Mass spectrometry-based proteomics strategies often are used in the discovery of putative disease biomarkers. Extensive sample prefractionation and the use of high-performance mass spectrometers have facilitated in-depth analysis of many highly complex human proteomes, particularly serum or plasma proteomes.1–3
As a result of the sensitivity of current proteomics methods, up to a hundred or more candidate biomarkers can be identified readily for diverse diseases either using model systems or small numbers of patient samples or pools of samples.4–7
In addition, large numbers of additional biomarkers can be obtained readily by surveying the scientific literature.8
However, verification and laboratory-scale initial validation of large numbers of candidate biomarkers in human serum or plasma are quite challenging and have become rate-limiting steps in the biomarker pipeline.7, 9
Sandwich enzyme-linked immunosorbent assays (ELISA) can be highly specific and capable of detecting blood proteins at the low pg/mL range with high throughput. However, appropriate ELISA assays are not available for many candidate biomarkers and these assays can be difficult to multiplex. Furthermore, preparing specific antibodies and developing sandwich ELISAs for novel target proteins are lengthy and costly processes. Based on recent experience in the field, it is reasonable to expect that only a modest portion of candidate biomarkers discovered using proteomics ultimately will prove to be clinically useful. As a result, cost- and time-effective methods are needed to quickly screen large numbers of candidate biomarkers to identify those proteins worth investing the resources required to develop sandwich ELISAs or equivalent higher throughput assays.
In recent years, MRM-MS has emerged as an attractive targeted MS technique for biomarker verification and initial validation.10, 11
High selectivity of MRM is achieved by isolating a specific peptide parent ion and a high-yield fragment ion (an MRM transition) in a triple quadrupole mass spectrometer, and extensive multiplexing can be achieved where many peptides are monitored in a single run.10, 12
MRM assays typically are coupled with stable isotope dilution (SID) using chemically identical synthesized peptides to achieve absolute and reproducible quantitation.13–16
SID-MRM, coupled with peptide fractionation or specific enrichment, has been shown to detect proteins in plasma or serum in the low ng/mL range with a broad dynamic range of up to five orders of magnitude.12, 17–19
As expected, SID-MRM quantitation is highly reproducible, even if the measurements are carried out in different laboratories.20
Since stable labeled peptides have identical retention times as targeted peptides, unambiguous confirmation of the targeted peptides can be achieved even in the presence of closely co-eluting peptides which are commonly encountered in MRM analyses of complex proteomes such as serum or plasma. Interferences can also be easily detected by monitoring multiple MRM transitions of the stable labeled peptides and comparing the transition intensity ratios with the ratios from targeted peptides. However, the cost and lead time for synthesizing, purifying, and evaluating stable isotope standard peptides for absolute quantitation, as well as setting up spike-in experiments and standard curves, can be substantial, especially at the verification and early-stage validation steps where screening of a large number of putative candidate biomarkers may be of interest.
Alternative strategies have been explored to generate labeled peptides in a more cost-effective manner. This includes expression of a synthetic gene in medium containing labeled amino acids to generate isotope-labeled, concatenated peptides or full-length proteins that can be purified and trypsin digested to produce stable isotope standard peptides.10, 21–23
Chemical modification techniques such as mTRAQ and 18
O-labeling also can be used to incorporate isotopic labels into peptides.24, 25
However, all these methods involve additional costs, time, and expertise. In addition, difficulties in expression of certain synthetic constructs have been encountered, and the release of concatenated tryptic peptides may differ from the natural protein.26
Despite the high selectivity of MRM, accurate quantitation of proteins in human serum can be compromised due to serum’s complexity and wide dynamic range of protein abundances, which span more than 10 orders of magnitude.7, 27
As a result, co-eluting ions may suppress desired peptide signals or incompletely resolved ions may interfere with specific MRM transitions.10, 17, 28
In addition, a protein can contain multiple isoforms or may undergo proteolytic processing or post-translational modifications that are physiologically relevant to a disease state but may not be apparent at the peptide level. Therefore, MRM quantitation strategies that could minimize or easily detect interferences and provide quantitation information on various forms of a protein are highly desirable.
In this study, we explored the use of label-free MRM quantitation of SDS gel-fractionated serum proteins (GeLC-MRM) as a rapid, first-level biomarker verification strategy. Label-free quantitation of ion peak areas recently has emerged as a promising strategy in discovery proteomics because it is simple to implement and allows relative quantitative comparisons across multiple samples in diverse experimental systems.29–33
However, label-free quantitation has only been used for LC-MRM assays in a few cases.10, 34, 35
More importantly, long-term reproducibility of label-free MRM analysis has not been demonstrated in previous studies. In this study, we observed that relative quantitation using label-free MRM is sufficiently reproducible and more appropriate as a first-level quantitation when screening large numbers of candidate biomarkers. By rigorously controlling experimental parameters, highly reproducible major protein immunodepletion, 1-D gel fractionation, and LC-MRM analysis could be achieved. Furthermore, GeLC-MRM could distinguish and quantitate different molecular forms of a protein, and reanalysis of the same sample on a different mass spectrometer yielded consistent results.