title: Investigation of asparagine deamidation in a SOD1-based biosynthetic human insulin precursor by MALDI-TOF mass spectrometry.
abstract: A biosynthetic human insulin precursor displayed enhanced susceptibility to deamidation at one particular site. The present study was undertaken to monitor progress of precursor deamidation at successive manufacturing stages. MALDI-TOF/TOF MS in combination with controlled endoproteinase Glu-C and endoproteinase Asp-N proteolysis was used for rapid and unambiguous determination of deamidated residue within the investigated structure. Close inspection of isotopic distribution patterns of peptides resulting from enzymatic digestion enabled determination of distinct precursor forms occurring during the production process. Asn, Asp, isoAsp and succinimide derivatives of the amino acid at position 26 were unambiguously identified. These modifications are related to the leader peptide of a precursor encompassing amino acid sequence corresponding to that of superoxide dismutase [Cu-Zn] (SOD1 1, EC=1.15.1.1). Monitoring of precursor deamidation process at successive manufacturing stages revealed that the protein folding stage was sufficient for a prominent replacement of asparagine by aspartic and isoaspartic acid and the deamidated human insulin precursor constituted the main manufactured product. Conversion proceeded through a succinimide intermediate. Significant deamidation is associated with the presence of SNG motif and confirms results achieved previously on model peptides. Our findings highlight an essential role of the specific amino acid sequence on accelerated rate of protein deamidation. To our knowledge, this is the first time that such a dramatic change in the relative abundance of Asp and isoAsp resulting from protein deamidation process is reported.
authors.affiliation: Institute of Biotechnology and Antibiotics, Warszawa, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Bierczyńska-Krzysik
authors.affiliation: Institute of Biotechnology and Antibiotics, Warszawa, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Łopaciuk
authors.affiliation: Bioton S.A., Macierzysz, Ozarow Mazowiecki, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Pawlak-Morka
authors.affiliation: Institute of Biotechnology and Antibiotics, Warszawa, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Stadnik
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D004345:Drug Industry; D007328:Insulin / Q000032:analysis / Q000096:biosynthesis*; D026581:Isoaspartic Acid / Q000737:chemistry / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000032:analysis / Q000096:biosynthesis*; D011786:Quality Control; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D000072105:Superoxide Dismutase-1
chemical_list: D007328:Insulin; D026581:Isoaspartic Acid; D011498:Protein Precursors; C000606290:SOD1 protein, human; D001224:Aspartic Acid; D001216:Asparagine; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N