PO-CON1457E A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG ASMS 14 ThP766 Rachel Lieberman 1, David Colquhoun 1, Jeremy Post 1, Brian Feild 1, Scott Kuzdzal 1, Fred Regnier, 1 Shimadzu Scientific Instruments, Columbia, MD, USA Novilytic L.L.C, North Webster, IN, USA
Novel Aspect Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-85 allows for improved analysis of Immunoglobulin G. Introduction Dried blood spot analysis (DBS) has provided clinical laboratories a simple method to collect, store and transport samples for a wide variety of analyses. However, sample stability, hematocrit effects and inconsistent spotting techniques have limited the ability for wide spread adoption in clinical applications. Dried plasma spots (DPS) offer new opportunities by providing stable samples that avoid variability caused by the hematocrit. This presentation focuses on an ultra-fast-immuno-ms platform that combines next generation plasma separator cards (Novilytic L.L.C., North Webster, IN) with fully automated immuno-affinity enrichment and rapid digestion as an upfront sample preparation strategy for mass spectrometric analysis of immunoglobulins. Sample Workflow Plasma Generation Affinity Selection Buffer Exchange Enzyme Digestion Desalting LC/MS/MS Noviplex TM Card Perfinity Workstation LCMS-85 Triple Quadrupole MS Rapid plasma extraction technology from whole blood (~ 18 minutes) -.5 ul of plasma collected (3 min) - Air dry for 15 minutes - Extract plamsa disc for analysis Automates and integrates key proteomic workflow steps: - Affinity Selection (15 min) - Trypsin digestion (1-8 min) - Online Desalting - Reversed phase LC Exceptional reproducibility (CV less than 1%) - Ultrafast MRM methods - Up to 555 MRM transitions per run - Heated electrospray source - Scan speeds up to 3, u/sec - Polarity switching 5 msec
Methods IgG was weighed out and then diluted in 5 μl of.5% BSA solution. Approximately15 ul of IgG standard was spiked into mouse whole blood and processed using the Noviplex card. The resulting plasma collection disc was extracted with 3 ul of buffer and each sample was reduced and alkylated to yield a total sample volume of 1 ul. IgG standards and QC samples were directly injected onto the Perfinity-LCMS-85 platform for affinity pulldown with a Protein G column followed by trypsin digestion and LC/MS/MS analysis. Level 1 3 4 5 6 7 Conc. (μg/ml) 465 315 14.5 17.5 1 6.5 Amount on column (μg) 34.88 3.63 1.69 9.56 7.65 4.5 1.69 Amount on column (pmol) 581.5 393.75 178.13 159.37 17.5 75. 8.1 Time (min). 8 9.5 1 1.5 1.51 16 %B 5 5 9 9 %B 1 8 6 4 4 6 8 1 1 14 16 Time (minutes) IgG concentrations for calibration levels. LCMS gradient conditions. Compound Name Transitions +/- Q1 Rod Bias (V) CE (V) Q3 Rod Bias (V) 937.7>836.5 937.7>73.95 + + -7-7 -8-3 -6-63.7>85.7 + - -16-13 MRM transitions on LCMS-85 for two IgG peptides monitored. Noviplex Cards () (3) (4) (1) Approximately 5 ul of the spiked whole blood was pipetted onto the Noviplex card test area (1). The spot was allowed to dry for 3 minutes (). The top layer of the card was then peeled back (3) to reveal the plamsa collection disc. The plasma collection disc was allowed to dry for an additional 15 minutes. Once the disc was dry (4), it was placed into an eppendorf tube for solvent extraction. 3
Results - Chromatograms 3 75 5 5 175 15 15 1 75 5 5..5 1. 1.5..5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 min Total Ion Chromatogram for IgG 15 1 75 Optimization of Collision Energies for peptides of interest Range CE: -5 to -1 V TTPPVLDSDGFFLYSK 5 5 6. 6.5 6.5 6.75 6.3 6.35 6.35 6.375 6.4 6.45 6.45 6.475 6.5 6.55 6.55 6.575 6.6 6.65 6.65 6.675 min Inten.. 1.75 938 [M+H] + 1.5 5 45 4 35 1.5 1..75.5 397 [P+H] + [P1+H] + 837 836 937 938 3 5.5. 836 35 74 397 47 83 95 54 337 369 561 915 1163 836 146 51 74 836 83 591 64 658 73 466 756 47 397 851 379 443 449 89 891 3 4 5 6 7 8 9 1 11 1 13 14 m/z 15 1 5..5 1. 1.5..5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 min MRM Chromatogram for Level 4 standard of spiked IgG in whole blood. Carryover Assessment 11 1 9 Control - Mouse blood 9 8 Blank Injection 8 7 7 6 6 5 5 4 4 3 3 1 1..5 1. 1.5..5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 min..5 1. 1.5..5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 min 4
Results - Calibration Curves Calibration Curve and MS Chromatograms 5 63.7>85.7(+) 937.7>836.5(+) 937.7>836.5(+) 63.7>85.7(+) 937.7>73.95(+) Level 1 937.7>73.95(+) Level 7 1 Level 1 6 Level 7 5 75 15 4 15 1 5 1 5 5 5 3 1 5.5 5.75 6. 6.5 6.5 5.5 5.75 6. 6.5 6.5 6. 6.5 6.5 6.75 6. 6.5 6.5 6.75 5 r =.979 3 5 r =.989 15 5 1 5 1 3 4 Conc. 1 3 4 Conc. Results - Tables and Replicates QC data and Calculations for IgG Peptides Sample Ret. Time Calc. Conc. Std. Conc. % Accuracy QC 1 6.49 3,49 5.84 465 18.1 QC 6.516 11,76 167.189 14.5 117.3 QC 3 6.514 8,57 115.155 1 11.9 QC 4 6.49,77 1.745.5 96.6 Sample Ret. Time Calc. Conc. Std. Conc. % Accuracy QC 1 6.9 61,55 416.447 465 89.6 QC 6.5 5,355 155.568 14.5 19. QC 3 6.47 16,9 94.58 1 9.7 QC 4 6.9 6,5 19.587.5 87.1 5
Skyline Data - Retention Time Replicates 6.65 y14-85.4385++ 6. y15-836.4169++ 6.6 6.15 6.55 6.5 6.45 Retention Time 6.1 6.5 6. 6.4 5.95 6.35 5.9 839 AM_614...L1...5 839 AM_614...L1...5 839 AM_614...L...4 839 AM_614...L3...3 839 AM_614...L4... 1433 PM_514...L5...8 1433 PM_514...L6...6 1433 PM_514...L7...4 839 AM_614...L...4 839 AM_614...L3...3 839 AM_614...L4... 1433 PM_514...L5...8 1433 PM_514...L6...6 Retention Time 1433 PM_514...L7...4 Replicate Replicate Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative figures showing the retention time reproducibility for each peptide monitored during the analysis. Conclusions Combining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection from whole blood, with the automated affinity selection and trypsin digestion of the Perfinity workstation coupled to LCMS-85, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore, this rapid immuno-ms platform can be applied to many other peptide/protein applications. First Edition: June, 14 www.shimadzu.com/an/ For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 14