Antibody based affinity capture LC-MS/MS in quantitative determination of proteins in biological matrices

Abstract

Determination of proteins in complex biological matrices has massive attention and is exploited in many different scientific disciplines. Routinely, proteins are determined using antibody based immuno-metric assays. Although these assays are easy to perform and widely used, interpretation of the results is challenging: cross reactivity, high dose hook-effect, presence of heterophile- or auto-antibodies give rise to false results, sometimes with dramatic consequences. In the quest for more robust assays a combination of antibody sample clean-up, tryptic digestion and mass spectrometric determination is gaining more attention. This review discusses the advantages of antibody based affinity capture and subsequent LC-MS/MS in protein analysis like less false results and possibilities like multiplexing and isoform differentiation. It also considers the interplay between the analytical, biological and biochemical factors, which still give rise to false results, even with mass spectrometry as the ultimate selective detection step. The intention of this review is to point out both strengths and weaknesses of antibody based affinity capture LC-MS/MS in quantitative determination of proteins in biological matrices.