title: 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.
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.
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Marie-Soleil
authors.initials: MS
authors.lastname: Gauthier
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Joëlle R
authors.initials: JR
authors.lastname: Pérusse
authors.affiliation: King Abdulaziz University, Jeddah, Saudi Arabia; McGill University, Montréal, QC H3A 1A1, Canada.
authors.forename: Zuhier
authors.initials: Z
authors.lastname: Awan
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Annie
authors.initials: A
authors.lastname: Bouchard
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Tessier
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Josée
authors.initials: J
authors.lastname: Champagne
authors.affiliation: Thermo Fisher Scientific, Biomarker Research Initiatives in Mass Spectrometry Center, 790 Memorial Drive, Cambridge, MA 02139, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Krastins
authors.affiliation: Thermo Fisher Scientific, Biomarker Research Initiatives in Mass Spectrometry Center, 790 Memorial Drive, Cambridge, MA 02139, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Byram
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Katherine
authors.initials: K
authors.lastname: Chabot
authors.affiliation: General Surgery Department, Hôpital du Sacré-Coeur de Montréal, Montréal, QC H4J 1C5, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Garneau
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Rémi
authors.initials: R
authors.lastname: Rabasa-Lhoret
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Denis
authors.initials: D
authors.lastname: Faubert
authors.affiliation: Thermo Fisher Scientific, Biomarker Research Initiatives in Mass Spectrometry Center, 790 Memorial Drive, Cambridge, MA 02139, USA.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Lopez
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada.
authors.forename: Nabil G
authors.initials: NG
authors.lastname: Seidah
authors.affiliation: Institut de Recherches Cliniques de Montréal (affiliated to the Université de Montréal), 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada; Department of Biochemistry, Université de Montréal, Montréal, QC H3T 1J4, Canada. Electronic address: benoit.coulombe@ircm.qc.ca.
authors.forename: Benoit
authors.initials: B
authors.lastname: Coulombe
mesh_terms: D000293:Adolescent; D000328:Adult; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007333:Insulin Resistance; D008077:Lipoproteins, LDL / Q000097:blood; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D009765:Obesity / Q000097:blood; D010641:Phenotype; D011247:Pregnancy; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000097:blood* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000097:blood* / Q000378:metabolism; D014280:Triglycerides / Q000097:blood; D055815:Young Adult
chemical_list: D008077:Lipoproteins, LDL; D014280:Triglycerides; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: Insulin resistance; LDL cholesterol; Mass spectrometric immunoassay (MSIA); PCSK9; Phosphorylation; Selected reaction monitoring (SRM)