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Proteins in a human body continuously undergo glycation reactions with reducing sugars, forming early as well as advanced glycation end-products (AGEs) which are highly disease-relevant. Specifically, N-1-(deoxyfructosyl) valine of β-hemoglobin (HbA1c) has been considered as a marker of diabetes, but the exact map of glycated Hb peptides corelated with diabetes in different stages is poorly studied. Here, the pseudotargeted parallel reaction monitoring (PRM) method combined with relative retention time (iRT) endogenous peptides was proposed for exploring the roles of deoxyfructosyl (DF-), carboxymethyl (CM-), and carboxyethyl (CE-) based Hb modifications in clinical prognosis and diagnosis of type 2 diabetes mellitus (T2DM) and its complication. For building the pseudotargeted list, data-dependent acquisition (DDA) combined with multiple enzyme digestion was employed for the comprehensive identification of the three types of modification in vitro Hb and in vivo Hb. The introduction of the endogenous iRT peptides during PRM analysis facilitates being able to obtain accurate quantitative results. When applying this new strategy to quantify the three kinds of glycated Hb peptides in clinical samples, patients with T2DM in different pathophysiological conditions were fully distinguished from the controls, indicating the necessity of adopting multiple glycation types for the improved diagnosis of T2DM. Taken together, the newly developed pseudotargeted PRM method not only expands the horizons of glycated Hb by reliably assessing the actual status of T2DM but also reveals that endogenous iRT might be a viable option for label-free quantitative analysis.

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