Capillary electrophoresis tandem mass spectrometry for proteomic analysis: optimization and comparison of two coupling interfaces

Abstract

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new proteic entities. As the samples are of high complexity and that some proteins could be present 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. For this purpose, optimization of basic parameters such as BGE composition was first carried out using a simple peptide mix. Several parameters were then optimized in order to maximize the sensitivity, such as the composition of the sheath liquid, the interface position and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). Finally, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. In our study, two commercialized interfaces were compared by analysing E. coli proteome digest. The coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) were both coupled with an IMS-qTOF-MS. Eventually, spray stability was found to be 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