In-Solution Digestion of Whole Milk

The in-solution digestion of whole milk was adapted from previous methods [14 (link)]. Briefly, a 20 μL milk sample was added to 80 μL of protein lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) with a protease inhibitor (Thermo Fisher Scientific, Rockford, IL, USA). The whole milk buffer was sonicated for 20 min in a water bath with cold water and put on ice for another 30 min. Impurities were removed by methanol/chloroform protein precipitation and the precipitate was air-dried. The pellet was further dissolved in an 8 M urea (Sigma, St. Louis, MO, USA)/50 mM ammonia bicarbonate (Alfa Aesar, Haverhill, MA, USA) solution, incubated with 10 mM dithiothreitol (Sigma, St. Louis, MO, USA) at 37 °C for 1 h and followed by alkylation with 20 mM iodoacetamide (Sigma, St. Louis, MO, USA) at 37 °C in the dark for 30 min. For digestion, the samples were incubated with a trypsin/lys-C mixture (w:w = 1:50; Promega, Madison, WI, USA) at 37 °C for 16 h and then the digest reaction was quenched with 1% formic acid (Thermo Fisher Scientific, Rockford, IL, USA). The resulting peptides were desalted using a C18 column (Thermo Fisher Scientific, Rockford, IL, USA) prior to lyophilization. The same tryptic peptides for proteomic analysis were directly applied to glycoproteomic analysis without further glycopeptide enrichment.