Applying High Sensitivity LC/MS and Automated Liquid Handling Technology to Peptide Quant

Transcription

1 Applying High Sensitivity LC/MS and Automated Liquid Handling Technology to Peptide Quant Christine Miller Senior Application Scientist March 2, 20

2 Overview Improvements in instrumentation Workflow enhancements Impact of flow rate and column id i.d. on sensitivity Robustness of analysis with complex samples

3 Improving QQQ Sensitivity: ifunnel Technology in the Agilent 6490 More efficient ionization Increased ion sampling Greater ion transfer Thermal confinement of ESI ion plume Efficient desolvation to create gas phase ions 6 capillary inlets Removes the gas but Samples 2x more ion rich captures the ions gas from the source Helps to remove source generated noise Heat Sink with Forced Air Cooling Nebulizer Heated Sheath Gas Thermal Gradient Focusing Region MS Inlet High Pressure Funnel Low Pressure Funnel 3

4 Using the 6490 QQQ with ifunnel Technology No ion funnel related optimization required and no additional method parameters from the dual ion funnel! Optimal collision energy for peptides appears to be about the same as for other 6400 QQQ and the 6500 Q-TOFs No need to optimize fragmentor with no skimmer, this value is set in tune and is not compound specific New enhanced resolution mode (±0.4 Da) for improved New enhanced resolution mode (±0.4 Da) for improved specificity in complex matrices

5 Triggered MRM An easy way to increase throughput with simultaneous confirmation and quantitation of large set of compounds in a single MS/MS run. Workflow When a primary MRM exceeds a certain threshold, additional or triggered MRMs can be activated (0 total primary + secondary MRMs). Find by MRM feature in Qual B.04 produces composite MRM spectrum similar to an MS/MS spectrum Time required for additional MRMs much less than for a true product ion scan so quant performance not compromised Sensitivity in MRM mode much better than full scan mode User specifies primary, secondary transitions & trigger threshold in Acquisition UI Firmware watches for specified threshold for each primary transition FW triggers secondary MRM acquisition for peptides which pass threshold criteria 5

6 MRM-triggered MRMs for Confirmation + Quantitation Spectrum Mill match for Q-TOF spectrum QQQ Acquisition setup for Triggered MRM

7 Triggered MRM Triggered cycle (above threshold) Peptide 3 triggers secondary transitions, collects n cycles & goes back to primary cycle Pi Primary cycle (below threshold) h Threshold Peptide 3 triggers 7

8 Qual B.04: Find by MRM for a tmrm Result Secondary Secondary Primary Secondary MRM chromatograms for each transition for this peptide Primary Secondary Composite Spectrum

9 Performance of the 6490

10 Impact of Ion Funnel on Sensitivity: Standard Flow LC/MS with JetStream Source Calibration Curves QQQ (6460) fmol on-column fmol 25 pmol QQQ with ion funnel 00 amol on-column 00 amol 25 pmol HSA Standard d Peptide (LVNEVTEFAK, ), Poroshell x 50 mm column at 0.5 ml/min

11 Sensitivity: 290/6490 vs. HPLC-Chip/ , µl injected, HPLC-Chip 300 nl/min Icd_02; 5.88 fmol 6490, µl injected, 290 Infinity LC 400 µl/min CitZ_03; 2.83 fmol Icd_02; 5.88 fmol Upp_02; 2.83 fmol CitZ_03; 2.83 fmol Upp_02; 2.83 fmol 290/6490 HPLC-Chip/6460: approx. 2 3 x improvement in signal intensity Page

12 Sensitivity: 290 vs. HPLC-Chip on the , µl injected, HPLC-Chip 300 nl/min Icd_02; 5.88 fmol CitZ_03; 2.83 fmol 6490, µl injected, 290 Infinity LC 400 µl/min Upp_02; 2.83 fmol CitZ_03; 2.83 fmol Upp_02; 2.83 fmol Icd_02; 5.88 fmol 290 HPLC-Chip: approx. 0 5 x improvement in signal intensity Page 2

13 JetStream Source vs. ESI at Capillary Flow Rates (7 µl/min) 0 fmol on-column Nebulizer Heated Sheath Gas Thermal Gradient Focusing Region x New AJS 2.6 New MS Inlet ESI Counts vs. Acquisition Time (min)

14 2. vs. 0.5 mm ID Columns: JetStream Source Shows Mass Dependent Behavior fmol on-column x mm column 2. mm column 2. mm column 300 µl/min 00 amol 0 pmol mm column 7 µl/min 00 amol pmol Counts vs. Acquisition Time (min)

15 Comparison of Standard (0.3 ml/min), Capillary (7 µl/min) and Nanospray (600 nl/min) 00 amol on-column fmol on-column x µm column x µm 3.2 column mm column 0.5 mm 0.6 column mm column 0.5 mm column Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min)

16 Robustness of LC/MS System Goal: Demonstrate the performance of the instrumentation for the analysis of peptides in a complex matrix Instrumentation: 290 Infinity LC system with 6490 QQQ Sample: Stable isotope-labeled standard (SIS) peptides (2 total) in undepleted plasma digest These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

17 Injecting Less Sample Reduces Matrix Effects Plasminogen: LFLEPTR 25 µg 2.5 µg x0 2 +ESI MRM Frag=380.0V CID@4.0 (4 x * Heavy x ESI MRM Frag=380.0V CID@20.0 (4 x0 2 * Heavy 2.5 +ESI MRM Frag=380.0V CID@4.0 (4 * ESI MRM Frag=380.0V CID@20.0 (4 * x x von Willebrand Factor: ILAGPAGDSNVVK 25 µg 2.5 µg +ESI MRM Frag=380.0V CID@23.0 (6 x0 * 6.83 Heavy ESI MRM Frag=380.0V CID@23.0 (6 * ESI MRM Frag=380.0V CID@23.0 (6 x0 2 +ESI MRM Frag=380.0V 0V CID@23.0 (6 * 6.83 * Heavy 0.6 x ESI MRM Frag=380.0V CID@0.0 (4 x Heavy +ESI MRM Frag=380.0V CID@0.0 (4 * x ESI MRM Frag=380.0V CID@23.0 (6 * 6.67 Heavy x ESI MRM Frag=380.0V CID@23.0 (6 * 6.92 x ESI MRM Frag=380.0V CID@4.0 (4 x0 4 +ESI MRM Frag=380.0V CID@4.0 (4 * * x0 2 +ESI MRM Frag=380.0V CID@23.0 (6 x0 * Light Light +ESI MRM Frag=380.0V CID@23.0 (6 * Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

18 Standard Flow LC/MS: Plasminogen Peptide in 2.5 µg of Plasma Digest Matrix 50 amol on-column 250 amol on-column 0 fmol on-column These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre 8

19 Standard Flow LC/MS: Quantitation of the Plasminogen Peptide in Plasma Linear plot Log-Log plot Calibration from amol/µl in 250 ng/ul plasma digest (n=3) 44.3> %Accuracy (average, n =3) Reproducibility (%RSD, n = 3) ESTD Calibration standards (amol/ul) Response factor Precision (%RSD, n = ) The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre 2.30

20 Reproducibility for 0 Injections (0 fmol SIS Peptides and 2.5 µg Plasma Digest On-column) Protein Response %RSD Ret. Time %RSD Adiponectin: IFYNQQNHYDGSTGK Antithrombin-III : DDLYVSDAFHK Apolipoprotein A-II precursor: SPELQAEAK 0.5 Apolipoprotein C-III: GWVTDGFSSLK 0.25 Ceruloplasmin : EYTDASFTNR 0. Heparin cofactor II: TLEAQLTPR 0.05 Histidine-rich glycoprotein: DGYLFQLLR Kininogen-: TVGSDTFYSFK 0.0 L-selectin: AEIEYLEK Plasminogen: LFLEPTR Vitamin D-binding protein: THLPEVFLSK 0.00 von Willebrand Factor: ILAGPAGDSNVVK Plasminogen_LFL_h - 6 Levels, 6 Levels Used, 5 Points, 5 Points Used, 0 QCs 5 y = E-004 * x E-004 R^2 = Relative Resp ponses Plasminogen LFLEPTR 7.9% RSD, n=4 2.3% RSD, n=4 2.2% RSD n=0 4.7% RSD, n= Relative Concentration The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

21 Reproducibility for 0 Injections: Overlay of TIC for Runs, 0, 20, 30, 40, 50,60,70,80 and 90 2

22 Standard Flow LC: Excellent RT Reproducibility in Complex Matrices Zoom view Overlay of TICs for depleted d (n=3) and non-depleted (n=3) plasma samples 92 peptides 22

23 SISCAPA: Enrich Target Peptides and Decrease Sample Complexity

24 Automated Sample Handling for Protein Biomarker Studies

25 SISCAPA Protocol The Bravo can be set up to perform. Enzymatic digestion, reduction and alkylation steps 2. The SISCAPA process in 2 scripts to fully utilize the magnetic beadbased antibody capture and to elute off the same beads An plex SISCAPA assay run on the Bravo and 6490 QQQ combination

26 -Plex SISCAPA Using Ion Funnel QQQ-MS at Standard Flow Rate Meso LPSBP AFP He er-2 PCI CA25 stfr Tg OPN FLC Tg2 Column: ZORBAX Eclipse Plus RRHD C8, 2.x50mm,.8 um, 95Å Injection volume: 0 L containing 200fmol each peptide Flow rate: 0.4 ml/min MS: 6490 QQQ with ifunnel technology MS Mode: Dynamic MRM

27 Comparison of Standard Flow Ion Funnel QQQ-MS to Nanoflow QQQ-MS Mesothelin forward and reverse curves (log/log) 00 Agilent 6490 (400ul/min) + Bravo 00 AB 4000 Qtrap (300nl/min) + Kingfisher L/H (fwd) or H/L (rev) Area Ra atio Bravo-Forward-Meso Bravo-Reverse-Meso Endogenous level: 3fmol/0ul = 6ng/ml L/H (fwd) or H/L (rev) Area Ra atio Meso:Xlink:Reverse Meso:Xlink:Forward Endogenous level: 3fmol/0ul = 6ng/ml fmol Spiked Varying Peptide Standard flow ion funnel QQQ-MS fmol Spiked Varying Peptide Nanoflow QQQ-MS Forward curve: IS=heavy, standard addition above endogenous level with light Reverse curve: IS=light, heavy peptide spiked in at known levels (no endogenous of heavy)

28 Effect of Temperature on Peptide Separation: Eclipse Plus EC-C8, C8 8µm.8 µm, 2x50mmColumn C 35 C 40 C 45 C

29 Conclusions The ion funnel system shows at least a 5x improvement in sensitivity compared to a standard QQQ ESI with thermal gradient focusing shows mass dependent behavior for capillary to standard LC flow rates Nanoflow is still significantly more sensitive than cap or standard flow. The QQQ + ifunnel system demonstrated robust performance The QQQ ifunnel system demonstrated robust performance for peptide quantitation in plasma digests using standard flow LC/MS

30 Acknowledgements University of Victoria Derek Smith Christoph H. Borchers Agilent Technologies Yanan Yang Moritz Wagner Plasma Proteome Inst. Leigh Anderson University of Victoria Terry Pearson Matt Pope Angela Jackson