An Antibody Cocktail-Based Immunoaffinity-LC-MS Method Enabled Ultra-Sensitive and Robust Quantification of...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Endocrinology

Background:

• Accurate measurement of circulating proinsulin proteoforms is vital for diabetes research.
• Previous assays lacked the specificity and sensitivity required for clinical applications.
• Limited ability to quantify specific proinsulin proteoforms hindered understanding of beta-cell function and diabetes progression.

Purpose of the Study:

• To develop a highly sensitive and specific LC-MS-based assay for quantifying intact proinsulin and its major proteoforms (des-31,32 and des-64,65 proinsulin) and C-peptide in circulation.
• To apply the developed assay for the first time to profile proinsulin proteoforms in clinical groups, including new-onset type-1 diabetes (T1D).
• To evaluate the assay's potential for differentiating disease states and providing insights into beta-cell function.

Main Methods:

• A quantitative affinity capture strategy using an optimized antibody cocktail to minimize bias.
• Lys-C digestion to generate unique signature peptides for each proinsulin proteoform.
• Trapping-nano-LC coupled with FAIMS/dCV-MS for ultra-sensitive and selective analysis with enhanced signal-to-noise ratio.

Main Results:

• The assay achieved exceptional sensitivity with serum limit of quantifications (LOQs) of 1.7 pg/mL for intact proinsulin, 2.3 pg/mL for des-31,32 proinsulin, and 3.6 pg/mL for des-64,65 proinsulin.
• The assay successfully differentiated new-onset T1D patients from control groups, a distinction missed by a parallel total-proinsulin ELISA.
• Distinct relative abundance ratios of proinsulin proteoforms were observed across clinical groups, suggesting unique expression patterns.

Conclusions:

• This novel LC-MS assay is the first capable of sensitively quantifying major circulating proinsulin proteoforms.
• The assay provides valuable insights into beta-cell function and the onset/progression of diabetes.
• The developed strategy is broadly applicable for targeted measurement of other biomarker proteoforms.