Targeted MRM assays have been used very successfully for quantifying small molecules (e.g., hormones, drugs and their metabolites) in pharmaceutical research and in clinical laboratories in applications such as screening newborns for disease11
. More recently, the merits of SID-MRM-MS for quantifying peptides derived from proteins in plasma have been demonstrated in several laboratories4–9,12
. These studies have, however, only addressed assay performance at a single laboratory, and thus were not able to demonstrate the multisite robustness needed in large-scale biomarker research and ultimately in preclinical and clinical applications. The main purpose of this study was to provide such a demonstration by performing an assessment of the analytical characteristics of a multiplexed, SID-MRM-MS assay across eight laboratories using seven target proteins with which to spike human plasma. A three-tiered experimental protocol was used that progressively introduced sample preparation variables likely to affect inter- and intralaboratory reproducibility, transferability, precision and sensitivity. Our results demonstrate that reproducible, quantitative measurements of proteins in plasma can be made by SID-MRM-MS in multiple laboratories using different instrument platforms through use of standardized protocols for sample preparation, data acquisition and data analysis. The robustness of such a targeted assay approach compensates for the greater variability in protein measurements inherent in shotgun (‘discovery’ proteomics) methods13,14
, enabling the development of an effective biomarker pipeline1
Reproducibility and precision of the quantitative measurements for nine of ten peptides tested across eight laboratories ranged from 4–14%, 4–13% and 10–23% interlaboratory CVs at or near the estimated LOQ for study I, II and III, respectively. Intralaboratory CVs were predominantly <15% and <25% at the identical concentration for studies I/II and III, respectively (Supplementary Table 2
). Although the current assay performance under real biomarker conditions (study III) is below that generally stated for clinical assays (typically <10–15%), the performance achieved is sufficient for the verification of candidate biomarkers2
present at more than ~2–6 µg/ml in plasma, with a linear dynamic range spanning three orders of magnitude. In all cases, interlaboratory and intralaboratory CVs improved with increasing analyte concentration. Such modest differences between interlaboratory and intralaboratory CVs underscore the excellent agreement between the eight participating laboratories. Likewise, the progressive increases in CVs from studies I to III indicate convincingly that sample preparation contributes more to assay variability than instrumental variability, further highlighting the data quality obtainable from SID-MRM-MS. Although most important parameters were governed by detailed SOPs, the transfer of MRM assays across LC-MS platforms did require optimization of the transitions being monitored to compensate for differing instrument-specific ion source and collision-induced dissociation parameters, and to ensure that each platform achieved optimum sensitivity (Supplementary Tables 1a–e
). Despite these variations concerning a small number of analyte peptides, interlaboratory variability and specificity of the assay were not affected ().
Differences emerged in assay performance for different peptides. Most peptides performed well at all eight sites, whereas a few exhibited variable or poor behavior. This result highlights the dependence of MRM assay performance in plasma on specific properties of the peptides selected as surrogates for the target proteins. Ideally the final selection of signature peptides for SID-MRM-MS biomarker assays should be based on multisite studies so as to ensure the most robust performance.
The most frequent cause of poor peptide performance was the presence of interference from the background plasma digest matrix, in either the analyte or internal-standard channels, which altered the ratios of these transitions. Monitoring a minimum of three transitions per analyte is critical in maintaining assay selectivity and recognizing such interferences when they occur. Most participating sites observed interferences in one or more peptides over the course of the three studies. In the case of CRP, we were able to establish that the flattening of the response curves was due to the presence of endogenous levels of CRP as all three transitions monitored were affected equally and the expected ratios of the transition-ion abundances to one another were maintained. Other interferences arose from problems with chromatography (e.g., large peak widths, shifting retentions times, or early elution and consequent sensitivity to intermittent or unstable electrospray conditions), which can be addressed by further refinement of protocols, particularly in LC operation and data acquisition.
Recovery of signature peptides generally decreased from study I to III, as proteolytic digestion and subsequent sample handling, such as desalting, were introduced into the experimental workflow. Digestion efficiency of proteins in the plasma matrix has only recently begun to be studied15
. If a signature peptide is not detected in an MRM assay, it is often unclear if this is because of (i) losses from sample handling, such as fractionation or desalting, (ii) poor enzymatic digestion, (iii) concentration below LOD, (iv) post-translational modification such as glycosylation and phosphorylation, (v) artifactual modifications to reactive amino acids, such as oxidation or carbamylation, or (vi) some combination thereof. The effect of decreasing control of sample preparation was reflected in the increased variability and lower peptide recoveries for a majority of peptides as sites progressed from study II to III ( and Supplementary Fig. 6
). In study III, one peptide was not recovered in any process replicate performed at all participating laboratories, and four peptides had <25% recovery ( and Supplementary Fig. 6
). Addition of labeled internal standard (IS) peptides at an early stage in sample processing (e.g., during enzymatic digestion) could help to account for peptide loss. However, lower recovery of signature peptides does not impede the use of these assays for verification where the goal is to precisely define the relative difference in abundance for candidate proteins between cases and controls rather than to determine the absolute concentration of each protein. Absent a general method ensuring stoichiometric digestion, absolute concentration measurements would likely require addition of isotopically labeled, recombinant protein standards at the start of sample processing.
The purpose of the present study was not to define the ultimate sensitivity possible for proteins by SID-MRM-MS, but rather to evaluate the transferability and robustness of the technology within and between laboratories. For this first study, we made no attempt to reduce the complexity of the plasma matrix by either depletion of abundant proteins or fractionation. The sensitivity of protein quantification by SID-MRM-MS in plasma is severely limited by the complexity and 1011
dynamic range of protein abundances in blood, and the susceptibility to interference from other peptides and their fragment ions is greatest in this matrix16
. Typical LODs and LOQs observed in prior studies of unfractionated plasma are in the high 100s of ng/ml to low µg/ml range of target protein6,8,17
. Results described here are consistent with these reports across sites and instrument platforms ( and Supplementary Fig. 4
). Although emphasis is often placed on discovery and verification of low-abundance candidate biomarkers (≤ ng/ml levels in serum), high-abundance serum proteins, such as CRP, transferrin, complement components, immunoglobulin classes and lipoproteins, are clinically relevant markers of disease and their levels in blood make them directly accessible by SID-MRM-MS using the approaches described here. The LODs and LOQs of MS-based assays have been extended into the low ng/ml range in plasma by using immunoaffinity depletion of high-abundance proteins, limited protein or peptide fractionation, or immunoaffinity enrichment at the protein or peptide level before SID-MRM-MS9,17–24
. The additional processing steps used are likely to introduce new sources of experimental variation that will have to be assessed in interlaboratory studies similar to those described here. Nevertheless, the assay performance reported in the present studies, measured at maximum levels of interfering high-abundance peptides in unfractionated plasma digests, suggests that similar or better intra- and interlaboratory assay performance may be achievable for quantitative, multiplexed measurement of proteins in the low ng/ml range in plasma by MS.
Our study demonstrates that targeted, quantitative and multiplexed MS-based assays can be rapidly configured and deployed in multiple laboratories to yield robust and reproducible assays for proteins down to low µg/ml levels in the context of unfractionated plasma. This is a critical first step toward potential widespread implementation of SID-MRM-MS assays for verification of novel protein biomarker candidates. The SID-MRM-MS technology has the potential to become the critical filter used to assess candidate biomarker performance in a sufficient number of patient samples before committing the very substantial time and resources required to create clinical-grade immunoassays. The performance required of such assays2
is not as stringent as that currently required for US Food and Drug Administration–approved clinical assays25
. Beyond candidate verification, SID-MRM-MS assays may eventually have potential to replace certain clinical immunoassays, especially in cases where interferences are known to exist23
or multiplex measurements are needed. By detecting a structural component of the protein, the signature peptide, with near-absolute structural specificity, SID-MRM-MS should avoid inter-assay differences that occur when different immunoassays for the same protein detect distinct, potentially labile epitopes. Furthermore, the simplicity of producing and characterizing peptide-based reference materials for SID-MRM-MS could help overcome well-known problems with ELISA assay standardization, which lead to varying results across multiple clinical laboratories26,27
The methods, reagents and multilaboratory data sets presented here should facilitate testing and implementation of MRM-based multiplex assays for quantifying target proteins in plasma by the proteomics community. Our results should foster greater acceptance by the clinical community of SID-MRM-MS technology as a generally applicable approach to verify candidate biomarkers in large clinical sample sets, and thus provide a critical component for a systematic biomarker-development pipeline.