Computational protocol: N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization

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Protocol publication

[…] Protein samples (1 mg each, three replicates) were digested into peptides by trypsinization. Hydrazide resin (Affi-Prep beads; Bio-Rad, Hercules, CA, USA) was used to conduct glycocapturing as previously described []. Validation of the identified N-GPs was carried out based on the consensus sequence N-X-S/T (Spectrum Mill, Agilent Technologies), a unique acceptor sequence conserved in N-glycosylation. Prepared samples were analyzed using an HPLC-chip/Q-TOF system as previously described. [...] To identify peptides, all tandem mass spectra were analyzed using Spectrum Mill (Agilent Technologies) with the Swiss-Prot database. For the database search, the enzyme was set to trypsin with two missed cleavages, and mass tolerance was limited to 20 and 50 ppm for precursor and product ions, respectively. The modification parameters included static modification for cysteine carbamidomethylation, a dynamic modification for methionine oxidation, and N-terminal carbamylation. Deamidation of asparagine was included for glycopeptide identification. MassHunter Mass Profiler Professional software (Agilent Technologies) was used for label-free quantification by investigating similarities and differences in features across multiple samples. MetaCore ver. 6.4 (GeneGo, St. Joseph, MI, USA) was used to visualize and analyze molecular and protein interaction networks. The significance of biological pathways was evaluated through multiple testing using Benjamini-Hochberg false discovery rate analysis. […]

Pipeline specifications

Software tools Spectrum Mill, MetaCore
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Infertility, Ovarian Neoplasms
Chemicals Vitamin D