Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high-organic-content capillary electrophoresis-mass spectrometry

A high-organic-content capillary electrophoresis-tandem mass spectrometry (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-meter long hydroxypropyl cellulose (HPC)-coated capillary, using 8% (V/V) formic acid (FA) in 70% (V/V) methanol and 22% water as the background electrolyte (BGE). A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to NanoLC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed NanoLC-MS with respect to the detection of low molecular weight peptides. This article is protected by copyright. All rights reserved