Elsevier

Methods

Volume 81, 15 June 2015, Pages 66-73
Methods

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

https://doi.org/10.1016/j.ymeth.2015.03.003Get rights and content

Highlights

  • Multiplexed quantitative, mass spectrometric immunoassay for variants of PCSK9.

  • Application of PCSK9 MSIA–SRM assay in cohorts of patients.

  • The majority of plasma PCSK9 molecules are phosphorylated at S688.

  • PCSK9 MSIA–SRM assay reveals novel relationships between PCSK9 and metabolic phenotypes.

Abstract

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.

Introduction

The secretory proprotein convertase subtilisin/kexin type 9 (PCSK9) [1] is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Its gene represents the third locus associated with familial hypercholesterolemia, following the low density lipoprotein receptor (LDLR) and apolipoprotein B (ApoB) [2]. Numerous loss and gain of function mutations have been described in the gene encoding PCSK9 (reviewed in [3]). The reduced cardiovascular risk in patients with loss-of-function alleles [4] has prompted the development of monoclonal antibodies for the treatment of hypercholesterolemia that are currently in clinical trials, some of them as far as in phase 3 (reviewed in [3], [5]).

PCSK9 affects levels of circulating LDL particles by decreasing the level of the LDLR protein at the plasma membrane [6], [7]. It does so through an extracellular pathway, where circulating PCSK9 binds the EGF-A domain of the LDLR protein leading to internalization and degradation of the complex, thereby rendering LDLR unavailable for the cellular uptake of LDL-C [8]. Intracellular pathways have also been proposed, where PCSK9 can enhance LDLR degradation prior to secretion [9] or promotes its exit from the endoplasmic reticulum [10].

A positive association between circulating PCSK9 and LDL-C has been described in various patients cohorts, with correlation coefficients ranging from 0.24 to 0.54 [11], [12], [13], [14], [15], [16], [17], [18]. These studies report PCSK9 abundance values obtained by ELISA assays, some of them being commercially available. There is, however, a lack of standardization amongst measurements obtained from different ELISA kits. Moreover, this assay intrinsically yields only one value per sample, so it lacks the discriminative power to resolve various protein forms and monitor posttranslational modifications (PTMs). Yet several PCSK9 protein species have been detected in plasma [19]. The shorter, Furin-cleavage product of PCSK9 is believed to be inactive [19], [20], [21], [22], or to show reduced activity [23]. PTMs of PCSK9 have also been observed such as sulfation at tyrosine 38 [19], phosphorylation at serines 47 and 688 [24] and N-glycosylation at asparagine 533 [1]. Depending upon the antibody’s specificity, an ELISA will report on either the mature, or both the mature and other protein species (including the shorter, furin-cleaved species), without discrimination or information on PTMs.

We describe herein a mass spectrometric immunoassay for the monitoring of PCSK9 abundance including one of its posttranslational modifications. Immuno-capture of the protein prior to mass spectrometric analysis addresses the issue of the low abundance of the protein (low ng/ml) compounded with the wide dynamic range of plasma proteins, a non-negligible challenge for current mass spectrometers. The specificity and multiplexing capability of SRM provides a means to assess distinct domains and PTMs. We have previously briefly described the development of a PCSK9 MSIA–SRM assay [25]. Herein we have extended our work with the inclusion of a phosphopeptide and have applied this assay to two distinct patient cohorts to study the relationship between PCSK9 abundance, blood lipid profile and insulin resistance.

Section snippets

Obese patient cohort

Twenty-nine obese patients enrolled between 2012 and 2014 in the ETAPP cohort were studied. The goal of the ETAPP cohort is to study factors involved in the complications associated with obesity before and after bariatric surgery. Only pre-surgical samples were included in the present analyses. Patients were enrolled if they were (1) obese with 35–55 kg/m2 BMI, (2) 18–60 years old, (3) sedentary (<3 h weekly physical activity). Patients were excluded from study participation if they had (1) type 2

Performance of the PCSK9 MSIA–SRM assay

Seven peptides were selected for monitoring based on their superior performance and coverage of the prodomain (peptide 1), the catalytic domain prior to the furin-cleavage site (R218) (peptide 2), the catalytic domain after the furin-cleavage site (peptides 3), the hinge (peptide 4), the cysteine and histidine rich domain (CHRD) (peptide 5), and the C-terminal domain encompassing S688 in both its phosphorylated and unmodified forms (peptides 6 and 7, respectively) (Fig. 2A). With the exception

Conclusions

We have developed and applied a MSIA–SRM assay that enables the quantification and characterization of PCSK9 in a small volume (75 μl) of plasma by mass spectrometry. This assay allows the multiplexed measurement of peptides from each of the PCSK9 domains as well as the C-terminal peptide phosphorylated at S688. When applied to two different human cohorts, the PCSK9 MSIA–SRM assay has allowed us to observe (1) associations between PCSK9 peptides and patients’ metabolic phenotypes that no other

Acknowledgements

We wish to thank Marguerite Boulos for technical assistance and Dr. Omid Hekmat for valuable advice. This work was supported by a grant from the Fondation Leduqc to B.C. and N.G.S. and the fonds IRCM-Banque Scotia to B.C. and R.R.L. B.C. holds the IRCM Bell-Bombardier Chair of Excellence. R.R.L. holds the IRCM clinical research J-A DeSève Chair of Excellence. We thank Martine Goulet and the bariatric surgery team of Hôpital du Sacré-Coeur de Montréal for their great help and collaboration.

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    These authors contributed equally to the work.

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