No abstract

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.

Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using 14 N-or 15 N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation R Met.Lab. ‫؍‬ 0.98R DiGE with r 2 ‫؍‬ 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss ( The proteome is generally defined as the total protein complement of a genome present in cells and/or tissue (1). Through splice variation and/or post-translational modifications the proteome is several orders of magnitude more complex than the genome. One extra factor that adds to the relative complexity of the proteome is that protein abundance varies over time, either as a reaction to changes in the environment or during development. To understand these dynamic processes, which may lead to indications why e.g. "healthy" cells are different from "diseased" or "stressed" cells or how cells change during differentiation, it is not only important to identify which proteins are involved but also to measure their differential expression levels. It is especially this latter notion that makes quantitative protein profiling an essential part of proteomics, which requires technologies that accurately, reproducibly, and comprehensively quantify the protein content in biological samples (2-4).Traditionally, and probably still, the most frequently used method to investigate differential protein abundances in large scale proteomic experiments on protein mixtures from cellular extracts or tissue is by two-dimensional (2D) 1 gel electrophoresis (5-9). In such experiments proteins are separated by their pI and molecular weight on a 2D gel and subsequently stained for visualization. ...

Modification through beta-elimination has proven to be a reliable first step in the approach for enrichment of serine/threonine-phopshorylated (Ser-/Thr) peptides. However, under harsh basic conditions, Ser-/Thr-glycosylated peptides are susceptible to beta-elimination as well. Therefore, we have optimized these conditions to achieve a beta-elimination that is highly selective for phosphorylated peptides. This is the first report of selective beta-elimination and enrichment of phosphorylated peptides in the presence of glycosylated peptides.

A DNA double-strand break (DSB) is highly cytotoxic; it emerges as the type of DNA damage that most severely affects the genomic integrity of the cell. It is essential that DNA DSBs are recognized and repaired efficiently, in particular, prior to mitosis, to prevent genomic instability and eventually, the development of cancer. To assess the pathways that are induced on DNA DSBs, 14 human lymphoblastoid cell lines were challenged with bleomycin for 30 and 240 minutes to establish the fast and more prolonged response, respectively. The proteomes of 14 lymphoblastoid cell lines were investigated to account for the variation among individuals. The primary DNA DSB response was expected to occur within the nucleus; therefore, the nuclear extracts were considered. Differential analysis was done using two-dimensional difference in gel electrophoresis; paired ANOVA statistics were used to recognize significant changes in time. Many proteins whose nuclear levels changed statistically significantly showed a fast response, i.e., within 30 minutes after bleomycin challenge. A significant number of these proteins could be assigned to known DNA DSB response processes, such as sensing DSBs (Ku70), DNA repair through effectors (high-mobility group protein 1), or cell cycle arrest at the G 2 -M phase checkpoint (14-3-3 Z). Interestingly, the nuclear levels of all three proteins in the INHAT complex were reduced after 30 minutes of bleomycin challenge, suggesting that this complex may have a role in changing the chromatin structure, allowing the DNA repair enzymes to gain access to the DNA lesions.