic CE optimization 18

19 Challenges No prediction for representative peptides/preferred charge state. Numerous runs for collision energy optimization based on the number of proteins and peptides. Questionable prediction for modified peptides. Mayo CMSL Mass Spec Overview Thursday, September 03,

20 Simplified MRM Method Development: Agilent All Ions MS/MS on a QTOF + Skyline SW mab Tryptic Digest CE=0 P 3+ P 2+ CE=25 V y3+ y5+ y12++ y14++ y7+ p++ y8+ y9+ y10+ y11+ Mayo CMSL Mass Spec Overview Thursday, September 03,

21 Method Development for Multiple Proteins Method development for multiple proteins can be done from the same All Ions result, simultaneously or at a later time. Import a different protein sequence and load the same All Ions data. Same procedure followed. Mayo CMSL Mass Spec Overview Thursday, September 03,

22 Skyline Prediction Workflow vs. All Ions-Skyline Workflow mab Tryptic Digest Skyline Prediction Workflow All Ions Skyline Workflow Number of peptides for CE optimization 40 3 (Selected by All Ions run) Injections required for CE optimization 40 (One inj. for one peptide) 3 Total time required for MRM method development = 400 min (3+1) 10=40 min (1 is for All Ions run) Save up to 90% of time and 90% of samples needed for method development Mayo CMSL Mass Spec Overview Thursday, September 03,

23 Triggered MRM Triggered cycle (above threshold) Peptide 3 triggers secondary transitions, collects n cycles & goes back to primary cycle Threshold Primary cycle (below threshold) Peptide 3 triggers 23

24 Quantitation with Confidence Qualifier to Quantifier Ratio Reference vs. experiment 24

25 Ultra High Sensitivity: Low Attomole LLOD 2.1 mm ID column w AJS and Hexabore = Cap LC Detection blank LLOD 3.0 amol on-column LLOQ 5.0 amol on-column 7.5 amol on-column Instrumentation: 1290 UHPLC + Agilent JetStream QQQ Sample: synthetic peptide standard (LVNEVTEFAK) spiked into enolase tryptic digest Injection volume: 1 µl 25

26 Excellent Precision at LLOQ, 2.1 mm ID column w AJS + Hexabore = Cap LC Amount measured DDetection Replicates %RSD t (99%) IDL 5.0 amol (LLOQ) n = 10 injections amol MDL = t x (%RSD/100) x Amount = x (14.0/100) x 5.0 amol = 2.0 amol Inj # Peak Area %RSD

27 Six Orders of Dynamic Range LVNEVTEFAK 5 amol 5 pmol on-column 6 orders of linear dynamic range R2 = Zoom-in amol LVNEVTEFAK 5.0 amol 7.5 amol Calibration Standards (amount on-column; 1 µl injected) 15 amol 30 amol %Accuracy Reproducibility (%RSD, n=10) 300 amol 3 fmol 30 fmol 300 fmol 3 pmol RT (%RSD, n=100) pmol 27

28 R e s p o n s e s mab Peptide in Serum Matrix Peptide External Calibration Concentration (1 µl injection) 10 amol 25 amol 50 amol 100 amol 250 amol 500 amol 1 fmol 5 fmol Accuracy (%, n=6) Cal. Conc. %RSD (n=6) Retention Time %RSD (n=48) 0.17% Peptide X - 8 Levels, 8 Levels Used, 47 Points, 47 Points Used, 0 QCs x10 4 y = * x R^2 = Type:Linear, Origin:Ignore, Weight:1/x Range: 10 amol to 5 fmol R 2 >0.998 Type: Linear, Origin: Ignore, Weight:1/x Zoom in: amol Concentration (ng/ml) 28

29 MassHunter Quant: Compound-at-a-glance Blank 10 amol 25 amol 50 amol 100 amol 29

30 Increased Mass Range Useful for Peptides QQQ MRM spectrum for glycopeptide: EEQYN[ ]STYR (G1F) 30

31 Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma Hexabore with six 0.6 mm ID Capillaries is more Robust and has better peak shape Vs a single large Diameter orifice % % % 80.00% 60.00% 40.00% 20.00% 0.00% 01/06/14 01/11/14 01/16/14 01/21/14 01/26/14 01/31/14 02/05/14 Apolipoprotein E L-selectin Plasminogen Albumin_serum Kininogen Transthyretin Hemopexin Selected peptides from 42 peptides in the QC sample normalized to Day 1 response Peptide QC samples analyzed daily after every ~25 plasma digest injections No significant signal degradation observed after 853 injections of 40 µg plasma digest per injection and 3.5 weeks of continuous operation Response %RSD:

32 Summary Automated sample prep: Automated sample cleanup, enrichment and protein digestion using AssayMap platform Sensitive: low attomole LLOQ for peptides with standard flow Wide dynamic range: six orders Reproducible: small CV at low levels Robust: less down time and less need for maintenance Simple MRM method development: automated optimization of CE; All Ions MSMS-Skyline workflow Quant with confidence: triggered MRM Easy to use quantitative analysis software 32