Deamidation of -Asn-Gly- Sequences during Sample Preparation for Proteomics:  Consequences for MALDI and HPLC-MALDI Analysis

We find that peptides containing -Asn-Gly- sequences typically show ∼70−80% degree of deamidation after standard overnight (∼12 h) tryptic digestion at 37 °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 (-βAsp-Gly-) residue. For the peptide SLNGEWR (54−60 β-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-Å sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -βAsp-/-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 ∼ 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -βAsp-/-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-Å pore size sorbents, or using formic acid as a modifier, increases the retention time of -βAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.