Until recently, only ELISA analyses have been able to detect and quantify cardiac troponins in the low μ
g/L range in plasma. Other approaches, such as immunoaffinity depletion/limited fractionation coupled to MRM, have been successful in quantifying low-abundance proteins in plasma with comparable precision, reproducibility, and multiplexing capability (4
). However, this methodology involves multistep plasma processing, which limits overall sample throughput. Here we demonstrated that SISCAPA can be multiplexed to quantify low-abundance cTnI and IL-33 in plasma with CV <15% in a linear range of 1.2 μ
g/L to 5 mg/L, a range where many protein biomarkers are expected (8
). This is also the first demonstration that SISCAPA MRM–based assays can quantify relative changes in concentration of cTnI pre- and postinjury in clinical samples. Similar trends in cTnI concentration were observed using SISCAPA and a commercial immunoassay in PMI patients in the 1–10 μ
g/L range, with an excellent correlation (R
= 0.89). Our results are consistent with the assay performance and LOQ recently described by Hoofnagle et al. for thyroglobulin (21
Our primary goal in these studies is not to replace existing immunoassays for proteins like cTnI that are already being measured in clinical practice (22
), but rather to leverage the unique advantages of targeted MS-based assay methods (10
) to configure assays for target proteins where high-quality antibodies suitable for an immunoassay are not available. We developed and characterized a SISCAPA assay for IL-33, a novel candidate marker of cardiovascular disease (14
) for which an immunoassay does not yet exist. Of note, IL-33 is the ligand for ST-2, a protein that is highly upregulated by myocardial cells subjected to mechanical stress (24
) and an emerging biomarker of acute and chronic myocardial injury (25
). The present study therefore paves the way for future investigation testing the additional clinical utility of simultaneous measurements of this ligand–receptor pair.
Sandwich immunoassays suffer from a variety of nonspecific interference that can be difficult to recognize and correct (26
). In SISCAPA, plasma proteins are denatured and enzymatically digested in the first processing step. These steps destroy specific and nonspecific protein–protein interactions that may produce false positives and interfere with the integrity of the measurements (21
). Because proteins are quantified by measuring peptides produced by the digestion of intact protein, MRM and SISCAPA-MRM assays can be used to measure truncated or degraded forms of the full-length protein, provided that the epitope recognized by the SISCAPA antipeptide antibody has not been compromised. Proteolytic degradation of cTnI in serum can cause measurement inaccuracies for ELISA assays that SISCAPA could, in principle, sidestep (29
). SISCAPA assays are also easier to multiplex than ELISA or sandwich immunoassays because off-target interactions are less likely to occur with peptides than proteins, and if they do occur the mass spectrometer can distinguish their presence and identity. In addition, MS-based SISCAPA assays are less expensive to develop than immunoassays, as only 1 antibody rather than a carefully matched pair is required because the mass spectrometer functions as the secondary antibody.
MS-based quantification is based on measuring the ion-counts for the fragment ions from the peptide analyte. Even slight differences in the composition of the matrix (digested plasma) at the time of elution and detection of the analyte can significantly alter the absolute signal of the analyte observed. This is why internal calibrants are routinely employed in LC-MS/MS–based assays (30
). In our assays, we employ the ideal internal standard, a 13
C and/or 15
N-labeled version of the same peptide that has identical physiochemical properties, including retention time, and differs from the analyte only in its parent and fragment masses. Any matrix constituent that causes suppression of the signal from the analyte also affects the internal standard equivalently, and therefore these effects are compensated for in the measurement. Whereas external calibration is typically used in immunoassay, internal calibration methods are beginning to be used, with clear benefits to assay precision being realized (31
Antiprotein antibody enrichment before SID-MRM-MS has also been used to establish high-quality assays (32
). These assays require the existence of affinity purification-grade antibodies that are not always available for the majority of proteins. Although antiprotein antibodies are widely used for rapid enrichment of proteins from complex mixtures for characterization by MS (35
), quantitative analyses require that the immunoprecipitation (IP) efficiency does not vary from sample to sample. Because the exogenous labeled peptide standard is added after protein IP, interassay reproducibility cannot be internally controlled. Addition of a suitably labeled protein standard (36
) could be used to correct for this variability in a manner similar to the use of stable isotopically labeled peptides described here. This approach could also be used to normalize for incomplete protein digestion. However, this method would be practically applicable only to cases in which a very small number of high-quality targets are to be measured.
There are limitations of SISCAPA-MRM assays worth noting. First, as demonstrated here, peptides derived from the same protein can have different assay performance. This may be caused by differences in the antibody affinity, the MS response of the respective peptides, or the recovery of the peptides owing to differences in the efficiency of release by digestion or differential loss upon desalting of the digest. Adding the internal standard peptide earlier in the process would account for losses that occur during desalting before antibody enrichment. Second, the results may not correlate well with immunoassay. The proportional bias observed here (slope = 0.28) may become the rule rather than the exception, since the immunoassay is measuring protein in its native state whereas SISCAPA is measuring a peptide recovered from that protein. More importantly, each assay was developed with separately qualified standards and calibrants. The LOQ of SISCAPA assays, presently in the low μ
g/L range for proteins in plasma, is limited by interferences from nonspecific binding of peptides to the antibody and resin, as well as traces of target peptide that remain bound to the antibody postpurification (21
). Investigations are underway using other types of magnetic beads and elution conditions to reduce nonspecific binding.
The LOD for our MS-based cTnI assay is well above the LOD of current commercial immunoassays (37
). Consistent with the expected literature levels of IL-33 in the blood (38
), IL-33 was not detected by this assay in patient samples. However, we expect that, as was the case for immunoassays, the sensitivity of the SISCAPA assays will improve over time. Provided that sample is not limiting, increasing the volume of plasma processed from 50μ
L used here to 500μ
L should provide a 10-fold increase in sensitivity without altering the assay format. Coupling immunoaffinity depletion to SISCAPA could also potentially improve the overall LOD (39
). Specific or nonspecific loss of target proteins can occur on depletion columns, however, and should be defined as part of assay configuration. For example, we have evaluated the recovery of cTnI following depletion of abundant proteins with both the MARS-7 and Genway-12 immunodepletion columns and found that about 90% of this protein was lost to the depletion columns (data not shown). Use of monoclonal antibodies in place of polyclonal antibodies could also increase capture efficiency and translate into an increase in sensitivity. It would also provide a continually renewable source of identical antibody. With higher affinities, less antibody and fewer magnetic beads would be required for the assay. As a result, nonspecific binding would be reduced and more target peptide could be loaded per analysis, ultimately increasing the signal and sensitivity of the SISCAPA assay. Regardless of the current sensitivity limitations, we have achieved the primary goal of clearly establishing that SISCAPA assays can multiplexed and used to measure proteins of clinical interest in the absence of commercial antibodies suitable for immunoaffinity/ELISA assays.
In conclusion, SISCAPA-MRM assays have the potential for high sensitivity, ease of automation, ability to be highly multiplexed, and high sample analysis throughput (10
). The present work further establishes that SISCAPA is a flexible alternative to sandwich and ELISA immunoassays, with the potential to triage large lists of candidate proteins and thereby help to alleviate the significant bottleneck that exists between unbiased discovery and clinical validation (2