Identification of the SELDI ProteinChip Human Serum Retentate by Microcapillary Liquid Chromatography-Tandem Mass Spectrometry

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has
been widely applied for conducting biomarker research with the goal of discovering patterns of proteins
and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers
of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein
profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have
generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the
features that underpin the diagnostic potential of the mass spectra is still largely unexplored. A detailed
investigation has been undertaken to identify the compliment of serum proteins that bind to the
commonly used weak cation exchange (WCX-2) SELDI protein chip. Following incubation and washing
of a standard serum sample on the WCX-2 sorbent, proteins were harvested, digested with trypsin,
fractionated by strong cation exchange liquid chromatography (LC), and subsequently analyzed by
microcapillary reversed-phase LC coupled online with an ion-trap mass spectrometer. This analysis
resulted in the identification of 383 unique proteins in the WCX-2 serum retentate. Among the proteins
identified, 50 (13%) are documented clinical biomarkers with 36 of these (72%) identified from multiple
peptides.
Keywords: SELDI • biomarker • serum proteomics • multidimensional fractionation • mass spectrometry