2.2. Hemoglobin glycation in vitro

Glycation of hemoglobin was incubated with glucose according to the previous protocol with slight modification [24]. For glycation of hemoglobin, 100 mM hemoglobin was incubated with 500 mM glucose in 10 mM PBS (pH 7.2) at 37 °C for 20 days, 40 days and 60 days in vitro, which screened the sensitive peptides and insensitive peptides to glucose. Three parallel samples were prepared at each incubation time in the hemoglobin glycation group, and the hemoglobin without the addition of d-glucose under the same conditions was used as a blank control. All solutions were added sodium azide as a preservative.