PURPOSE: The objective of this study was to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay.

EXPERIMENTAL DESIGN: A 2D-LC-MS/MS-based SRM and PRM assay was developed for quantitative measurements of HSP90α in serum. Forty-three control sera were compared by SRM, PRM and ELISA following the manufacturer's instructions. Serum samples were trypsin-digested and fractionated by SCX chromatography prior to SRM and PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability and reproducibility of the SRM, PRM and ELISA were determined.

RESULTS: PRM data obtained by high-resolution mass spectrometry correlated better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, ELISA) were able to quantify HSP90α in serum at the ng/mL level, the use of PRM on a high-resolution mass spectrometer reduced variation and showed comparable sensitivity to immunoassay.

CONCLUSIONS AND CLINICAL RELEVANCE: Using fractionation, it is possible to measure ng/mL levels of HSP90α in a reproducible, selective and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders. This article is protected by copyright. All rights reserved.