Computational protocol: Glycomic Analysis of HighDensity Lipoprotein Showsa Highly Sialylated Particle

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Protocol publication

[…] All tryptic MS/MS data were analyzed using X! Tandem ( Trypsin digesion would help identify the HDL-associated proteins. X! Tandem was set up to search the Swissprot human complete proteome set database. X! Tandem was searched with a fragment ion mass tolerance of 80 ppm and a parent ion tolerance of 100 ppm. Iodoacetamide derivative of cysteine was specified in X! Tandem as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine and tryptophan were specified in X! Tandem as variable modifications.Data analyses for N-glycans, glycopeptides, and gangliosides were performed with the MassHunter Qualitative Analysis software ver. B.03.01 (Agilent Technologies, Inc., Santa Clara, CA).For the N-glycan analysis, within a 20 ppm accurate mass criterion molecular feature extraction (MFE) was performed through a mode of the “Find by Molecular Feature” function for nontargeted profiling. The deconvoluted glycan mass, retention time, and abundance were extracted. The glycan compositions were assigned based on the accurate mass and the corresponding MS/MS data.Glycopeptide assignments were achieved by the combination of the tandem MS information and the accurate glycopeptide precursor ion mass via in-house software GP Finder. The mass list of the glycopeptides precursor ions was analyzed with accurate mass, protein sequence, biological filters of glycans, and a MS tolerance level (≤20 ppm). The tandem mass spectra were then taken into account for further inspection with glycan and peptide fragmentation patterns with a mass tolerance for fragment ions (≤80 ppm). The unique significance of GP Finder is to use a false discovery rate by generating a decoy library to the scoring system and to serve as a more reliable means of making assignments.For gangliosides, MFE was performed to generate a peak list (m/z, retention time, and peak area) taking all ions into account exceeding 1000 counts. Focused postprocessing precursor ion scan analysis was performed through a mode of the “Find by Auto MS/MS”. The NeuAc ions (m/z 290.095) were the fragment ions used to determine the precursor ion masses representing gangliosides. […]

Pipeline specifications

Software tools X! Tandem, GP Finder
Application MS-based untargeted proteomics
Diseases Multiple Sclerosis
Chemicals Gangliosides, Neuraminic Acids, Sialic Acids