Influence of different proteomic protocols on degree of high-coverage identification of nonspecific lipid transfer...

Both top-down (combining protein separation with MS analysis of intact proteins) and bottom-up (MS analysis of digested proteins) proteomic approaches were used for detailed characterization of nonspecific lipid transfer protein from barley malt. The aim was obtaining high-coverage of the primary structure of the proteins and the determination of PTMs such as lipid adduction and glycation. Here we present an influence of 15 proteomic protocols (differing in applied separation technique, enzyme and digestion procedure) on the extent of the coverage of the protein primary structure. The most successful protocols were in-gel digestion with trypsin of alkylated protein and in-solution digestions with trypsin or trypsin/chymotrypsin mixture of the nonalkylated protein. Totally, full sequence coverage based on the PMF and 85% sequence coverage based on the peptide fragmentation including PTMs was obtained.