Post-translational modifications (PTMs) collectively pertain to a phase in protein biosynthesis that involves changes in a polypeptide chain, resulting in a fully functional protein product.
The completion of the Human Genome Project has generated an extensive compilation of genotypic information, yet these sequences further undergo adaptations during translation, producing a wide variety of proteins that play specific roles in the normal physiology of the cell. Current proteomic efforts have thus focused on understanding how cellular activities are governed by proteins.
Proteomics, or global protein analysis, appears to be more challenging than genomic analysis. First, the isolation of intact proteins and the analysis of each amino acid within a polypeptide chain are often plagued by issues relating to its biochemical properties. Advances in instrumentation have identified mass spectrometry as the most promising technology in protein analysis.
Coupling this technique with computational tools has allowed scientists to further enhance the speed and accuracy of protein identification, characterization, and quantification. Also known as “next-generation proteomics,” this approach has been developed to complement the existing next-generation DNA sequencing technology.
The application of mass spec to large-scale PTM profiling of O-GlcNAcylation and phosphorylation proteins in the mitochondria as well as the elucidation of their important roles in the development of diabetes and heart failure has relied on the use of chemoenzymatic labeling for the enrichment of protein samples for better detection and analysis.
“The detection of O-GlcNAc modifications remains a bottleneck that restricts further research in this field,” explains Junfeng Ma, Ph.D., a postdoctoral research fellow at the laboratory of Gerald W. Hart, Ph.D., at the department of biological chemistry, Johns Hopkins University School of Medicine. The detection of O-GlcNAc modification can be done by classical biochemical assays (e.g., O-GlcNAc-specific antibodies) and by mass spec. However, mass spec is the only powerful and high-throughput tool for site mapping.
“The difficulties of unambiguous assignment of O-GlcNAc modification sites in proteins lie in two major aspects: 1) the glycosidic bonds are liable in gas phase and therefore the O-GlcNAc moiety is lost with the traditional collision-induced dissociation (CID) mass spectrometry; and 2) the O-GlcNAc modification is generally of rather low abundance, making it even more challenging to detect,” says Dr. Ma.
Dr. Ma and others tried to address these issues. After successful trials of several methods, they developed a more robust and reliable method that can be applied to diverse samples by more laboratories worldwide. Dr. Ma describes a newly refined method for the enrichment and identification of O-GlcNAc proteins.
For this method, 1) O-GlcNAc groups in proteins/peptides are tagged with azido sugars by using a mutant galactosyltransferase; 2) with the use of a multifunctional reagent, the tagged peptides are captured via the click chemistry on solid phase followed by mild release with chemical cleavage; 3) the released peptides are readily detected by electron transfer dissociation mass spec, which can keep the O-GlcNAc moiety intact and therefore is very useful for O-GlcNAc detection, as demonstrated by collaborator Weihan Wang, Ph.D., at the chemistry department, University of Virginia.
In comparison to previous methods (e.g., antibody-based enrichment and hydrophilic chromatography), the newly developed capture-and-release approach shows higher selectivity, specificity, and sensitivity. The applicability of this method has been tested with individual proteins, mitochondrial samples, and others. Of particular note is that for the first time, they have identified tens of O-GlcNAc proteins in mitochondria.
“By combining the new O-GlcNAc enrichment method and the well-established phosphopeptide enrichment methods, we are doing a larger-scale PTM profiling of O-GlcNAcylated and phosphorylated proteins in mitochondria, which might provide a novel insight for the elucidation of the etiology and development of diabetes and the related diabetic cardiovascular diseases,” Dr. Ma concludes.