Application of Isotope Dilution Gas Chromatography-Mass Spectrometry Using Selected Ion-Monitoring to Evaluate...

Mass spectrometry (MS)-based approach for the detection and quantification of nonenzymatic posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome damage with high throughput and deep coverage. Here, we describe a gas chromatography-mass spectrometry with a selected ion-monitoring mode (SIM-GC/MS) method for protein damage profiling of biological and clinical samples. This approach can be carried out on materials ranging from isolated proteins to tissue/biofluid samples. In particular, the SIM-GC/MS method is applied to detect and quantify diverse protein damage markers in the human cerebrospinal fluid (CSF) proteome. The protocol includes protein extraction, digestion, derivatization, PTMs detection and quantification by GC/MS, followed by computational data processing. Different protein damage markers of oxidation, glycoxidation, lipoxidation and succination can be confidently detected and quantified simultaneously. This protocol provides a general protein nonenzymatic modification measurement tool, enabling the dissection of protein and proteome modifications in a particular biofluid, the CSF.