title: Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis.
abstract: We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba 799 JBRC, 715 McDermot Avenue, Winnipeg, MB, R3E 3P4, Canada. krokhino@cc.umanitoba.ca
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Krokhin
authors.forename: Mihaela
authors.initials: M
authors.lastname: Antonovici
authors.forename: Werner
authors.initials: W
authors.lastname: Ens
authors.forename: John A
authors.initials: JA
authors.lastname: Wilkins
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Standing
mesh_terms: D000577:Amides / Q000032:analysis / Q000138:chemical synthesis*; D001216:Asparagine / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005998:Glycine / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000577:Amides; D010455:Peptides; D001216:Asparagine; D005998:Glycine