Mapping Post-Translational Modifications

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Tuesday 08/06/2013

The Proteomics Facility is frequently asked to locate sites of protein post-translational modifications.  When purified proteins can be isolated (e.g. affinity purification, in-vitro kinase experiments) peptide mapping is often utilized.  A protease, typically trypsin, is used to digest the modified protein into peptides for analysis by mass spectrometry.  The identified peptides then produce a “map” of the sequence coverage and hopefully the map is extensive enough to locate sites of modifications.  Depending on the modified protein’s sequence, some of the proteolytic peptides may not be of suitable size for mass spectrometry. 

​Using a combination of proteases to perform serial digestions of proteins enhances the peptide mapping sequence coverage when a modified protein’s sequence warrants it (based on theoretical protease digestions).  Typically trypsin is used first and the resulting digestion mixture is split in half.  One half is analyzed by mass spectrometry while the other half is further digested with a second protease (Glu-C, chymotrypsin, thermolysin) and analyzed by mass spectrometry.  The two mass spectrometry data files are analyzed using automated database searching to generate the peptide map.  The dual protease digestion format dramatically increases the sequence coverage and it can increase the number of modifications identified compared with a single protease.  To date we have used this methodology to extensively map sites of phosphorylation, ubiquitinylation, acetylation, and methylation.

Precise localization of phosphorylation sites can be particularly challenging since multiple serine, threonine, and tyrosine residues can exist on a single peptide.  To unambiguously locate a site of phosphorylation, tandem mass spectrometry fragment ions must be identified that uniquely assign a phosphate group to a specific site.  Sufficient fragment ions may not be present in the tandem mass spectrum to precisely locate the phosphosite and automated database search algorithms are not optimized for phosphosite localization.  To address the phosphosite localization issue, the facility has started to use software called PhosphoRS1 which uses tandem mass spectra data along with peptide sequences to calculate site probabilities for all potential phosphorylation sites.  This software provides a more thorough analysis of phosphosite localization than solely using data search algorithms.  Ultimately, users are provided with information on which phosphorylation sites were localized and at what degree of certainty, and equally important which phosphorylation sites were not localized due to insufficient mass spectral data.

J. Proteome Res. 10:5354-62 (2011).

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Rajesh K. Uthamanthil, DVM, PhD, DACLAM

Associate Vice President, Shared Resources