Cyanoborohydride

Bone samples (in triplicate) designated for glycoxidation were placed into vials with cyanoborohydride coupling buffer (0.20M disodium phosphate, pH 8.0–8.5, 0.20M NaCl, 3.0 g/L sodium cyanoborohydride; all buffer components were purchased from Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 0.15M glyoxal or 0.15M glyoxylic acid (Fig. 1A) and 0.5mM CaCl₂, and incubated at 37°C for 24, 48, or 72 hours. The matching control bone samples were incubated at 37°C for 24, 48, or 72 hours in the 0.20M disodium phosphate pH 8.0 to 8.5, 0.20M NaCl, and 0.5mM CaCl₂ buffer. After completion of incubations, all samples underwent dialysis against phosphate buffered saline of pH 7.4. In the next experimental step, the samples were lyophilized overnight and stored at −80°C until their analysis.

GLP-1 7–36 amide, ¹³C₆¹⁵N₂-L-lysine, ¹³C₆¹⁵N₄-L-arginine, formaldehyde, ¹³C-D₂-formaldehyde and cyanoborohydride were purchased from Sigma Aldrich (St. Louis, MO). Dialyzed heat-inactivated fetal bovine serum (FBS) was purchased from Biochrom AG (Merck, Germany). TiO₂ beads were purchased from GL sciences (Tokyo, Japan).