Top-down mass spectrometric immunoassay for human insulin and its therapeutic analogs

J Proteomics. 2018 Mar 20;175:27-33. doi: 10.1016/j.jprot.2017.08.001. Epub 2017 Aug 3.


Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays. Reviewed here is an insulin mass spectrometric immunoassay (MSIA) that combines micro-scale immunoaffinity capture with sensitive MS detection of insulin and its therapeutic analogs. An antibody reactive to all insulin analogs was used to affinity capture the insulin analogs. Following elution, insulins were detected with MALDI-TOF MS or LC-MS analysis. Isotopic resolution for insulin was achieved for both MS techniques, and several insulin analogs were detected at unique m/z signals. Porcine insulin, spiked in all samples, served as an internal reference standard for quantification. Linear standard curves spanning three orders of magnitude were obtained, with limits of detection of 15pM for the MALDI-TOF MS and 1.5pM for the LC-MS. This insulin assay was capable of detecting and quantifying not only human endogenous insulin, but also most of the therapeutic insulin analogs, which could find use in diagnosis of severe hypoglycemia and in sports anti-doping.

Significance: Insulin replacement therapy consists of injection of long- or fast-acting insulin analogs with slightly modified primary sequences compared to human insulin. Assays that are capable of detecting all insulin analogs are desired, not only for medical management of diabetes and severe hypoglycemia but also for sports anti-doping and toxicology.

Keywords: Human plasma; Immunoaffinity; Insulin; Mass spectrometry; Proteomics; Top-down.

Publication types

  • Review

MeSH terms

  • Animals
  • Antibodies
  • Computational Biology
  • Diabetes Mellitus / drug therapy
  • Doping in Sports
  • Humans
  • Hypoglycemia / drug therapy
  • Immunoassay / methods*
  • Insulin / analogs & derivatives
  • Insulin / analysis*
  • Insulin / standards
  • Insulin, Regular, Pork / analysis
  • Mass Spectrometry / methods*
  • Protein Multimerization
  • Swine


  • Antibodies
  • Insulin
  • Insulin, Regular, Pork