Computational protocol: Glycoform Analysis of Recombinant and Human ImmunodeficiencyVirus Envelope Protein gp120 via Higher Energy Collisional Dissociationand Spectral-Aligning Strategy

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Protocol publication

[…] HIV gp120 peptides were identified by using Proteome Discoverer software (Thermo Fisher Scientific, version 1.4). The database used was recombinant gp120 HxBc2 strain or virion gp120 proteins from LAV strain sequences in a NCBI RefSeq database (downloaded from the NCBI Web site on August, 19, 2013, 53 738 entries). The precursor mass tolerance was set at 20 ppm and the MS/MS tolerance at 0.06 Da. Parameters of the search were modified as follows: oxidized methionines (add Met with 15.995 Da) and a (PNGase F-catalyzed) conversion of Asn to Asp (add Asn with 0.984 Da) set as dynamic modifications and Cys modification (add cysteine with 57 Da) set as a fixed modification. A maximum of two missed tryptic cleavage sites were allowed. MaxQuant version with default setting was also used for database search with default settings., [...] An in-house software tool (manuscript in preparation) was developed to identify the glycopeptides from MS and MS/MS data. The MS raw files were converted to mzXML files by Trans-Proteomic Pipeline (TPP). MatLab version R2013b (MathWorks, Inc.) was then used to extract information from mzXML files and code for the search strategy. The database was gp120 tryptic peptides containing the site of glycosylation. The search strategy is defined in the following steps: (1) identification of deglycosylated peptides—the sequences and glycosylation sites were identified from MS/MS analysis of PNGase F treated tryptic peptide samples using Proteome Discoverer (Thermo Fisher Scientific, version 1.4) of SEQUEST search; (2) detecting the b- and y-ions of glycopeptides—sequences of deglycopeptide were used to pick the b- and y-ions in charge states 1, 2, and 3 within a 20 ppm tolerance; (3) extraction of MS/MS spectra containing glycopeptides—MS/MS spectra containing oxonium ions from Hex, HexNAc, HexHexNAc, and NeuAc at m/z 163, 204, 366, 274, 292 were extracted as putative glycopeptide spectra; (4) assigning glycopeptides—the putative glycopeptide spectra were used to search for b- and y-ions detected from deglycosylated peptides. The ratio of glycopeptide b- and y-ions versus those detected from deglycosylated peptide was calculated. To use the data for each glycopeptide backbone as putative glycopeptides, the ratio above 20% for y+ ion was kept; (5) evaluate the match of glycopeptides—to evaluate glycopeptides matched to deglycosylated peptide sequences containing specific glycosylation sites, the MS1 scan number, MS2 scan number, precursor m/z, precursor charge states, precursor mass, ratio and presence of b- and y-ions between glycopeptides and deglycosylated peptides, other peptide b- and y-ions detected in spectra of glycopeptides but not in deglycosylated peptides, Δglycan mass, potential glycan composition consisting HexNAc, Hexose, Fucose, and NeuAc, Y-ions in charge states 1, 2, and 3 were extracted and computed in the program. These were considered to shortlist the putative spectra for glycopeptides. The best-matched spectrum for individual glycopeptide in different peptide backbones was manually confirmed and used as spectral template to cross-match the putative glycopeptide spectra using b-, y-, peptide, Y-ions, and software Xcalibur version 2.2 (ThermoFisher Scientific). Platelet glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and analyzed by the same algorithm to evaluate the false discovery rate (FDR). Glycan stuctures reported previously, and ExPASy tools GlycoMod, PeptideMass, and GlycanMass were used to identify glycan compositions and glycan types., MS analysis infers the glycan compositions rather than actual glycan structures, hence the glycan structures reported here were the representatives of the glycans with the same composition. Xcalibur was used to inspect the correct monoisotopic peak for glycopeptides on the MS1 level. […]

Pipeline specifications

Software tools Proteome Discoverer, MaxQuant, Comet, GlycoMod, PeptideMass, GlycanMass
Databases ExPASy
Applications MS-based untargeted proteomics, Protein sequence analysis
Diseases HIV Infections, Genetic Diseases, X-Linked
Chemicals Glycopeptides, Mannose