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.

The Combination of Lectin Affinity Chromatography, Gel Electrophoresis and Mass Spectrometry in the Study of Plant Glycoproteome: Preliminary Insights

“…Previously, we dealt with a detailed analysis of the water-soluble proteome of barley grain in a different degree of malting with respect to non-enzymatic glycation [22][23][24][25][26]. In the present study, attention has been devoted to the plant proteins (especially from barley and Arabidopsis thaliana) that can possibly be glycosylated.…”

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

“…Signals at m/z equal to 7109, 9689, 9985 and 10279 Da correspond to ns-LTP2, ns-LTP1, ns-LTP1b, and ns-LTP1c [12]. The most intense ns-LTP1 signal in the malt spectrum was 9689 Da, indicating loss of the oxylipin modification of LTP1b (9985) during malting [12,9,13]. Identity of ns-LTP1 and ns-LTP2 was proved by MS/MS sequencing of proteins purified by reversed phase chromatography on a C18 column (gradient of 20-80% ACN/0.1% TFA in 25 min).…”

“…There were several attempts to develop relatively fast and simple method for protein identification and determination of glycation in malt and beer. So far, most of them considered an application of gel electrophoresis (GE), in-gel degradation, and mass spectrometry (MS) [9].…”

A Review: The Role of Barley Seed Pathogenesis-Related Proteins (PRs) in Beer Production

“…Ns-LTP1 can be used as a marker of glycation level during malting 12 . Different proteomic approaches (bottom-up and top-down), used for detailed characterization of ns-LTP1 modification, confirmed lipid-adduction and glycation 23 . Both glycated ns-LTP1 and ns-LTP1b were recovered during mashing (Table II) 81 .…”