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Analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics.

ABSTRACT: A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. To meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins must be developed. Although the proteome wide examination of carbohydrate modification has become an area of keen interest, the intricacy of protein glycosylation has frustrated the progress of large-scale, systems oriented research on site-specific protein-glycan relationships. Indeed, the analytical obstacles in this area have been more instrumental in shaping the current glycoproteomic paradigm than have the diverse functional roles and ubiquitous nature of glycans. This report describes the ongoing development and analytically salient features of bead immobilized pronase for glycosylation site footprinting. The present work bears on the ultimate goal of providing analytical tools capable of addressing the diversity of protein glycosylation in a more comprehensive and efficient manner. In particular, this approach has been assessed with respect to reproducibility, sensitivity, and tolerance to sample complexity. The efficiency of pronase immobilization, attainable pronase loading density, and the corresponding effects on glycoprotein digestion rate were also evaluated. In addition to being highly reproducible, the immobilized enzymes retained a high degree of proteolytic activity after repeat usage for up to 6 weeks. This method also afforded a low level of chemical background and provided favorable levels of sensitivity with respect to traditional glycoproteomic strategies. Thus, the application of immobilized pronase shows potential to contribute to the advancement of more comprehensive glycoproteomic research methods that are capable of providing site-specific glycosylation and microheterogeneity information across many proteins.

SUBMITTER: Dodds ED

PROVIDER: S-EPMC2637306 | biostudies-other | 2009 Feb

REPOSITORIES: biostudies-other

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Analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics.

Dodds Eric D ED Seipert Richard R RR Clowers Brian H BH German J Bruce JB Lebrilla Carlito B CB

Journal of proteome research 20090201 2

A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. To meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins mus ...[more]

PMID: 19072223

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Project description:Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains - known as condensation domains - is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

Dataset Information

Analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics.

Publications

Analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics.

Similar Datasets

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