Leading the Way in Life Science Technologies

GEN Exclusives

More »

Feature Articles

More »
May 15, 2013 (Vol. 33, No. 10)

Mass Spec Provides Peek into PTMs

  • 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.

  • Glycosylation

    Click Image To Enlarge +
    A frozen human prostate tissue slice (10 µm; distal nontumor) was digested with protein N-glycanaseF to release N-glycans: Expression profiles of two representative released N-glycans specific to stroma (m/z 1851.055) or glandular regions (m/z 1906.119) are shown individually or as an overlay in comparison to an H&E stain of an adjacent prostate tissue slice. [MUSC]

    An on-tissue mass spec imaging method has facilitated analysis of glycosylation PTMs associated with different cancers for E. Ellen Jones, Ph.D., a postdoctoral fellow in the laboratory of Richard Drake, Ph.D., professor and director of the Medical University of South Carolina (MUSC) Proteomics Center, and her colleagues.

    Through a collaboration with Anand Mehta, Ph.D., of the Drexel Institute for Biotechnology and Virology Research, and Thomas Powers, a graduate student at MUSC, they have extensively characterized the precise glycan structures and sites of glycosylation.

    “This new mass spec-based glycan imaging approach combined with on-tissue protein N-glycanase F (PNGaseF) digestion allows us to spatially profile released N-linked glycans in their local microenvironment. The method has been designed to facilitate the tissue analysis of the cell surface glycan changes that occur with cancer progression and other diseases, while maintaining pathology-compatible preparation workflows,” says Dr. Jones.

    She further explains that matrix-assisted laser desorption/ionization mass spec imaging (MALDI-MSI) has primarily been utilized to spatially profile proteins, lipids, drug, and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis.

    “Using this basic approach, global snapshots of major cellular N-linked glycoforms are obtained, including their tissue localization and distribution, structure, and relative concentrations. Depending on the tissue type used, generally 20–30 N-glycan species are detected simultaneously.

    “The method has been applied to multiple frozen tissues, with emphasis on human prostate cancer, renal cancer, and different therapeutic mouse xenograft cancer tissues. Our initial data suggests that this method can be used to identify multiple N-glycan species reflective of the many changes that occur during the transition of organ-confined tumors to the metastatic phenotype, linked directly to histopathology data,” discusses Dr. Jones.

    She also explains that off-tissue extraction of glycans from similarly processed tissues, permethylation, and further mass spectrometry analysis has confirmed these structural designations.

    “The glycan profiles are also readily applied to MALDI-MSI imaging data of proteins and lipids in the same tissues, moving toward a more complete biomolecular spatial profile of targeted tissues. These translatable workflows using MALDI-MSI and on-tissue PNGaseF digestions are in continued development, and can readily be applied to any tissue type of interest,” mentions Dr. Jones.

    Efforts are also ongoing to use the mass of the identified glycans to identify the specific glycoproteins that were carrying the glycans using high-resolution glycopeptide analysis approaches on a Thermo Scientific Orbitrap Elite mass spectrometer.

  • Data and Algorithms

    In terms of using algorithms and software in investigating PTMs, several scientists have embarked on different approaches in capturing as much information across various tissues and conditions. Nuno Bandeira, Ph.D., assistant professor at the Skaggs School of Pharmacy and Pharmaceutical Sciences and the Department of Computer Science and Engineering at UC San Diego, focuses his research on the premise that various PTMs generate characteristic signatures in mass spectrometry data.

Related content

Be sure to take the GEN Poll

Cancer vs. Zika: What Worries You Most?

While Zika continues to garner a lot of news coverage, a Mayo Clinic survey reveals that Americans believe the country’s most significant healthcare challenge is cancer. Compared to other diseases, does the possibility of developing cancer worry you the most?

More »