MALDI versus ESI: The Impact of the Ion Source on Peptide Identification.

For mass spectrometry-based proteomic analyses, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are the commonly used ionization techniques. To investigate the influence of the ion source on peptide detection in large-scale proteomics, an optimized GeLC/MS workflow was developed and applied either with ESI/MS or with MALDI/MS for the proteomic analysis of different human cell lines of pancreatic origin. Statistical analysis of the resulting data set with more than 72 000 peptides emphasized the complementary character of the two methods, as the percentage of peptides identified with both approaches was as low as 39%. Significant differences between the resulting peptide sets were observed with respect to amino acid composition, charge-related parameters, hydrophobicity, and modifications of the detected peptides and could be linked to factors governing the respective ion yields in ESI and MALDI.

Ion source-dependent performance of 4-vinylpyridine, iodoacetamide, and N-maleoyl derivatives for the detection of cysteine-containing peptides in complex proteomics.

Cysteine is unique among the proteinogenic amino acids due to its ability to form disulfide bonds. While this property is of vital importance for protein structures and biological processes, it causes difficulties for the mass spectrometric identification of cysteine-containing peptides. A common approach to overcome these problems in bottom-up proteomics is the reduction and covalent modification of sulfhydryl groups prior to enzymatic digestion. In this study, established alkylating agents and N-maleoyl amino acids with variable hydrophobicity were characterized with respect to a variety of relevant parameters and subsequently evaluated in a large-scale analysis using different ion sources. Depending on the compound, the ion source had a profound impact on the relative and absolute identification of cysteine-containing peptides. The best results were obtained by derivatization of the cysteine residues with 4-vinylpyridine and subsequent matrix-assisted laser desorption ionization (MALDI). Modification with 4-vinylpyridine increased the number of cysteine-containing peptides identified with any other compound using LC-MALDI/MS at least by a factor of 2. This experimental observation is mirrored by differences in the gas-phase basicities, which were computed for methyl thiolate derivatives of the compounds using density functional theory. With electrospray ionization (ESI), complementary use of reagents from three different compound classes, e.g., iodoacetamide, 4-vinylpyridine, and N-maleoyl beta-alanine, was beneficial compared to the application of a single reagent.