N octyl β d glucopyranoside og

Incorporation of membrane proteins into the vesicles was achieved by diluting a detergent-solubilized protein/vesicle mixture and reducing the concentration of the detergent below the critical micelle concentration (CMC). We used the detergent n-octyl-β-D-glucopyranoside (OG) (Sigma, USA). For the formation of SLBs, VAMP2-to-lipid ratios were 1:500. In contrast, the protein-to-lipid ratios were 1:200 for an in vitro lipid mixing assay. To form pre-t-SNARE complexes, syntaxin HT and SNAP-25 (molar ratio of 1:2) were incubated with agitation for 1 h at room temperature for preventing the formation of dead-end t-SNARE complexes. DiI-labeled vesicles were then mixed with pre-t-SNARE complexes and OG to form t-vesicles with agitation for 30 min at 4°C. VAMP2 was added to DiD-labeled vesicles with OG to form v-vesicles with agitation for 30 min at 4°C. Two different vesicles were rapidly diluted in 25 mM HEPES buffer with 100 mM KCl (pH 7.4) to reduce the concentration of OG below its CMC.

Bovine α_s-casein (α-CN), bovine β-casein (β-CN), bovine α-lactalbumin (α-Lac), bovine β-lactoglobulin A (β-LgA), bovine β-lactoglobulin B (β-LgB), histamine, urea, and n-octyl-β-D-glucopyranoside (OG) were from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were from Tedia Company (Fairfield, OH, USA). All other reagents were of analytical grade or higher grade. Silica gel (ZEX-II, 5 µm, 300 Å) was from Qingdao Meigao Chemical Co., Ltd. (Qingdao, China). All aqueous solutions were prepared using ultrapure water from an MK-459 Millipore Milli-Q Plus ultrapure water system. Commercial infant milk powder samples were from local shops.
Stock standard solutions of α-CN, α-Lac, β-CN, β-LgB, and β-LgA were prepared in a buffer consisting of 6 M urea and 0.2% OG (pH 3.3) [13] (link), at concentrations of 10 mg/mL. Working solutions containing all analytes were % mM KCl, 2.54 mM CaCl₂, 1.19 mM KH₂PO₄, 10 mM HEPES, 5 mM glucose, 0.1% (w/v) BSA, pH 7.3). Stock internal standard solutions of histamine-d4 at 30 ng/mL were prepared in ACN.