title: Influence of different proteomic protocols on degree of high-coverage identification of nonspecific lipid transfer protein 1 modified during malting.
abstract: 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.
authors.affiliation: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
authors.forename: Josef
authors.initials: J
authors.lastname: Chmelik
authors.forename: Jitka
authors.initials: J
authors.lastname: Zidkova
authors.forename: Pavel
authors.initials: P
authors.lastname: Rehulka
authors.forename: Inga
authors.initials: I
authors.lastname: Petry-Podgorska
authors.forename: Janette
authors.initials: J
authors.lastname: Bobalova
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry; D002523:Edible Grain / Q000737:chemistry; D050556:Fatty Acid-Binding Proteins; D001467:Hordeum; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; C422456:Ltp1 protein, barley; D010940:Plant Proteins; D002918:Chymotrypsin; D014357:Trypsin