Preparative capillary electrophoresis (CE) fractionation of protein digests improves protein and peptide identification in bottom-up proteomics

• Published in: Analytical methods Vol. 14; no. 11; p. 1103
• Main Authors: Weaver, Simon D, Schuster-Little, Naviya, Whelan, Rebecca J
• Format: Journal Article
• Language: English
• Published: England 17-03-2022
• Subjects: Chromatography, Liquid; Electrophoresis, Capillary - methods; Escherichia coli; Peptides - chemistry; Proteins - chemistry; Proteomics - methods; Tandem Mass Spectrometry - methods
• ISSN: 1759-9679
• DOI: 10.1039/d1ay02145a

Reversed-phase liquid chromatography (RPLC) is widely used to reduce sample complexity prior to mass spectrometry (MS) analysis in bottom-up proteomics. Improving peptide separation in complex samples enables lower-abundance proteins to be identified. Multidimensional separations that combine orthogonal separation modes improve protein and peptide identifications over RPLC alone. Here we report a preparative capillary electrophoresis (CE) fractionation method that combines CE and RPLC separations. Using this method, we demonstrate improved protein and peptide identification in a tryptic digest of
cell lysate, with 132 ± 33% more protein identifications and 185 ± 65% more peptide identifications over non-fractionated samples. Fractionation enables detection of lower-abundance proteins in this complex sample. We demonstrate improved coverage of ovarian cancer biomarker MUC16 isolated from conditioned cell media, with 6.73% sequence coverage using CE fractionation compared to 2.74% coverage without preparative fractionation. This new method will allow researchers performing bottom-up proteomics to harness the advantages of CE separations while using widely available LC-MS/MS instrumentation.