Preparation and Analysis of Homocysteinylated Myoglobin

Horseradish peroxidase streptavidin (SA-5004) was from Vector Laboratories (Burlingame, CA, USA). SuperSignal West Pico chemiluminescent substrate was from Thermo Scientific (Rockford, IL, USA). Myoglobin from equine skeletal muscle (M0630) was from Sigma-Aldrich. N-Homocysteinylated myoglobin was prepared as we previously reported [64 (link)]. Biotinyl-Asp-Glu-Val-Asp-aldehyde (CAS registry number: 178603-73-1) was from Bachem Americas (N-1470, Torrance, CA, USA; see Figure S1 for its structure in Support Information). Rhodamine-aldehyde (10 mM) in 50% 200-proof ethanol was synthesized as previously described [64 (link)]. EZ-Run prestained protein ladder (BP3603) was from Fisher Scientific. Natural unstained protein ladder (161-0317) was from Bio-Rad. Biotinylated protein markers were from Sigma-Aldrich (B2787) and Bio-Rad (161-0319), respectively. All reagents were ACS grade and used as received without further purification. All incubations were carried out in an Eppendorf Thermomixer^® (Eppendorf North America, Hauppauge, NY, USA) at 25 °C unless specified otherwise.