ovalbumin and 2-5-dihydroxybenzoic-acid

The use of a novel 2,5-dihydroxybenzoic acid/N,N-dimethylaniline (DHB/DMA) matrix-assisted laser desorption/ionization (MALDI) matrix for detection and quantitative analysis of native N-linked oligosaccharides was investigated in this study. Substantial improvements in sensitivity were observed relative to the signals obtained with a traditional DHB matrix. Moreover, the morphology of the matrix crystal layer was very uniform, unlike that of DHB. This resulted in highly homogeneous sample distribution throughout the spot, allowing reproducible and consistent mass spectra to be obtained without spot-to-spot variations in signal. Here, we also demonstrate an approach for performing sensitive and accurate quantitative analysis of native N-linked glycans with this novel matrix using an internal standard method.

Topics: Aniline Compounds; Animals; Chickens; Gentisates; Oligosaccharides; Ovalbumin; Polysaccharides; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.

Topics: Amino Acid Sequence; Animals; Carbonic Anhydrases; Caseins; Cattle; Chickens; Gentisates; Molecular Sequence Data; Ovalbumin; Phosphopeptides; Serum Albumin; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Titanium