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

The sites and levels of Asn deamidation in obtained from enzymatic digestion. However, deamidation that proteins are often determined by LC-MS analysis of peptides occurs during sample preparation steps results in over-estimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in O-18 water. When using (H2O)-O-18, 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 O-18 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 O-18 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 O-18 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 O-18-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 O-18-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.