Large-scale inter-laboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma
S.E. Abbatiello, B. Schilling, D.R. Mani, L.J. Zimmerman, S.C. Hall, B. MacLean, M. Albertolle, S. Allen, M. Burgess, M.P. Cusack, M. Ghosh, V. Hedrick, J.M. Held, H.D. Inerowicz, A. Jackson, H. Keshishian, C.R. Kinsinger, J. Lyssand, L. Makowski, M. Mesri, H. Rodriguez, P. Rudnick, P. Sadowski, N. Sedransk, K. Shaddox, S.J. Skates, E. Kuhn, D. Smith, J.R. Whiteaker, C. Whitwell, S. Zhang, C.H. Borchers, S.J. Fisher, B.W. Gibson, D.C. Liebler, M.J. MacCoss, T.A. Neubert, A.G. Paulovich, F.E. Regnier, P. Tempst, S.A. Carr “Large-scale inter-laboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma”, Mol. Cell. Proteomics, 2015, published online on Feb 18. mcp.M114.047050.
- Organism: Homo sapiens
- Instrument: Triple Quadrupole (Agilent, SCIEX, Thermo, Waters)
There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.
The study utilized 8 different LC-MS instrument configurations in 11 separate laboratories on a total of 14 individual systems to target and quantitatively measure >100 peptides from a total of 34 proteins, including 27 that are cancer relevant. In our study, sensitivity for proteins in plasma was increased into the low-to-sub nanogram/mL level by one-step immunoaffinity depletion as well as gradient optimization to maximize the chromatographic resolution in the sample matrix. Use of heavy-labeled protein internal standards added to samples prior to processing greatly improved the accuracy of protein-level quantification. Intra- and inter-laboratory reproducibility sufficient for most biological studies as well as for candidate protein biomarker verification was achieved. Overall, this study demonstrates that highly multiplexed MRM-MS based assays can, with appropriate attention to experimental design, analytical validation, and suitable quality control measures, be implemented by multiple laboratories to provide sensitive, specific, reproducible and quantitative measurements of proteins and peptides of clinical and biological interest in complex biological matrices, specifically plasma.
Study Phase II (Study 9.1): The samples analyzed in this phase of the study were prepared centrally. Briefly, samples used to generate a nine-point response curve were prepared in human MARS-14 depleted K2EDTA plasma. The depleted plasma was denatured, reduced and alkylated, digested with Lys-C and trypsin and desalted according to a standard operating procedure. The resulting digested, depleted plasma was spiked with 125 synthetic 12C/14N and corresponding 13C/15N-isotopically labeled internal standard (IS) peptides. Nine concentration point samples were prepared by serial dilution to generate calibration curves spanning a concentration range of 1 amol/µL to 100 fmol/µL (with 1 µL volume on-column) of all light peptides spiked into a 0.5 µg/µL background of the depleted plasma digestion with a constant concentration of 10 fmol/µL of all 13C/15N-isotopically labeled IS peptides. The individual concentrations of the light peptides were 100.00, 23.71, 5.62, 1.33, 0.316, 0.075, 0.018, 0.004, 0.001 fmol/µL (loading 1 µL of sample on-column). The four calibration curves were generated individually. In addition, six samples, referred to as blinded samples, with light peptide concentrations unknown to the instrument operator were provided to each site. The blinded samples were analyzed at the end of each of the four singlicate response curves . Phase II sample kits were shipped to the 9 participating laboratories (representing 14 LC MSMS instrument configurations).
Study Phase III (Study 9.2): For Phase III , response curves and blinded samples were generated by spiking 27 unlabeled undigested ANL cancer relevant target proteins and 6 unlabeled undigested previously characterized proteins into depleted, undigested human K2EDTA plasma. The 9-point response curve for the 27 unlabeled cancer relevant proteins spanned a concentration range of 10 amol/µL to 100 fmol/µL ((loading 1 µL of sample on-column) in a background of depleted human K2EDTA plasma (0.5 µg/µL). The six additional unlabeled proteins, used as a digestion reproducibility control, were spiked at a constant concentration such that following digestion and dilution, a final on-column amount of 2.5 fmol equivalent was achieved. In addition, 27 U-15N-labeled proteins were spiked into depleted plasma at a constant concentration, so that after digestion and dilution yielded 115 U-15N target peptides at 25 fmol equivalent on-column. Samples were prepared such that after protein digestion the individual concentrations of the proteolytically generated light peptides were calculated to be 100, 24, 5.6, 1.3, 0.82, 0.32, 0.075, 0.018, and 0.010 fmol/µL (1 µL injection volume). In addition, six blinded samples with light protein concentrations unknown to the instrument operator at each site were provided and analyzed at the end of each of the four singlicate response curves (see Supplemental Table 3). Phase III sample kits with undigested light and heavy proteins spiked into undigested depleted plasma were prepared at Vanderbilt University and sent to the 7 participating laboratories (representing 11 LC-MRM-MS instrument configurations). The samples were digested and desalted at the individual sites. MS operators digested 3 independent protein calibration sets and blinded samples. Data for the third protein calibration curve were acquired as a technical MS duplicate, so that as in Phase II, there was a total of 4 LC-MRM-MS acquisition replicates. All instruments operators spiked the 13C/15N-isotopically labeled peptides, post-desalt, to yield a final concentration of 10 fmol/µL. At 3 participating sites the 13C/15N-isotopically labeled peptides were spiked in pre- and post-desalt as independent experiments to assess sample loss during desalting. Protein digestion efficiencies were estimated using U-15N-labeled proteins that had been spiked into depleted plasma.
Created on 2/6/15, 4:52 PM