Facile identification by electrospray mass spectrometry of the insulin fragment A^14–21‐B^17–30 produced by insulin proteinase

Abstract

We confirm the cleavage at position B16-17 of porcine insulin which occurs during in vitro digestion by insulin proteinase. The fragment A14-21-B17-30 was purified by reversed-phase high performance liquid chromatography and characterized by electrospray ionization mass spectrometry. Fast-atom bombardment mass spectrometry, on the other hand, failed to detect the presence of this fragment.

AP/MALDI‐MS complete characterization of the proteolytic fragments produced by the interaction of insulin degrading enzyme with bovine insulin

“…Although insulin is one of the most widely studied protein hormones due to its use in the treatment of insulin‐dependent diabetes, it still remains one of the most problematic proteins to be studied experimentally 33. MS investigations of insulin solutions have been usually carried out by electrospray ionization (ESI),34 nanoflow ES,25, 35 or MALDI‐time of flight (TOF),36 while in the past the interaction of the hormone with IDE was addressed by fast atom bombardment (FAB)20 or ESI coupled to HPLC 19. Insulin structure consists of two chains, usually named A and B, linked by disulfide bonds between the cysteines at positions A7 and B7 and between positions A20 and B19 (Fig.…”

“…Confirmation of the cleavage sites involved in the interaction of IDE with insulin have been searched since the discovery of such interaction and, overall, several different experimental approaches16–18 have been applied in order to identify them. Mass spectrometry (MS), often coupled to high‐performance liquid chromatography (HPLC), has demonstrated to be one of the most valuable tool in this scenario 19, 20. Nevertheless, so far, some cleavage sites involved in the interaction of IDE with its substrates were elusive to the MS investigation and in order to have a global and complete assessment of the peptide fragments generated by the interaction of IDE with insulin, a recourse to different approaches21 or to complicated and time‐consuming experimental procedures utilizing [125I]iodoinsulin were necessary 22.…”

Short-Term Insulin-Induced Glycogen Formation in Primary Hepatocytes as a Screening Bioassay for Insulin Action

“…The sample was washed with 0.1% TFA in water and then eluted with 40% acetonitrile/0.1% TFA in water. Acetonitrile was removed under N 2 , and the sample was purified by preparative HPLC as described previously (19). The four major peaks of the digest were collected, desalted by Sep-Pak as above, and characterized by analytical HPLC as described previously (31) and ESI-MS. Fragment concentration was determined by absorbance at 280 nm (32).…”

“…This enzyme may not be specific to insulin since it has been shown to degrade several other peptide hormones, including glucagon (13,14). The fragments produced upon insulin degradation by insulin protease have been well characterized with distinct cleavage sites between residues A [13][14] , A 14 -15 , B 9 -10 , B 10 -11 , B [13][14] , B 16 -17 , B 24 -25 , and B [25][26] (7,(15)(16)(17)(18)(19). Interestingly, partially degraded forms of insulin have been found in the circulation of rats (20), mice (21), and humans (22), where some maintain the capacity to bind to the membrane insulin receptor.…”

A Stable Isotope Dilution Assay for the In Vivo Determination of Insulin Levels in Humans by Mass Spectrometry

“…Proinsulin-to-insulin conversion intermediates (1)(2)(3)(4)(5), especially des 31,32 split proinsulin, have been the subject of several studies (6)(7)(8)(9). Much remains also to be discovered concerning the processing and degradation of insulin once it is specifically bound to its receptor (10)(11)(12)(13)(14).…”