Quantitative proteomic analysis of proteins and post‐translational modifications during the metaphase to anaphase transition after altered O‐GlcNAcylation

Abstract

Post‐translational modifications (PTMs) are essential in regulating the function, stability, and localization of proteins. One such modification is the O‐GlcNAc. O‐GlcNAc is the attachment of a single N‐acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. The modification is dynamically processed on proteins by the enzymes O‐GlcNAc transferase (O‐GlcNAc addition) or O‐GlcNAcase (O‐GlcNAc removal). Our objective is to explore how alterations in O‐GlcNAcylation alter several different types of PTM's during mitosis. We employed SILAC (Stable Isotope Labeling of Amino Acids in Cell culture) labeling to quantitate differences in cells with either an O‐GlcNAc transferase or O‐GlcNAcase gain of function. These samples were combined, digested, and fractionated to enrich for O‐GlcNAc, phosphate, acetyl, or ubiquitinated peptides. By mass spectrometry analysis, we have identified numerous proteins and PTMs altered by aberrant O‐GlcNAcylation. Multiple bioinformatic and systems biology tools has allowed a comprehensive analysis of the interplay between multiple post‐translational modifications providing valuable insight into how these modifications control cellular functions such as mitotic progression. NIH‐NHLBI contract N01 HV‐00239 (current CPC), S10 RR020946 (Thermo LTQ‐Orbitrap), and P41 RR010888 (Mass Spectrometry Resource) to C.E.C.