Femtogram proteomics: We report an ultrasensitive capillary zone electrophoresis-mass spectrometry system based on an improved nanospray interface. This system is used for analysis of picogram to femtogram amounts of E. coli digests. Over 100 proteins were identified based on tandem mass spectra from 16 pg digests; over 60 proteins were identified from 400 fg digests based on accurate mass and time tags in 10 min.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has fallen far below that of reversed phase liquid chromatography (RPLC)-MS/MS. Here, we report the use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (~300). This method is coupled to an Orbitrap Fusion mass spectrometer via an electro-kinetically pumped sheath flow interface for analysis of complex proteome digests. Single-shot CZE-MS/MS identified over 10 000 peptides and 2 100 proteins from a HeLa cell proteome digest in ~100 min. This performance is nearly an order of magnitude superior to earlier CZE studies and is within a factor of 2 to 4 of state-of-the-art nano ultrahigh pressure LC system.

We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified.

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Capillary zone electrophoresis (CZE) with an electrokinetically pumped sheath-flow nanospray interface was coupled with a high-resolution Q-Exactive mass spectrometer for the analysis of culture filtrates from Mycobacterium marinum. We confidently identified 22 gene products from the wildtype M. marinum secretome in a single CZE–tandem mass spectrometry (MS/MS) run. A total of 58 proteoforms were observed with post-translational modifications including signal peptide removal, N-terminal methionine excision, and acetylation. The conductivities of aqueous acetic acid and formic acid solutions were measured from 0.1% to 100% concentration (v/v). Acetic acid (70%) provided lower conductivity than 0.25% formic acid and was evaluated as low ionic-strength and a CZE–MS compatible sample buffer with good protein solubility.

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Femtogram proteomics: We report an ultrasensitive capillary zone electrophoresis-mass spectrometry system based on an improved nanospray interface. This system is used for analysis of picogram to femtogram amounts of E. coli digests. Over 100 proteins were identified based on tandem mass spectra from 16 pg digests; over 60 proteins were identified from 400 fg digests based on accurate mass and time tags in 10 min.

No abstract