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Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a -3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases and proteomic pipelines.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress.

The article discusses the technique of shotgun/ bottom-up proteomics for the analysis of protein. It states that bottom-up process characterizes protein by analyzing the peptides released from the protein through proteolysis. It discusses several techniques related to shotgun proteomics which include two dimensional polyacrylamide gel electrophoresis (20-PAGE), chromatographic methods and post-translational modification (PTM) analysis by Mass Spectrometry.