Determination of Deamidation Artifacts Introduced by Sample Preparation Using ¹⁸O-Labeling and Tandem Mass Spectrometry Analysis

The sites and levels of Asn deamidation in proteins are often determined by LC–MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in ¹⁸O water. When using H₂¹⁸O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the ¹⁸O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two ¹⁸O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of ¹⁸O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using ¹⁸O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of ¹⁸O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions