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. 2018 Jul;17(7):1448-1456.
doi: 10.1074/mcp.RA118.000590. Epub 2018 Apr 3.

Software for Peak Finding and Elemental Composition Assignment for Glycosaminoglycan Tandem Mass Spectra

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Free PMC article

Software for Peak Finding and Elemental Composition Assignment for Glycosaminoglycan Tandem Mass Spectra

John D Hogan et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Glycosaminoglycans (GAGs) covalently linked to proteoglycans (PGs) are characterized by repeating disaccharide units and variable sulfation patterns along the chain. GAG length and sulfation patterns impact disease etiology, cellular signaling, and structural support for cells. We and others have demonstrated the usefulness of tandem mass spectrometry (MS2) for assigning the structures of GAG saccharides; however, manual interpretation of tandem mass spectra is time-consuming, so computational methods must be employed. In the proteomics domain, the identification of monoisotopic peaks and charge states relies on algorithms that use averagine, or the average building block of the compound class being analyzed. Although these methods perform well for protein and peptide spectra, they perform poorly on GAG tandem mass spectra, because a single average building block does not characterize the variable sulfation of GAG disaccharide units. In addition, it is necessary to assign product ion isotope patterns to interpret the tandem mass spectra of GAG saccharides. To address these problems, we developed GAGfinder, the first tandem mass spectrum peak finding algorithm developed specifically for GAGs. We define peak finding as assigning experimental isotopic peaks directly to a given product ion composition, as opposed to deconvolution or peak picking, which are terms more accurately describing the existing methods previously mentioned. GAGfinder is a targeted, brute force approach to spectrum analysis that uses precursor composition information to generate all theoretical fragments. GAGfinder also performs peak isotope composition annotation, which is typically a subsequent step for averagine-based methods. Data are available via ProteomeXchange with identifier PXD009101.

Keywords: Bioinformatics; Bioinformatics software; Glycomics; Glycosylation; Mass Spectrometry; glycosaminoglycan; heparan sulfate; peak finding.

Figures

Fig. 1.
Fig. 1.
Comparison of expected isotopic distributions for oligosaccharides with varying sulfation. A, Expected isotopic distribution of non-sulfated N-acetylglucosamine (GlcNAc) compared with 3,6-O-sulfated, N-sulfated glucosamine (GlcNS3S6S). B, Expected isotopic distribution of a non-sulfated octasaccharide with acetyl groups at all four N positions compared with a hexadecasulfated octasaccharide with sulfate groups at every possible position. Notice the higher intensity at the A+2 peak for each fully sulfated oligosaccharide; for the octasaccharide, the A+2 peak has the highest intensity, making monoisotopic peak detection more difficult. Intensity is relative to the total intensity for the whole isotopic distribution. Key for octasaccharide: [ΔHexA, HexA, GlcN, Ac, SO3].
Fig. 2.
Fig. 2.
Plot of log10 of the number of possible structures given oligomer length. The number of unmodified protein sequences of a given oligomer length grows at a much faster rate than those of HS, CS, or KS. The slower combinatorial growth rate allows GAGfinder's brute force search to be feasible.
Fig. 3.
Fig. 3.
Workflow for GAGfinder. The steps in GAGfinder's algorithm.
Fig. 4.
Fig. 4.
Flowchart describing steps in determining terminal sugars. In several cases, GAGfinder can determine the reducing and non-reducing end sugars based on biosynthetic rules. In cases where the sugars cannot be distinguished from the composition, both monosaccharides of the class of GAG are considered as the terminal sugars. RE = reducing end; NRE = non-reducing end.
Fig. 5.
Fig. 5.
Structures of the ten synthetic standards used for testing purposes. #1 has charge state of 4- and dissociation method of NETD. #2 has charge state of 8- and dissociation method of NETD. #3 has charge state of 5- and dissociation method of NETD. #4 has charge state of 4- and dissociation method of EDD. #5 has charge state of 6- and dissociation method of EDD. #6 has charge state of 4- and dissociation method of NETD. #7 has charge state of 4- and dissociation method of NETD. #8 has charge state of 3- and dissociation method of EDD. #9 has charge state of 3- and dissociation method of NETD. #10 has charge state of 4- and dissociation method of EDD. These standards were selected randomly because of their range of modifications, length, and different dissociation methods.

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