Increasing Throughput and Efficiency with Exactive LC/MS and Triple Quadrupole LC/MS/MS

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1 Increasing Throughput and Efficiency with Exactive LC/MS and Triple Quadrupole LC/MS/MS Nicholas Duczak Thermo Scientific Annual Mass Spectrometry Users Meeting Somerset, NJ October 12th, 2011

2 Lead Finding Background Lead finding Screening Traditionally performed using plate reading devices 100K s of compounds screening Rapid sample analysis required Low cost per well/sample preferred Label Free Detection Reduction in False positives Removal of unrelated interferences Simplified sample prep and assay development MS Screening Methods Triple Quadrupole Instruments used for current analyses Lower throughput than plate reading devices Higher quality data 2

3 Why LC-MS for HTS Screening? Separation Sensitivity Reduced Sample Prep Improved Data Quality 3

4 HTS-Aria Acquisition Module Rapid sample introduction- injections every seconds with chromatography Capability to collect/control multiple injections into a single data file Operates as a typical multiplexing system Transcend Liquid Chromatography System Combine the power of online extraction and the speed of multidimensional chromatography with the only truly independent, parallel, multi-channel UHPLC system in the industry TurboFlow Technology minimizes sample prep., reduces ion suppression, simplifies method development Enables up to four independent channels, permitting multiple methods. 4

5 Process Overview Autosampler L Pump E Pump TFLC HPLC L Pump L Pump E Pump E Pump TFLC TFLC HPLC HPLC Selector Valve Detector L Pump E Pump TFLC HPLC Four parallel systems, synchronized by Aria software, all lead into the MS and work independently from each other 5

6 Data Collection Key Benefits Eliminate MS file downloading time between samples Data grouping controlled by operator Fully integrated data review in with QuickCalc Software RT: Relative Abundance NL: 1.56E7 TIC F: + c ESI SRM ms [ ] MS Imipramine NL: 4.16E6 TIC F: + c ESI SRM ms [ ] MS Imipramine Time (min) Scan Range window is proportional to overall data file size 6

7 Imipramine001 4/9/2010 4:33:42 PM Imipramine001 None RT: Relative Abundance NL: 1.63E7 TIC F: + c ESI SRM ms [ ] MS Imipramine001 NL: 4.39E6 TIC F: + c ESI SRM ms [ ] MS Imipramine Time (min) RT: Relative Abundance Time (min) 7

8 How to handle all the data? -Data review software to perform discovery calculations -Results are stored to a database -Customizable Reports -Supports Low Throughput and High Throughput acquisition modes Supports HTS data acquisition on Thermo MS systems (Exactive and all TSQ series) -Aria HTS: utilizes batch file information to populate the sequence list for a sample by sample review. Aria batch information is automatically associated to the data file or sequence 8

9 General Chromatography Viewer Compound I.S. The user has the capability to view the data in several different formats and to customize the views to show only what is important to the scientist 9

10 10 High Through Acquisition Display

11 Calibration Curve Viewer The user has the capability to view the calibration curve, assign the best fit, modify sample types, and exclude data points. 11

12 Excel Format The customizable output displays only the desired columns and graphics Compound Information Data Calibration Curve 12

13 QuickCalc Integration with Aria Files created by Aria are easily pulled into QuickCalc Files contain injection times and detailed sample information All information is automatically attached to each injection and listed in table form 13

14 HTS-Data Review Tools Initial view may be set to full chromatogram Click on a sample to zoom in on peak 14

15 15 Heat Map Tools for Data Review

16 Aria-HTS with TSQ 16 Courtesy: S. Stout, Merck, Rahway, NJ

17 Calibration Curve Results 17 Courtesy: S. Stout, Merck, Rahway, NJ

18 System Reproducibility 18 Courtesy: S. Stout, Merck, Rahway, NJ

19 System Reproducibility 19 Courtesy: S. Stout, Merck, Rahway, NJ

20 Exactive HTS Mass Spec Screening Platform LX4 plus Exactive Orbitrap 20

21 Background CREBtide (AnaSpec, Fremont, CA) Cat # H - Lys - Arg - Arg - Glu - Ile - Leu - Ser - Arg - Arg - Pro - pser - Tyr - Arg - Lys - Cys OH Cat # H - Lys - Arg - Arg - Glu - Ile - Leu - Ser - Arg - Arg - Pro - Ser - ptyr - Arg - Lys - OH Crebtide (KRREILSRRPSYR) - is a substrate peptide derived from protein Creb, which is widely utilized as a synthetic substrate for PKA and PKC kinase family Poor fragmentation for standard QQQ Analysis 21

22 Method Development LC Goals Speed ( > 1 min gradient cycle time) Chromatographic separation Reproducible peak shape and RT Total Sample time 15-20sec Robust under neat and buffered sample matrixes LC Conditions Step Gradient ACN:Water 1.5 ml/min flow rate 10uL partial loop injection ISTD correction C18, 4.6 x 20 mm, 3.5 µm column 0.05% TFA MS Conditions Mass range of Resolution 25,000 Maximum inject time 250ms Vaporizer temp.350 C Capillary temp230 C Sheath gas 60 AUX gas 30 22

23 Where LC is needed for HTS: CREB1 200ng/mL neat Peptide standard in neat solution for 1 min LC Method RT: Relative Abundance CREB_200ng-mL_9104 #59 RT: 0.47 AV: 1 NL: 2.52E5 T: FTMS {1,1} + p ESI Full ms [ ] Relative Abundance TIC Analyte and ISTD 0.53 Time (min) Analyte and ISTD NL: 1.20E7 TIC MS CREB_200ng-mL_9104 NL: 7.05E5 m/z= F: FTMS {1,1} + p ESI Full ms [ ] MS CREB_200ng-mL_ m/z CREB_200ng-mL_9104 #64 RT: 0.49 AV: 1 NL: 1.26E6 T: FTMS {1,1} + p ESI Full ms [ ] Relative Abundance Interferences m/z 23

24 Method Development cont. Pinpoint 1.1 Calculate Exact Peptide Mass Calculate Expected and Unexpected Modifications Optional Calculation of Peptide SRM Transitions 24

25 25 Method Development cont.

26 Method Development cont. CREBtide Infusion Formic Acid Modifier CREBtide Infusion TFA Modifier (used for chromatography CREB_250ng-mL_1001 #3136 RT: AV: 1 NL: 2.04E4 T: FTMS {1,1} + p ESI Full ms [ ] z= z= z=? z=? z=? z=? 5 Relative Abundance z=? z=? z=? z=? z= m/z z=? CREB1_300ng_1001 #3206 RT: AV: 1 NL: 8.59E4 T: FTMS {1,1} + p ESI Full ms [ ] z= z= z= z= z=1 z= z= z=1 z=3 z=? z=1 z= z= z= z=? z=? z=? m/z Relative Abundance

27 Experimental Results Sample analysis Neat (ACN:Water) Solution ~3000 injections Buffer Solution (HEPES and MgCl 2 ) ~3000 injections Results LOD & LOQ from 10nM to 250nM Linear Response %CV Z Value Unexpected Results 27

28 Experimental Results. Data collection into single data file format (17 seconds) RT: Relative Abundance SM: 7B Time (min) 28

29 Experimental Results cont. Isotopic charge envelopes for CREBtide (left) and CREBtide (right) in the 3+ charge state in neat solution. CREB1_50ng_3001 #6179 RT: AV: 1 NL: 1.11E4 T: FTMS {1,1} + p ESI Full ms [ ] Relative Abundance m/z CREB1_50ng_3001 #6179 RT: AV: 1 NL: 6.92E3 T: FTMS {1,1} + p ESI Full ms [ ] Relative Abundance m/z 29

30 Experimental Results cont. Calibration Curve, %CV, and Z Value for neat sample injections y = 0.020x R² = nM 25nM 50nM 100nM 150nM 250nM # of Injections %CV Z' Value

31 Experimental Results cont. Sample analysis in buffer solution No Analyte signal present at high concentration RT: SM: 3B Relative Abundance Time (min) 31

32 Experimental Results cont. Pinpoint used to calculate additional modifications Cat # H - Lys - Arg - Arg - Glu - Ile - Leu - Ser - Arg - Arg - Pro - pser - Tyr - Arg - Lys - Cys OH CREB1_500ng_1001 #6717 RT: AV: 1 NL: 7.76E3 T: FTMS {1,1} + p ESI Full ms [ ] Relative Abundance m/z 32

33 Experimental Results cont. Data Re-examined for new modifications 100nM 150nM 250nM # of Injections %CV

34 Conclusion Sample Cycle time < 20 sec Reasonable Sensitivity and LOQ Linear Response Across Conc. Range Z Value Indicates Excellent Assay Reproducibility 25K Resolution Provides Separation for Samples Tested Existing Software Provides Simplified Data Review and Sample Calculation 34

35 Future Considerations LC Condition Improvement Improved LOQ Additional Sample Matrixes Sample Analysis using Q Exactive 35

36 Acknowledgements Steve Stout, Merck, Rahway, NJ Thermo Team, Franklin, MA Thermo Team, San Jose, CA Dr. John Peltier, Novartis, Cambridge, MA 36