Integrated sample preparation methodology for proteomics: analysis of native proteins.

An innovative sample preparation strategy is reported for protein identification in complex mixtures based on integration of affinity chromatographic selection and accelerated trypsin digestion using a continuous flow immobilized enzyme reactor (cf-IMER). Affinity selected glycoproteins were released to a cf-IMER column which converted native proteins to peptides in 5 min at elevated temperature. Digestion with the cf-IMER was compared to the traditional 16 h solution-based trypsin digestion of reduced and alkylated proteins. With immobilized antibody selection of Lewis x (Lex) glycan bearing glycoproteins from plasma, 66 proteins were identified in total with the two methods while approximately 1/3 of the total proteins and peptides were only observed with the cf-IMER. This suggests that proteomics based on protein identification by reduction and alkylation with solution-based trypsin digestion alone may not be identifying large numbers of proteins or peptides present at detectable levels in samples. Fur...