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Modern mass spectrometry (MS)-based approaches are capable of identifying and quantifying thousands of proteins and phosphorylation events in a single biological experiment. Here we present a (phospho)proteomic workflow based on in-solution proteome digestion of samples labeled by stable isotope labeling by amino acids in cell culture (SILAC) and phosphopeptide enrichment using strong cation exchange (SCX) and TiO 2 chromatographies. These procedures are followed by high-accuracy MS measurement on an Orbitrap mass spectrometer and subsequent bioinformatic processing using MaxQuant software., (© 2017 Cold Spring Harbor Laboratory Press.)

Stable isotope labeling by amino acids in cell culture (SILAC) enables the relative quantification of protein amounts and posttranslational modifications in complex biological samples through the use of stable heavy isotope-labeled amino acids. Here we describe methods for the application of SILAC to fission yeast Schizosaccharomyces pombe using either labeled lysine or a combination of labeled lysine and labeled arginine. The latter approach is more complicated than the use of labeled lysine alone but may yield a more comprehensive (phospho)proteomic analysis. The protocol includes methods for construction of SILAC-compatible strains, growth of cultures in labeled medium, cell harvesting, and protein extraction., (© 2017 Cold Spring Harbor Laboratory Press.)

Shotgun proteomics combined with stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach to quantify proteins and posttranslational modifications across the entire proteome. SILAC technology in Schizosaccharomyces pombe must cope with the "arginine conversion problem," in which isotope-labeled arginine is converted to other amino acids. This can be circumvented by either using stable isotope-marked lysine only (as opposed to the more standard lysine/arginine double labeling) or using yeast genetics to create strains that only very inefficiently convert arginine. Both strategies have been used successfully in large-scale (phospho)proteomics projects in S. pombe Here we introduce methods for performing a typical SILAC-based experiment in fission yeast, including generation of SILAC-compatible strains, sample preparation, and measurement by mass spectrometry., (© 2017 Cold Spring Harbor Laboratory Press.)

Fission yeast shugoshin Sgo1 is meiosis specific and cooperates with protein phosphatase 2A to protect centromeric cohesin at meiosis I. The other shugoshin-like protein Sgo2, which requires the heterochromatin protein Swi6/HP1 for full viability, plays a crucial role for proper chromosome segregation at both mitosis and meiosis; however, the underlying mechanisms are totally elusive. We here demonstrate that, unlike Sgo1, Sgo2 is dispensable for centromeric protection of cohesin. Instead, Sgo2 interacts with Bir1/Survivin and promotes Aurora kinase complex localization to the pericentromeric region, to correct erroneous attachment of kinetochores and thereby enable tension-generating attachment. Forced localization of Bir1 to centromeres partly restored the defects of sgo2Delta. This newly identified interaction of shugoshin with Survivin is conserved between mitosis and meiosis and presumably across eukaryotes. We propose that ensuring bipolar attachment of kinetochores is the primary role of shugoshin and the role of cohesion protection might have codeveloped to facilitate this process.