Total and isoform‐specific quantitative assessment of circulating fibulin‐1 using selected reaction monitoring MS and time‐resolved immunofluorometry. Issue 7 (12th January 2015)

• Record Type: Journal Article
• Authors: Overgaard, Martin; Cangemi, Claudia; Jensen, Martin L.; Argraves, William S.; Rasmussen, Lars M.; Cutler, Paul; Voshol, Hans
• Is Part Of: Proteomics. Volume 9:Issue 7/8(2015)
• Page Start: 767
• Page End: 775
• Publication Date: 2015-01-12
• Subjects: Proteomics -- Periodicals; 572.605
• DOI: 10.1002/prca.201400070
• Languages: English
• ISSNs: 1862-8346

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="prca1589-sec-0010" sec-type="section"> <title>Purpose</title> <p>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.</p> </sec> <sec id="prca1589-sec-0020" sec-type="section"> <title>Experimental design</title> <p>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.</p> </sec> <sec id="prca1589-sec-0030" sec-type="section"> <title>Results</title> <p>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.</p> </sec> <sec id="prca1589-sec-0040" sec-type="section"> <title>Conclusions and clinical relevance</title> <p>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.</p> </sec> </abstract>