Pierce colorimetric peptide assay

Oligosaccharide fractions were isolated from human milk as previously described⁵⁹ . In short, pooled human milk samples (of unknown secretor status) were kindly donated by Irvinestown Human Milk Bank (Co. Fermanagh, Ireland) and stored at − 80 °C on arrival. Lipids and proteins were removed before applying samples to a BioGel P2 size exclusion column (92 × 5 cm; Bio-Rad Laboratories, Inc., Hercules, CA, USA) to separate lactose and HMO components. High pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used, as described below, to quantify lactose in the eluted fractions while a Pierce colorimetric peptide assay (Thermo Fisher Scientific, Reinach, Switzerland) was used to measure levels of peptides. Peptide-free and low-trace lactose (< 80 mg/L) fractions were pooled and freeze-dried and the resultant HMO-enriched fraction was stored at 4 °C prior to use in experiments.

NP swab samples were thawed and immediately treated with HALT phosphatase inhibitor cocktail (Thermo Scientific). As the NP swabs used for this experiment were residual clinical samples, the volume remaining for each was variable and ranged between 1 and 2 ml of PBS each. Each sample was aliquoted into multiple microcentrifuge tubes (200 μl aliquots), and protein was precipitated with the addition of methanol to a final concentration of 90%. Samples were then centrifuged at 15,000g for 10 min, and protein pellets from each sample were resuspended in a total of 200 μl of 8 M urea in 100 mM Tris, pH 8.0. Samples were then probe-sonicated to ensure each pellet was completely dissolved, and the protein content was estimated by BCA assay (Pierce). Disulfide bonds were reduced and alkylated with 10 mM TCEP and 10 mM IAA at room temperature for 30 min while protected from light. Trypsin/Lys-C Mix (Promega) was added at a 1:50 enzyme-to-protein ratio, and samples were digested overnight (16 h) at 37 °C. The enzymatic digest was terminated by adding TFA to a final concentration of 0.2%, and peptides were desalted using a 10 mg Strata-X PSVDB cartridge (Phenomenex), dried, and reconstituted in 100 μl of 50 mM TEAB. Peptide concentration was determined using a Pierce Colorimetric peptide assay, and 95 μg from each sample was labeled with TMTpro (Thermo Scientific) for 1 h.