Enhanced on-plate digestion of proteins using a MALDI-digestion chamber

Elucidation of chemical modifier reactivity towards peptides and proteins and the analysis of specific fragmentation by matrix‐assisted laser desorption/ionization collision‐induced dissociation tandem mass spectrometry

“…The amino acids cysteine, histidine, lysine and serine were selected because of their pronounced nucleophilicity resulting in a higher chance for modification by Michael‐like acceptors compared to other amino acids. After the incubation on the MALDI target for 1 h at 37°C in a humidity chamber according to Rühl et al MALDI‐MS analysis was performed and the spectra were analyzed for the particular Michael‐like chemical modification product masses or the masses of the chemical modification after oxidation of an ortho‐ or paraquinone, respectively. In this screening experiment chemical modifications were found for all compounds at different amino acid residues.…”

“…Afterwards, matrix‐analyte cocrystals were washed with 5% chilled formic acid and recrystallized with matrix solvent (70% ACN, 0.1% TFA). For more details, see Rühl et al…”

Detergent‐assisted sample preparation for MALDI‐MS: Investigation of octylglucoside and docecylmaltoside for matrix crystallization, on‐plate digestion, and trypsin activity

“…Prior to all experiments, the 384‐well stainless steel target (Thermo Fisher Scientific, Bremen, Germany) was cleaned (for detailed cleaning procedure, see supporting information section 1). The digestion of proteins was performed directly on the MALDI‐MS target according to Rühl et al after detergent solution was pipetted and dried on the MALDI‐MS target. The proteins were dissolved in 50 mM ammoniumbicarbonate to a protein concentration of 1 μM.…”

“…In this study, we investigated 2 commonly used nonionic detergents for biochemical approaches: n‐octylglucoside and laurylmaltoside. Octylglucoside has been used in previous studies for on‐plate digestion and was already described by Chait et al to improve MALDI‐MS analysis. Laurylmaltoside was chosen because of the lower critical micelle concentration value and the expected higher potency to improve lipophilic peptide detection.…”

“…Trypsin activity was investigated to detect the effects of n‐octylglucoside and laurylmaltoside on the enzyme itself, and differences between detergent‐assisted and nonassisted on‐plate digestion were investigated.…”