Abstract
Mass spectrometry (MS) has become a mainstream platform for comprehensive profiling of proteome, especially with the improvement of multiplexed tandem mass tag labeling coupled with two-dimensional liquid chromatography and tandem mass spectrometry (TMT-LC/LC-MS/MS). Recently, we have established a robust method for direct profiling of undepleted cerebrospinal fluid (CSF) proteome with the 16-plex TMTpro method, in which we optimized parameters in experimental steps of sample preparation, TMT labeling, LC/LC fractionation, tandem mass spectrometry, and computational data processing. The extensive LC fractionation not only enhances proteome coverage of the CSF but also alleviates ratio distortion of TMT quantification. The crucial quality control steps and improvements specific for the TMT16 analysis are highlighted. More than 3000 proteins can be quantified in a single experiment from 16 different CSF samples. This multiplexed method offers a powerful tool for profiling a variety of complex biofluids samples such as CSF, serum/plasma, and other clinical specimens.