Orbitrap Illustration

PTM Phosphorylation



Nearly all proteins undergo chemical modifications after translation. These post-translational modifications (PTMs) play crucial roles in functional proteomics, regulating the protein structure, activity, and expression. PTMs regulate interaction with cellular molecules such as nucleic acids, lipids and cofactors, as well as other proteins. PTMs can occur at any moment in the "life cycle" of a protein, influencing their biological function in processes such as initiating catalytic activity, governing protein-protein interactions, or causing protein degradation. Glycosylation and phosphorylation are of particular interest to researchers because they are critical pathways for signaling, activation, and often give insight into disease states.

Analysis of PTMs by mass spectrometry using multiple fragmentation techniques yields the most comprehensive structural characterization of modified proteins. Here we describe useful workflows for analysis of glycosylated and phosphorylated proteins.

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Workflow Overview for Phosphorylation


Reversible protein phosphorylation occurring on serine, threonine, or tyrosine residues is one of the most important and most-studied PTMs. Phosphorylation plays a central role in regulating many cellular processes including cell cycle, growth and apoptosis, as well as participating in signal transduction pathways. Given the influence that phosphorylation has on biological processes, a huge emphasis has been placed on understanding the biological role of protein phosphorylation in the context of human disease. Using sample enrichment followed by MS analysis with complementary fragmentation techniques CID, HCD and ETD, sensitive and conclusive structural elucidation of phosphorylation sites can be achieved.









Literature Highlights


Large-scale phosphorylation analysis of mouse liver

Villén J, Beausoleil SA, et al.
Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1488-93.
 

Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

Beli P, Lukashchuk N, et al.
Mol Cell. 2012 Apr 27;46(2):212-25.