title: Total and isoform-specific quantitative assessment of circulating fibulin-1 using selected reaction monitoring MS and time-resolved immunofluorometry.
abstract: OBJECTIVE
  Targeted proteomics using SRM-MS combined with stable-isotope dilution has emerged as a promising quantitative technique for the study of circulating protein biomarkers. The purpose of this study was to develop and characterize robust quantitative assays for the emerging cardiovascular biomarker fibulin-1 and its circulating isoforms in human plasma.
  
  
  METHODS
  We used bioinformatics analysis to predict total and isoform-specific tryptic peptides for absolute quantitation using SRM-MS. Fibulin-1 was quantitated in plasma by nanoflow-LC-SRM-MS in undepleted plasma and time-resolved immunofluorometric assay (TRIFMA). Both methods were validated and compared to a commercial ELISA (CircuLex). Molecular size determination was performed under native conditions by SEC analysis coupled to SRM-MS and TRIFMA.
  
  
  RESULTS
  Absolute quantitation of total fibulin-1, isoforms -1C, and -1D was performed by SRM-MS. Fibulin-1C was the most abundant isoform in plasma. Circulating fibulin-1 isoforms were homo -or hetero multimeric complexes (range 318-364 kDa). Good correlation was obtained between SRM-MS and TRIFMA but not CircuLex.
  
  
  CONCLUSIONS
  For biomarker studies using smaller cohorts, SRM-MS provides an alternative measure of total and specific fibulin-1 isoforms in undepleted plasma. For larger cohorts TRIFMA provides a faster platform for fibulin-1 quantitation in plasma. While the correlation between these methods was acceptable, low correlation was obtained between the commercial CircuLex assay and SRM-MS or TRIFMA.
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.|Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Martin
authors.identifier: http://orcid.org/0000-0003-2277-590X
authors.initials: M
authors.lastname: Overgaard
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cangemi
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Jensen
authors.affiliation: Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
authors.forename: William S
authors.initials: WS
authors.lastname: Argraves
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.|Centre for Individualized Medicine in Arterial Diseases, Odense University Hospital, Odense, Denmark.|Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Rasmussen
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000737:chemistry; D002850:Chromatography, Gel; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D013997:Time Factors
chemical_list: D002135:Calcium-Binding Proteins; D010455:Peptides; D020033:Protein Isoforms; C060820:fibulin
keywords: Fibulin-1; Immunoassay; Isoform; Plasma biomarker; SRM