Zhang Jennifer, Katta Viswanatham
Protein Analytical Chemistry, Genentech Inc, South San Francisco, CA, USA.
Methods Mol Biol. 2012;899:365-77. doi: 10.1007/978-1-61779-921-1_23.
Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.
天冬氨酸（Asp）通过琥珀酰亚胺中间体异构化为异天冬氨酸（isoAsp）是蛋白质常见的降解途径，可能影响其结构完整性。由于Asp/isoAsp质量数相同，通过液相色谱-串联质谱（LC-MS/MS）肽图谱分析很难确定修饰位点。在此，我们描述了一种在蛋白质水平标记参与异构化的Asp残基的方法，即通过在H₂¹⁸O中水解琥珀酰亚胺中间体。对这种标记的蛋白质进行胰蛋白酶消化，由于水解过程中掺入了¹⁸O，含异构化位点的肽段将比含¹⁶O的对应肽段重2 Da。比较掺入和未掺入¹⁸O的异构化肽段的串联质谱，可轻松鉴定出参与的Asp残基。该方法在鉴定Asp-Asp基序中发生异构化的位点时特别有用。
