Asbestos exposure is known to cause lung cancer and mesothelioma. The exceptionally long latency periods of most asbestos-related diseases have hampered its preventative treatment. New biomarkers are needed to detect asbestos exposure at an earlier stage and to individualize treatment. Fiblin-3 was recently reported as a new potential biomarker for pleural malignant mesothelioma. However, controversy results were reported due to unsatifactory bioanalytical methodology or biological variability. In this study, we developed an immunoprecipitation approach coupled with nanoLC-high resolution mass spectrometry (NanoLC-HRMS) method for quantifying fibulin-3 in human plasma as a biomarker for asbestos exposure and evaluated its prognostic value.<br/>Sheep Anti-fibulin-3 polyclonal antibody was selected out of five antibodies as it provides the highest specificity and pull-down efficiency. Dimethyl pimelimidate (DMP) was used to cross-link the antibody to protein A/G beads which enable fewer antibody contamination and less matrix effect on MS. Recombinant fibulin-3 was expressed from HEK293 cells using stable isotope labeling by amino acids in cell culture (SILAC) strategy, then it was spiked into plasma samples at initial step of sample preparation as an internal standard. Three signature peptides were selected from native fibulin-3 and SILAC-labeled fibulin-3. Parallel reaction monitoring (PRM) was used on Q Exactive HF (Thermo) for providing high selectivity and high sensitivity. Absolute quantification was based on the ratio of endogenous protein and SILAC-labeled protein. The low limit of quantification of current method reaches to attomole level in human plasma. This improved sensitivity and specificity was obtained by antibody-based immunoprecipitation step, as well as the use of HRMS under PRM mode. The enhanced sensitivity and specificity offered by the current method will allow for a more complete analysis of fibulin-3 and fibulin family, shedding light on previously unknown mechanisms of asbestos exposure.<br/>

Absolute quantification of plasma fibulin-3 as a biomarker for asbestos exposure by immunoprecipitation-high resolution mass spectrometry

254th National Meeting (2017)

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