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Updated: Aug 8, 2026

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis
Published on: May 23, 2021
Comprehensive two-dimensional separation in coupling of reversed-phase chromatography with capillary isoelectric
Wenjia Yu1, Yan Li, Chunhui Deng
1Department of Chemistry & Research Center of Proteome, Fudan University, Shanghai, PR China.
Abstract:
A coupling of capillary RP LC as the first dimension with CIEF as the second dimension followed by MALDI-MS identification was demonstrated. Based on 2-D separation system developed by our group (Electrophoresis 2003, 24, 3289-3295), this paper focused on incorporating tryptic digestion into the top-down proteomics methodology, retaining the benefits of the top-down method. Hydrophobic layer of packing-material C18 coated with SE-30 on the MALDI-target surface was used to permit the CIEF fractions to be easily concentrated and free of ampholytes using on-target washing. Following the removal of ampholytes, on-target tryptic digestion was performed to generate PMF for protein identification. Using the proteome analytical strategy, we could obtain not only intact protein pI value but also PMF for accurate protein identification. The feasibility of the strategy was first tested with a mixture of model proteins with different pIs and molecular masses. Proteome of rat liver tissue extracts was further analyzed using the system for verification. The results have shown that the system is effective for complex proteomic analysis.
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