A simple and efficient approach for preparing cationic coating with tunable electroosmotic flow for capillary zone electrophoresis-mass spectrometry-based top-down proteomics

ABSTRACT: Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has become a valuable analytical technique in top-down proteomics (TDP) in recent years. CZE-MS/MS-based TDP typically employs separation capillaries with neutral coatings (i.e., linear polyacrylamide, LPA). However, issues related to separation resolution and reproducibility remain with the LPA-coated capillaries due to the unavoidable non-specific protein adsorption onto the capillary wall. Cationic coatings can be critical alternatives to LPA coating for CZE-MS/MS-based TDP due to the electrostatic repulsion between the positively charged capillary inner wall and proteoform molecules in the acidic separation buffer. Unfortunately, there are only very few studies using cationic coating-based CZE-MS/MS for TDP studies. In this work, we aimed to develop a simple and efficient approach for preparing separation capillaries with cationic coating, i.e., poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride, PAMAPTAC for CZE-MS/MS-based TDP. The PAMAPTAC coating-based CZE-MS produced significantly better separation resolution of proteoforms compared to traditionally used LPA-coated approach. It achieved reproducible separation and measurement of simple proteoform mixture and a complex proteome sample (i.e., a yeast cell lysate) regarding migration time, proteoform intensity, and the number of proteoform identifications. The PAMAPTAC coating-based CZE-MS enabled the detection of large proteoforms (≥30 kDa) from the yeast cell lysate reproducibly without any size-based prefractionation. Interestingly, the electrophoretic mobility of proteoforms using the PAMAPTAC coating can be predicted accurately using a simple semiempirical model. The results render the PAMAPTAC coating as a valuable alternative to the LPA coating to advance CZE-MS-based TDP towards high-resolution separation and highly reproducible measurement of proteoforms in complex samples.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD051862 | Pride | 2024-10-15

REPOSITORIES: Pride