A semi-automated mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) reveals novel relationships between circulating PCSK9 and metabolic phenotypes in patient cohorts.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.

Hybrid data acquisition and processing strategies with increased throughput and selectivity: pSMART analysis for global qualitative and quantitative analysis.

Data-dependent acquisition (DDA) and data-independent acquisition strategies (DIA) have both resulted in improved understanding of proteomics samples. Both strategies have advantages and disadvantages that are well-published, where DDA is typically applied for deep discovery and DIA may be used to create sample records. In this paper, we present a hybrid data acquisition and processing strategy (pSMART) that combines the strengths of both techniques and provides significant benefits for qualitative and quantitative peptide analysis. The performance of pSMART is compared to published DIA strategies in an experiment that allows the objective assessment of DIA performance with respect to interrogation of previously acquired MS data. The results of this experiment demonstrate that pSMART creates fewer decoy hits than a standard DIA strategy. Moreover, we show that pSMART is more selective, sensitive, and reproducible than either standard DIA or DDA strategies alone.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum.

OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.