Broad Audience Title

Protein Post-Translational Modification Characterization

Scientific Title

On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry

By Ian Carrick
Biomedicine/Biosystems
iCons Year 4
2018
Executive Summary 

As post-translational modification (PTM) biomarkers become increasingly relevant in the field of biomedicine, the need for a quick and accurate method for characterizing protein modifications has become apparent. Of PTMs, glycosylation holds significant potential for use as a biomarker, due to its known modulation in many pathologies, including cancer, Alzheimer’s, immune disorders, and diabetes. The traditional analytical technique for measuring protein glycosylation is to enzymatically cleave the glycans from the protein, and detect the glycans using mass spectrometry. This “bottom-up” approach has associated drawbacks, including an increased risk of artifact introduction. A more favorable approach is to obtain information about a PTM by observing the mass distribution of the whole protein of interest. This “top-down” approach has limitations; large proteins with extensive heterogeneity cannot be resolved at the level required for PTM characterization. In the case of glycosylated proteins, extensive heterogeneity is common due to the enzymatic nature of the glycosylation process. Fortunately, many glycosylated proteins that are relevant as biomarkers consist of subunits connected by disulfide bonds. In this work, top-down characterization of the plasma protein haptoglobin is performed to measure its fucosylation levels. This novel method that is presented employs a cross-path reactive scheme, where the injection of protein into a size exclusion chromatography column is delayed relative to the injection of a reactive plug. This allows reactions (reduction of disulfides in this case) to be performed inside the column for a limited and controlled amount of time.

Problem Keywords 
PTM biomarker
cancer
diabetes
Scientific Keywords 
mass spectrometry
chromatography

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