Combination of Bottom-up 2D-LC-MS and Semi-top-down GelFree-LC-MS Enhances Coverage of Proteome and Low Molecular Weight Short Open Reading Frame Encoded Peptides of the Archaeon Methanosarcina mazei.

The recent discovery of an increasing number of small open reading frames (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. For biological and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. Consequently in the present study we evaluated analytical approaches that will allow for simultaneous analysis of widest parts of the proteome together with the predicted sORF.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation.

C-Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C-Termini by C-Terminomics.

The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the negatively charged C-termini with ethanolamine (EA) has been suggested to improve the detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study, the derivatization with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)guanidine (AG) leading to a positively charged C-terminus was investigated.

Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain.

Identification and relative quantification of phosphopeptides by a combination of multi-protease digestion and isobaric labeling.

Rationale: The identification and the determination of the extent of protein phosphorylation are major prerequisites for the comparative analysis of this important posttranslational modification of proteins in different biological situations. High sequence coverages and the availability of straightforward quantification methods are necessary to achieve these goals.

Methods: Phosphoproteins and non-phosphorylated analogues separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were digested using four different proteases (trypsin, chymotrypsin, elastase and GluC) and the digests were isobarically labeled using eight-plex iTRAQ.

Systems genomics evaluation of the SH-SY5Y neuroblastoma cell line as a model for Parkinson's disease.

Background: The human neuroblastoma cell line, SH-SY5Y, is a commonly used cell line in studies related to neurotoxicity, oxidative stress, and neurodegenerative diseases. Although this cell line is often used as a cellular model for Parkinson's disease, the relevance of this cellular model in the context of Parkinson's disease (PD) and other neurodegenerative diseases has not yet been systematically evaluated.

Results: We have used a systems genomics approach to characterize the SH-SY5Y cell line using whole-genome sequencing to determine the genetic content of the cell line and used transcriptomics and proteomics data to determine molecular correlations.

LC-MS based cleavage site profiling of the proteases ADAM10 and ADAM17 using proteome-derived peptide libraries.

A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity.

Paleoproteomic study of the Iceman's brain tissue.

The Tyrolean Iceman, a Copper-age ice mummy, is one of the best-studied human individuals. While the genome of the Iceman has largely been decoded, tissue-specific proteomes have not yet been investigated. We studied the proteome of two distinct brain samples using gel-based and liquid chromatography-mass spectrometry-based proteomics technologies together with a multiple-databases and -search algorithms-driven data-analysis approach.

Optimized fragmentation conditions for iTRAQ-labeled phosphopeptides.

Protein phosphorylation is an important post-translational modification that plays a regulatory role within numerous biological processes. The simultaneous identification, localization, and quantification of phosphorylated proteins is vital for understanding this dynamic control mechanism. The application of isobaric labeling strategies, for example, iTRAQ, for quantitative phosphopeptide analysis requires simultaneous monitoring of peptide backbone fragmentation, loss of phosphoryl moieties, and the cleavage of isobaric labeling reporter ions.