Abstract
Heparin and heparan sulfate are very large linear polysaccharides that undergo a complex variety of modifications and are known to play important roles in human development, cell-cell communication and disease. Sequencing of highly sulfated glycosaminoglycan oligosaccharides like heparin and heparan sulfate by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains challenging because of the presence of multiple isomeric sequences in a complex mixture of oligosaccharides, the difficulties in separation of these isomers, and the facile loss of sulfates in MS/MS. We have previously introduced a method for structural sequencing of heparin/heparan sulfate oligosaccharides involving chemical derivatizations that replace labile sulfates with stable acetyl groups. This chemical derivatization scheme allows the use of reversed phase LC for high-resolution separation and MS/MS for sequencing of isomeric heparan sulfate oligosaccharides. However, because of the large number of analytes present in complex mixtures of heparin/HS oligosaccharides, the resulting LC-MS/MS data sets are large and cannot be annotated with existing glycomics software because of the specifically designed chemical derivatization strategy. We have developed a tool, called GAG-ID, to automate the interpretation of derivatized heparin/heparan sulfate LC-MS/MS data based on a modified multivariate hypergeometric distribution to weight the annotation of more intense peaks. The software is tested on a LC-MS/MS data set collected from a mixture of 21 synthesized heparan sulfate tetrasaccharides. By testing the discrimination of scoring with this system, we show that stratifying peaks into different intensity classes benefits the discrimination of scoring, and GAG-ID is able to properly assign all 21 synthetic tetrasaccharides in a defined mixture from a single LC-MS/MS run.
Highlights
Heparin and heparan sulfate (HS)1 are involved in numerous physiological [1] and pathophysiological [2] processes, including cellular and organ development [3, 4], cancer [5, 6], and angiogenesis [7]
The ability to automatically annotate spectra with confidence from heparin/HS GAG mixture data sets was a primary goal for the development of GAG-ID
The theoretical GAG-DB was created and used to test a data set generated from LC-MS/MS analysis of a defined mixture of 21 synthetic, derivatized tetrasaccharides
Summary
GAG-ID: Heparan Sulfate (HS) and Heparin Glycosaminoglycan High-Throughput Identification Software*□S. We have developed a tool, called GAG-ID, to automate the interpretation of derivatized heparin/heparan sulfate LC-MS/MS data based on a modified multivariate hypergeometric distribution to weight the annotation of more intense peaks. Tabb and coworkers [31] have introduced an open-source program called MyriMatch, which uses a statistical model to score peptide matches and is based on multivariate hypergeometric distribution analysis This program highlights the limitation of existing database search algorithms that count matched peaks without differentiating them by intensity. Saad and Leary [34] refined this basic approach and introduced a program called heparin oligosaccharide sequencing tool (HOST) for automated sequencing using the results of tandem mass spectrometry for disaccharides produced by sequential enzyme digestion from the target oligosaccharide The use of such a method is limited in application to structurally homogeneous samples and requires digestion with several heparin lyases. Our GAG-ID software coupled with our previously published heparin/HS derivatization LCMS/MS method developed in our lab makes high-throughput sequencing of heparin/HS oligosaccharide mixtures possible
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
