αs cn

The milk protein samples, which included κ-CN, α_S-CN, β-CN, BSA, β-lactoglobulin and α-lactalbumin, were obtained from Sigma Co. (St. Louis, MO, USA). MTGase was obtained from Ajinomoto Co., Inc. (1.0 units/mg). To investigate the MTGase-induced cross-linking of milk proteins, each milk protein sample and 2.0 units/mL MTGase were added into a 0.05 M phosphate buffer solution (pH 6.8) and dissolved. Each milk protein sample without/with 2.0 units/mL MTGase was incubated in a dry bath incubator for 0, 1, 2, or 3 h at 30 °C. Milk protein samples were heated to 80 °C for 3 min to inactivate the MTGase. Each sample was analyzed in triplicate.

The following certified bovine protein standards (all purchased from Sigma–Aldrich Corp., St. Louis, MO, USA) were used to prepare a milk protein standard mixture (MPSM): α lactalbumin (α-LA, purity ≥ 85%), lactoferrin (LF, purity ≥ 85%), bovine serum albumin (BSA, purity ≥ 98%), β lactoglobulin A + B (β-LG, purity ≥ 90%), immunoglobulin G (IgG, purity ≥ 95%), α_s-casein (α_s-CN, purity ≥ 70%), β-casein (β-CN, purity ≥ 98%), and κ-casein (κ-CN, purity ≥ 70%). Phosphate buffer (10 mM, pH 6.7, prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate, c.f., Section 2.1.3) was used to dissolve the protein standards. The MPSM was aliquoted and stored at −25 °C. Table 2 shows the protein concentrations for the MPSM (prepared following Farrell Jr. et al. [31 (link)]). Twenty-three synthetic peptide standards were purchased from Synpeptide Co., Ltd. (Shanghai, China) according to their sequences (Table 3).