title: Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.
abstract: Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH's (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.
authors.affiliation: Department of Chemistry and Chemical Biology, Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, 02115, USA.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Chemical Biology, Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, 02115, USA.
authors.forename: Kevin Ryan
authors.initials: KR
authors.lastname: Moulton
authors.affiliation: Department of Chemistry and Chemical Biology, Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, 02115, USA.
authors.forename: Jared Robert
authors.initials: JR
authors.lastname: Auclair
authors.affiliation: Department of Chemistry and Chemical Biology, Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, 02115, USA. z.zhou@neu.edu.
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000737:chemistry; D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D016477:Artifacts; D001216:Asparagine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D002021:Buffers; D002147:Calmodulin / Q000737:chemistry; D002417:Cattle; D000077270:Exenatide; D006863:Hydrogen-Ion Concentration; D026581:Isoaspartic Acid / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000737:chemistry*; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014688:Venoms / Q000737:chemistry
chemical_list: D000577:Amides; D002021:Buffers; D002147:Calmodulin; D026581:Isoaspartic Acid; D010455:Peptides; D012996:Solutions; D014688:Venoms; D001224:Aspartic Acid; D001216:Asparagine; D000324:Adrenocorticotropic Hormone; D000077270:Exenatide; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase
keywords: Adrenocorticotropic hormone; Artifact; Calmodulin; Deamidation; Exenatide; Glu-C; Isoaspartic acid; Isomerization; Mass spectrometry