title: Determination of deamidation artifacts introduced by sample preparation using 18O-labeling and tandem mass spectrometry analysis.
abstract: 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 (18)O water. When using H(2)(18)O, 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 (18)O 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 (18)O 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 (18)O 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 (18)O-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 (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.
authors.affiliation: Merck Research Laboratory, 1011 Morris Avenue, Union, New Jersey 07083, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Du
authors.forename: Fengqiang
authors.initials: F
authors.lastname: Wang
authors.forename: Kimberly
authors.initials: K
authors.lastname: May
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D016477:Artifacts; D001216:Asparagine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007553:Isotope Labeling / Q000592:standards*; D010103:Oxygen Isotopes / Q000737:chemistry; D010455:Peptides / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000592:standards*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D010103:Oxygen Isotopes; D010455:Peptides; D001216:Asparagine; D014357:Trypsin