Identification by fast atom bombardment mass spectrometry of insulin fragments produced by insulin proteinase

Abstract

We describe the isolation by reversed-phase h.p.l.c. of a number of products of the degradation of insulin by insulin proteinase and their direct analysis by fast atom bombardment mass spectrometry (f.a.b.-m.s.). Various semisynthetically labelled insulins were used, including [[2H2]GlyA1]insulin and [18O]LysB29]insulin. The results obtained confirm and extend the results obtained by non-mass-spectrometric methods [Davies, Muir, Rose & Offord (1988) Biochem. J. 249, 209-214, and papers cited therein]. Cleavage sites were identified between positions A13-A14, A14-A15, B9-B10, B13-B14, B24-B25 and B25-B26. The advantages and disadvantages of the application of f.a.b.-m.s. to such studies are discussed.

Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF)

“…[21]; amino acid analysis and N-terminal analysis [20]; protein thiol analysis [22]; and protein disulphide analysis [23]. Circular dichroism Spectra were measured using a Jobin-Yvon Dichrographe IV linked to a BBC microcomputer for recording data.…”

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

“…Fragment A 14 -21 B 10 -30 is one of the larger known degradation fragments of insulin (16,17), maintaining the region B 24 -B 26 , which is important for the binding of insulin to its receptor (27). Preparations of this fragment isolated from digests of insulin displayed consistent activity between different assays and different preparations (whereas the three other fragments prepared at the same time were always inactive).…”

“…We demonstrate this assay by determination of the dose-response curve of porcine insulin and by examination of the four major fragments formed upon porcine insulin degradation by pig skeletal muscle insulin protease: A (16,17).…”

Protein Design and the Development of New Therapeutics and Vaccines

“…In order to study the biological degradation of insulin and its possible aberrations, we prepared a number of isotopically labeled insulins. These were, in addition to our original [(3H)Phe BI ]insulin (Halban and Offord, 1975), [(3H)-Gly ]Al ]insulin (Davies and Offord, 1985), [(3H)Ala B3 0]insulin , [(lsO)LysB29]insulin (Rose et al, 1984;Savoy et al, 1988), and [eH)GlyAI]insulin (Savoy et al, 1988). All of these analogs had the porcine sequence; the 180-labeled material was also made with the human sequence.…”