proteomics applicationsContinuing our series on the 14 articles on proteomics research published in a 30-day span in Nature and Nature Methods magazine. If you are wondering why this series, do read the first blog post on this subject.

Here are the next five articles from Christian Ravnsborg’s list. Happy reading!

Article Title:Mining the O-glycoproteome using zinc-finger nuclease–glycoengineered SimpleCell lines

Synopsis: In this article, the authors describe using Zinc-finger nuclease (ZFN) gene targeting of COSMC to glycoengineer stable human cell lines with simple O-glycosylation displaying only truncated Tn and STn O-glycans, called SimpleCell lines. The SimpleCell lines was used to interrogate the O-glycoproteome of human cells using a modified LWAC lectin chromatography and nLC-MS/MS strategy, and uncover many O-glycoproteins and glycosylation sites including a previously unidentified linkage to tyrosine residues. The authors identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The importance of ETD for glycopeptide work is highlighted as well as the HR/AM needed for detection of glycan oxonium ions.

Article Title:The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements

Synopsis: This article describes how the authors, in order to investigate the role of Piwi-catalysed endonucleolytic activity, engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the MiliDAH and Miwi2DAH alleles, respectively. The analysis of Mili-bound piRNAs from homozygous MiliDAH fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. MS analysis of Mili complexes from wild type and MiliDAH P10 testis helped understand some of these process.

Article Title:Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

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Synopsis: The authors describe routes of multiphosphorylation events that are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1. Mass spectrometry (MS) was used in this study to asses quantitative determination of Cks1-dependent phosphorylation at T48.

Article Title:Metabolic priming by a secreted fungal effector

Synopsis: In this article, the authors demonstrate the secretion of Cmu1 during plant colonization by proteome analysis of apoplastic fluids isolated after infection of maize with a mixture of compatible U. maydis strains.

Article Title:Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase

Synopsis: This article describes use mass spectrometry to measure the initial mass of the wild-type THI4p (wtTHI4p), recombinantly expressed in Escherichia coli. This showed that the mass was 34 Da lower than the calculated mass of the protein. The active site mutants ofTHI4p8, which did not co-purify with any bound metabolites and did not show any activity, were unmodified, indicating that the 34 Da mass loss was in some way related to the catalytic activity of the protein. To localize the site of this modification, chymotrypsin digestion of modified wtTHI4p was carried out, followed by MALDI and FT-MS analysis of the peptide fragments.

Look for the next blog post listing the last of these great articles. Also, do tell us if you have read any others in the comments field below.