A new sight to explore site-specific N-glycosylation in donkey colostrum milk fat globule membrane proteins with glycoproteomics analysis

New insights into site‐specific N‐glycosylation profiling differences of serum proteomes between colostrum and mature breast milk

“…The digestion of the serum protein fraction was performed following the previously described method by Guan et al. (2022) with slight adaptations. Briefly, the resulting serum protein fractions (300 μg) were suspended in 300 μL of 100 mmol/L NH 4 HCO 3 (pH 8.0) followed by the addition of 4 μL of 500 mmol/L dithiothreitol for 60 min at 57°C to reduce disulfide bonds.…”

“…The database search parameters were set as follows: N ‐glycan database, 309 entries; precursor tolerance, 10.0 ppm; fragment tolerance 0.01 Da; maximum number of missed cleavages, two; fixed modification, carbamidomethylation (C, +57.02 Da); variable modification, oxidation (M, +15.99 Da). To ensure data quality, the resulting data were manually filtered according to the criteria previously established by our group, retaining entries with a Byonic score ≥100 and 2D‐FDR score ≤0.01 (Guan et al., 2022). The intact glycopeptides identified by Byonic were quantified using Panda software (Chang et al., 2019) with the following parameters: mode, extracted‐ion chromatography method; label type, label‐free quantification.…”

Comparative Site-Specific O-Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk

“…6 This strategy not only provides glycopeptide sequences but also elucidates glycan structures and their corresponding conjugation glycosites. 6 Our group has already used this strategy to construct site-specific N-glycosylation profiles of the donkey MFGM proteome; 11,12 In the current study, a label-free site-specific glycoproteomics technology pipeline was employed for the high-throughput characterization of donkey colostrum (DC) and donkey mature milk (DM) MFGM O-glycoproteins. Mapping the site-specific O-glycosylation landscape of the donkey MFGM proteome will contribute to our understanding of structure−activity relationships and provide a scientific background for developing donkey MFGM O-glycoprotein-related functional foods.…”

“…This strategy not only provides glycopeptide sequences but also elucidates glycan structures and their corresponding conjugation glycosites . Our group has already used this strategy to construct site-specific N -glycosylation profiles of the donkey MFGM proteome; , however, site-specific O -glycosylation patterns remain unclear. Given the biological effects of O -glycosylation on milk proteins, characterization of the donkey MFGM O -proteomic profile during lactation is warranted.…”