To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.
Keywords: Digestion; ISET; Immunoaffinity; MALDI; MRM; MS; PSA; Proteomics; RP; SPE; SPR; integrated selective enrichment target; mass spectrometry; matrix-assisted laser desorption/ionization; multiple reaction monitoring; prostate specific antigen; reverse phase; solid-phase extraction; surface plasmon resonance.
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