Epitope Fishing in Digested Biological Samples

Immunocapture LC-MS/MS is a promising technique to ensure high sensitivity and selectivity of low-abundant protein biomarkers. For this purpose, the use of monoclonal antibodies (mAb) is especially attractive as they are renewable reagents that can be standardized. In this article we investigated the possibility of using mAbs developed against intact proteins to capture proteotypic epitope peptides. Three mAbs were tested, and all selectively extracted proteotypic epitope peptides from a complex sample. Compared to intact protein extraction, this concept which we call epitope fishing provided cleaner extracts, which further improved the sensitivity. Analysis of three patient samples demonstrated that epitope fishing can be used for the determination of different endogenous protein levels.

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 14.5px Arial}

Human serum from healthy subjects was obtained from Oslo University Hospital, Ullevål (Oslo, Norway), and serum samples from cancer patients were supplied by the Norwegian Radium Hospital, Oslo University Hospital. All serum samples were stored at −30 °C. The use of patient samples for our research purposes was approved by the Norwegian Regional Committee for Medical Research Ethics (REK, http://helseforskning.etikkom.no). Informed consent was obtained from all subjects. Methods used to analyze all serum samples were in accordance with relevant guidelines and regulations.