Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms.
Rachele A LubeckyjElijah N McCoolXiaojing ShenQiang KouXiaowen LiuLiangliang SunPublished in: Analytical chemistry (2017)
Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 μL loading capacity, 90 min separation window, and high peak capacity (∼280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal, and some post-translational modifications including oxidation and acetylation were detected.
Keyphrases
- ms ms
- ultra high performance liquid chromatography
- tandem mass spectrometry
- liquid chromatography
- mass spectrometry
- escherichia coli
- high performance liquid chromatography
- liquid chromatography tandem mass spectrometry
- simultaneous determination
- gas chromatography
- high resolution mass spectrometry
- solid phase extraction
- high resolution
- high throughput
- multiple sclerosis
- small molecule
- klebsiella pneumoniae
- biofilm formation
- cystic fibrosis
- multidrug resistant