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Capillary zone electrophoresis–electrospray ionization–mass spectrometry (CZE–ESI–MS) was used for analysis of reduced antibodies. We first developed a simple protocol to condition commercial linear-polyacrylamide coated capillaries for use in top–down proteomics. We then suspended reduced antibodies in a solution of 35% acetic acid, 50% acetonitrile in water. Heavy and light chains were baseline resolved within 10 min and with 3–30 µg/mL detection limits using a 0.1% aqueous formic acid background electrolyte. Quintuplicate runs of a two-antibody mixture produced relative standard deviations of ∼1% in migration time and 10% in peak amplitudes. Resolution was further improved for the two-antibody mixture by using 5% acetic acid as the background electrolyte, highlighting the potential of capillary electrophoresis–mass spectrometry for analysis of antibody mixtures. [ABSTRACT FROM AUTHOR]

Capillary zone electrophoresis (CZE) with an electrokinetically pumped sheath-flow nanospray interface was coupled with a high-resolution QjExactive 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 0.196 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. [ABSTRACT FROM AUTHOR]

Femtogram proteomics: An ultrasensitive capillary zone electrophoresis–mass spectrometry system that is based on an improved nanospray interface has been developed. This system is used for the analysis of picogram to femtogram amounts of E. coli digests; for example, over 100 proteins were identified from 16 pg digests by tandem mass spectrometry. AMTs=accurate mass and time tags. [ABSTRACT FROM AUTHOR]

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed-phase liquid chromatography (RPLC)-MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath-flow interface for the analysis of complex proteome digests. Single-shot CZE-MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state-of-the-art nano ultrahigh-pressure LC system. [ABSTRACT FROM AUTHOR]

We coupled capillary zone electrophoresis (CZE) with an ultrasensitive electrokinetically pumped nanospray ionization source for tandem mass spectrometry (MS/MS) analysis of complex proteomes. We first used the system for the parallel reaction monitoring (PRM) analysis of angiotensin II spiked in 0.45 mg/mL of bovine serum albumin (BSA) digest. A calibration curve was generated between the loading amount of angiotensin II and intensity of angiotensin II fragment ions. CZE-PRM generated a linear calibration curve across over 4.5 orders of magnitude dynamic range corresponding to angiotensin II loading amount from 2 amole to 150 fmole. The relative standard deviations (RSDs) of migration time were <4% and the RSDs of fragment ion intensity were ∼20% or less except 150 fmole angiotensin II loading amount data (∼36% RSD). We further applied the system for the first bottom up proteomic analysis of a human cell line using CZE-MS/MS. We generated 283 protein identifications from a 1 h long, single-shot CZE MS/MS analysis of the MCF7 breast cancer cell line digest, corresponding to ∼80 ng loading amount. The MCF7 digest was fractionated using a C18 solid phase extraction column; single-shot analysis of a single fraction resulted in 468 protein identifications, which is by far the largest number of protein identifications reported for a mammalian proteomic sample using CZE. [ABSTRACT FROM AUTHOR]