Dataset Information

Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.

ABSTRACT: Chemical cross-linking of proteins combined with mass spectrometry provides an attractive and novel method for the analysis of native protein structures and protein complexes. Analysis of the data however is complex. Only a small number of cross-linked peptides are produced during sample preparation and must be identified against a background of more abundant native peptides. To facilitate the search and identification of cross-linked peptides, we have developed a novel software suite, named Hekate. Hekate is a suite of tools that address the challenges involved in analyzing protein cross-linking experiments when combined with mass spectrometry. The software is an integrated pipeline for the automation of the data analysis workflow and provides a novel scoring system based on principles of linear peptide analysis. In addition, it provides a tool for the visualization of identified cross-links using three-dimensional models, which is particularly useful when combining chemical cross-linking with other structural techniques. Hekate was validated by the comparative analysis of cytochrome c (bovine heart) against previously reported data. Further validation was carried out on known structural elements of DNA polymerase III, the catalytic ?-subunit of the Escherichia coli DNA replisome along with new insight into the previously uncharacterized C-terminal domain of the protein.

SUBMITTER: Holding AN

PROVIDER: S-EPMC3859183 | biostudies-other | 2013 Dec

REPOSITORIES: biostudies-other

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Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.

Holding Andrew N AN Lamers Meindert H MH Stephens Elaine E Skehel J Mark JM

Journal of proteome research 20131004 12

Chemical cross-linking of proteins combined with mass spectrometry provides an attractive and novel method for the analysis of native protein structures and protein complexes. Analysis of the data however is complex. Only a small number of cross-linked peptides are produced during sample preparation and must be identified against a background of more abundant native peptides. To facilitate the search and identification of cross-linked peptides, we have developed a novel software suite, named Hek ...[more]

PMID: 24010795

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Project description:Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3-6 monosaccharides attached to specific serine residues can be extended by 20-200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-022-00092-3.

Dataset Information

Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.

Publications

Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.

Similar Datasets

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