The tryptic peptides of hemoglobin for diagnosis of type 2 diabetes mellitus using label-free and standard-free LC-ESI-DMRM
REDOX BIOLOGY(2021)
Abstract
N-1-(deoxyfructosyl) valine of beta-hemoglobin, commonly referred to as HbA1c, is the "gold standard" for clinical detection of diabetes. Instead of quantifying the full-length HbA1c glycated protein, in the present study, we proposed the peptide-based strategy to quantify the depletion of the tryptic peptides of hemoglobin for the diagnosis of type 2 diabetes mellitus (T2DM). The peptides were discovered and validated as T2DM biomarkers by label-free LC-ESI-DMRM method without reference material. The glucose could react with hemoglobin's free amino group of N-terminus and epsilon-amino group of lysine residues and leave the modification on the hemoglobin tryptic peptides. Thus, there are two types of peptides in the hemoglobin: sensitive peptides and insensitive peptides to glucose due to the differential sensitivity of lysine residues to glycation. To discover two types of peptides of hemoglobin, we first developed the assay of liquid chromatography-electrospray ionization mass spectrometry coupled with dynamic multiple reaction monitoring. The protein coverage reaches 94.2%. Moreover, the hemoglobin was incubated with the 500 mmol/L glucose for 20 days, 40 days and 60 days in vitro to screen the sensitive peptides and insensitive peptides to glucose. A total of 14 sensitive peptides and 4 insensitive peptides were discovered. Furthermore, the LC-ESI-DMRM method was also utilized to validated the glucosesensitive peptides by 40 clinical samples with healthy control individuals (n = 20) and type 2 diabetes mellitus patients (n = 20). Three putative sensitive peptides (LLGNVLVCVLAHHFGK, VVAGVANALAHKYH, LRVDPVNFK) from the hemoglobin showed excellent sensitivity and specificity based on receiver operating characteristic analysis and were verified as the promising biomarkers for the diagnosis of diabetes mellitus. And one peptide (LLVVYPWTQR) was found as glucose-insensitive peptide. Taken together, the findings of this study suggest that quantification of hemoglobin tryptic peptides using label-free and standard-free LC-ESI-DMRM is an alternative method for the diagnosis of T2DM, which could be combined with other MS-based blood biomarkers for diagnosis of multiple diseases in MS single shot.
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Key words
Diabetes mellitus,LC-ESI-DMRM,Peptide biomarkers
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