Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma

Abstract

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 seven 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 subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory 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 interlaboratory 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 control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Fig. 1.

Schematic of the experimental design of the three phases of the study. Phase I consisted of method development and optimization of the sample handling, LC, and MS parameters for peptide detection. Phase II was generation of the peptide-level response curve in which 125 light peptides were spiked into depleted, digested plasma at nine concentrations and 125 ¹³C/¹⁵N peptides were spiked in as internal standards and 750 transitions were monitored on the different LC-MRM-MS platforms. Phase III introduced unlabeled (light) and uniformly ¹⁵N-labeled proteins into the workflow, which were spiked into depleted plasma to generate a nine-point response curve. Samples were further processed at the individual sites to denature, reduce, alkylate, desalt, and reconstitute the samples with ¹³C/¹⁵N peptide standards for LC-MRM-MS analysis, resulting in a total of 1,095 transitions for each method. Skyline was integral from Phase I through Phase III for transition selection, method building, retention time scheduling, and data integration across the different vendor platforms.

Fig. 2.

Limit of detection distributions for the peptides monitored at each site. The black bar in each box represents the median peptide LOD at that site, the box represents the interquartile range and the whiskers represents 3x the interquartile range. Outlier peptides are shown as black dots. Panel A represents data from Phase II for the 13 instruments completing the study. Panel B shows the LOD distribution for the eight instruments that completed Phase III, with the synthetic ¹³C/¹⁵N peptides used as internal standards. Panel C represents the same Phase III data, except the U¹⁵N-peptides, derived from the U¹⁵N-proteins, were used as internal standards.

Fig. 3.

Comparison of the LODs for the eight peptides from the Addona et al., 2009 study and the current study (Phase II). The box and whisker chart represents the distribution of LODs from the participating sites in both studies for the peptide-level spike experiment.

Fig. 4.

Evaluation of the accuracy of determined concentrations for 125 peptides in the blinded samples. Sets of samples were spiked with peptide (125 peptides in Phase II) and protein (27 proteins in Phase III) analytes at concentrations blinded to the study participants. Blinded samples were analyzed at the sites after each response curve replicate in Phases II and III. Panel A shows the four blinded sample concentrations and the range of peptide concentrations detected at each site in Phase II. Panels B and C represent the Phase III blinded sample concentrations determined when using the ¹³C/¹⁵N peptides (panel B) or the U¹⁵N-proteins (panel C) as internal standards. The light blue lines represent the actual concentrations of spiked proteins. Note that in panel B all measured concentrations are well below the actual concentrations when calculating concentration based on spiked heavy peptides. Concentration values are much closer to the actual values in panel C where concentration values were relative to peptides derived from the digestion of U¹⁵N-labeled internal standard proteins.

Fig. 5.

Reproducibility plots for Phases II and III at each sample concentration. The median peak area %CV for 115 peptides is shown for Phase II (panel A) and Phase III (panel B) for all sites.

Fig. 6.

Technical and process variability assessed from digestion controls and SIS peptide spikes for Phase III. Six unlabeled (light) proteins were spiked into all samples predigestion at a fixed concentration (2.5 fmol/μl). The black bars represent the CV of the raw peak areas arising from the light peptides and reflect the process variability (due to digestion, desalt, and sample handling) of the assay for 40 individual samples. Eight ¹³C/¹⁵N peptides were spiked into all samples post-desalt at 10 fmol/μl. The gray bars represent the CV of the raw peak areas from the ¹³C/¹⁵N peptides and reflect the technical variability of the LC-MRM-MS measurements. Here, we see the process variability exceeds the technical variability for all peptides and is 35% or less, based on raw peak area. The technical variability is 25% or less for all peptides over the measurement of 40 different samples, and is 20% or less for six of the eight peptides. This is an example from Phase III, site 56B90, plotted in Skyline.