C-peptide microheterogeneity in type 2 diabetes populations

Abstract

Purpose: The purpose of this study was to investigate naturally occurring C-peptide microheterogeneity in healthy and type 2 diabetes (T2D) populations.

Experimental design: MS immunoassays capable of simultaneously detecting intact C-peptide and variant forms were applied to plasma samples from 48 healthy individuals and 48 individuals diagnosed with T2D.

Results: Common throughout the entire sample set were three previously unreported variations of C-peptide. The relative contribution of one variant, subsequently identified as C-peptide (3-31), was found to be more abundant in the T2D population as compared to the healthy population. Dipeptidyl peptidase IV is suspected to be responsible for this particular cleavage product, which is consistent with the pathophysiology of T2D.

Conclusions and clinical relevance: C-peptide does not exist in the human body as a single molecular species. It is qualitatively more heterogeneous than previously thought. These results lay a foundation for future studies devoted to a comprehensive understanding of C-peptide and its variants in healthy and diabetic populations.

Figure 1

C-peptide MSIA spectrum qualitatively representative of the subjects in this study. The mass spectrum contains the intact form of C-peptide as well as C-peptide (2–31) (calc. monoisotopic m/z 2888.47), C-peptide(3–31) (calc. monoisotopic m/z 2817.44), and C-peptide(4–31) (calc. monoisotopic m/z 2688.39). These N-terminally truncated variants had signals registering at m/z =2888.49, 2817.45, and 2688.41 (labeled as I, II, and III, respectively). The loss of ammonia from the N-terminus is observed on all native and variant forms and is frequently observed under reflector-TOF-MS conditions.

Figure 2

Expanding on the last row of Table 1, these histograms provide the frequency of occurrence within the healthy and T2D populations for the RPA of variant II. A broad distribution averaging 10% RPA (average of all individuals in the cohort) was observed for the T2D cohort, as compared with a narrow distribution averaging 5% observed for the healthy cohort.

Figure 3

(A) MALDI-TOF/TOF-MS spectrum of intact C-peptide (positive mode m/z =3019.50; calc. mono-isotopic m/z =3019.52). (B) MALDI-TOF/TOF-MS spectrum of variant II aligned to the sequence of C-peptide(3–31). The y-ion fragmentation series registers as the same pattern and mass as the intact series (note the three overlaid enlarged glycine registers), which is expected for N-terminally truncated peptide forms. The signal for the b-ion of C-peptide(1–31) terminating at Q22 is observed at m/z 2093.1, but the b-ion of the putatively truncated C-peptide terminating at the same glutamine residue is shifted in mass by −200.2 Da, which corresponds to the mass difference associated with the loss of the N-terminal Glu-Ala dipeptide.