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Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Published on: July 31, 2011
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An Antibody Cocktail-Based Immunoaffinity-LC-MS Method Enabled Ultra-Sensitive and Robust Quantification of
Qingqing Shen1, Wang Cao1, Ming Zhang1,2
1The Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, New York 14214, United States.
Analytical Chemistry
|August 28, 2025
Summary
A new LC-MS assay accurately measures circulating proinsulin proteoforms, crucial for diabetes research. This method distinguishes new-onset type 1 diabetes (T1D) patients from controls, offering insights into beta-cell function.
Area of Science:
- Biomarker Discovery and Validation
- Analytical Chemistry
- Endocrinology and Metabolism
Background:
- Accurate measurement of circulating proinsulin proteoforms is vital for diabetes mellitus investigation.
- Previous assays lacked the specificity and sensitivity required for precise quantification of multiple proinsulin proteoforms.
- This limitation hindered a comprehensive understanding of beta-cell function and disease progression.
Purpose of the Study:
- To develop a highly sensitive and specific liquid chromatography-mass spectrometry (LC-MS) assay for quantifying intact proinsulin and its major proteoforms (des-31,32 and des-64,65 proinsulin) and C-peptide in circulation.
- To apply this novel assay for the first time to profile proinsulin proteoforms in clinical groups, including new-onset type 1 diabetes (T1D).
- To assess the utility of this assay in differentiating disease states and understanding beta-cell dynamics.
Main Methods:
- A quantitative affinity capture strategy using an optimized antibody cocktail to mitigate bias across proteoforms.
- Lysine-C (Lys-C) digestion to generate unique signature peptides for each proinsulin proteoform.
- Trapping-nano-LC coupled with Field Asymmetric Ion Mobility Spectrometry/differential Cyclic Voltammetry-Mass Spectrometry (FAIMS/dCV-MS) for ultrasensitive and selective analysis with enhanced signal-to-noise ratio.
Main Results:
- The developed assay demonstrated exceptional sensitivity, with serum lower limits of quantification (LOQs) as low as 1.7 pg/mL for intact proinsulin.
- The assay successfully differentiated control subjects from new-onset T1D patients, a distinction missed by a parallel total-proinsulin ELISA.
- Distinct relative abundance ratios of proinsulin proteoforms were observed across clinical groups, suggesting differential beta-cell processing or secretion.
Conclusions:
- This novel LC-MS assay is the first capable of sensitively quantifying major circulating proinsulin proteoforms, overcoming previous analytical limitations.
- The assay provides valuable insights into beta-cell function and the pathophysiology of diabetes, particularly T1D.
- The developed strategy is broadly applicable for targeted proteoform measurement of other critical biomarkers.
