Pavel Rehulka

Comparative analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure

Cadmium (Cd) is classified as a serious pollutant due to its high toxicity, high carcinogenicity, and widespread presence in the environment. Phytoremediation represents an effective low‐cost approach for removing pollutants from contaminated soils, and a crop with significant phytoremediation potential is flax. However, significant differences in Cd accumulation and tolerance were previously found among commercial flax cultivars. Notably, cv. Jitka showed substantially higher tolerance to elevated Cd levels in soil and plant tissues than cv. Tábor. Here, significant changes in the expression of 14 proteins (related to disease/defense, metabolism, protein destination and storage, signal transduction, energy and cell structure) were detected by image and mass spectrometric analysis of two‐dimensionally separated proteins extracted from Cd‐treated cell suspension cultures derived from these contrasting cultivars. Further, two proteins, ferritin and glutamine synthetase (a key enzyme in glutathione biosynthesis), were only up‐regulated by Cd in cv. Jitka, indicating that Cd tolerance mechanisms in this cultivar may include maintenance of low Cd levels at sensitive sites by ferritin and low‐molecular weight thiol peptides binding Cd. The identified changes could facilitate marker‐assisted breeding for Cd tolerance and the development of transgenic flax lines with enhanced Cd tolerance and accumulation capacities for phytoremediating Cd‐contaminated soils.

Analysis of Oxidized Phospholipids by MALDI Mass Spectrometry Using 6-Aza-2-thiothymine Together with Matrix Additives and Disposable Target Surfaces

6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stubiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) as reference compound for phospholipid analysis, and specific matrix additives was performed. Thereby, ATT turned out to be the reagent of choice for MALDI analysis of major biologically relevant OxPL classes (e.g., OxPC, OxPE, and OxPS) in positive and negative ionization mode. ATT used together with specific chaotropic reagents at low concentration (0.5-2 mM) acting as OxPL ionization enhancers revealed an excellent comatrix system for application with MALDI instrument types employing UV- and Nd:YAG laser systems (337 and 355 nm). Moreover, disposable MALDI targets surfaces with specific physicochemical properties (e.g., metallized glass or polymeric substrates) were revealed as superior over stainless steel in terms of reduced chemical background noise ( approximately 10-fold better S/N ratios), increased mass spectral reproducibility, and enhanced sensitivity (LOD approximately 250-500 fg on target). The combination of these parameters offers a significant advantage for highly sensitive OxPL profiling by MALDI-MS of biological samples (e.g., human plasma) at trace levels.

Influence of different proteomic protocols on degree of high-coverage identification of nonspecific lipid transfer protein 1 modified during malting

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.