Total and isoform-specific quantitative assessment of circulating fibulin-1 using selected reaction monitoring MS and
Martin Overgaard1,2, Claudia Cangemi1, Martin L Jensen1
1Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.
Purpose:
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.
Experimental Design:
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 And Clinical Relevance:
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.
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