CE-MS/MS for bottom-up proteomics: comparaison of two coupling interfaces with ion mobility qTOF

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein mixtures. As the samples are of high complexity and that some proteins can be at very low concentrations, efficient and sensitive instruments have to be used in order to maximize peptide identification. Nowadays, capillary electrophoresis tandem mass spectrometry (CE-MS/MS) has gained interest in proteomic analysis as it is considered as complementary to the gold standard method, namely reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS).
However, the coupling of CE with MS is not straightforward. Indeed robust interface is needed in order to conserve the high-resolution in-capillary separation while ensuring a stable spray. Among the commercialized interfaces, the coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) have been tested for the analysis of BSA and E. coli proteome digests. Both interfaces were coupled with an IMS-qTOF-MS. Several parameters were optimized in order to maximize the sensitivity, such as the composition of the sheath liquid and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). In our study, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. At the end, spray stability was found as the main strength of the triple tube interface, whereas the EMASS-II interface was found to provide higher sensitivity thanks to the reduced flow rate of the sheath liquid.