MALDI-TOF mass spectrometry-based quantification of C-peptide in diabetes patients

Abstract

Background Serum C-peptide concentrations reflect insulin secretion and beta cell function and can be used to diagnose and distinguish type-1 and type-2 diabetes. C-peptide is a more accurate indicator of insulin status than direct insulin measurement for monitoring patients with diabetes. However, the current methods available for C-peptide quantification exhibit poor reproducibility, are costly, and require highly trained laboratory personnel. Here, we have developed and evaluated a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay to standardize C-peptide measurements, providing highly accurate and comparable results across testing systems and laboratories. Methods C-peptide from human serum was enriched using antibody-conjugated magnetic beads. The eluted isolates were further modified with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to enhance the ionization of naturally acidic C-peptide. After desalting with ZipTips, the samples were subjected to MALDI-TOF MS analysis. Recombinant human C-peptide was used to develop the assay, and a heavy isotope labeled human C-peptide was used as an internal standard for quantification. Results The MALDI-TOF MS method was validated in accordance with the restrictions of the device, with a limit of quantitation of 25 pmol/L. A correlation between the MAL-DI-TOF MS assay and a reference method was conducted using patient samples. The resulting regression revealed good agreement. Conclusions A simple, high-throughput, cost effective and quantitative MALDI-TOF MS C-peptide assay has been successfully developed and validated in clinical serum samples.

The potential for isotope dilution-LC-MS/MS to improve laboratory measurement of C-peptide: Reasons and critical determinants

“…Compared to immunoassays it is more expensive and tedious. [45] Urine [ 13 C 6 Leu 26 , 30 ] Cp PP/ SPE (Waters MCX) Orbitrap Exploris 480 HR-MS (Thermo) ESI(+) ID-LC-MS/MS N/A This procedure is non-invasive. The SP procedure is simple and inexpensive.…”

“… Derivatization . This approach is commonly used for target compounds in GC–MS measurement procedures, but derivatization is also quite useful to enhance signals in LC-MS/MS for C-peptide [45] . In 2014, Kinumi et al used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to modify the N-terminal amino group of C-peptide, which dramatically increased fragmentation efficiency by adding a positive charge to the peptide.…”

Enhanced-Precision Measurement of Glutathionyl Hemoglobin by MALDI-ToF MS

“…The published method gives multiple warnings on critical steps throughout the procedure and on the several sources of possible error [ 9 ]. Other recent studies address the quantification in human blood of peptides and proteins, such as a phosphorylated amyloid peptide [ 10 ], monoclonal free light chains [ 11 ], C-peptide [ 12 ], isoforms of glutathione-S-transferase [ 13 ] and glycated hemoglobin [ 14 , 15 ].…”