Capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics using attapulgite nanoparticles functionalized separation capillaries.

ABSTRACT: Attapulgite nanoparticles have good chemical properties and can be modified easily for broad applications. In this work, for the first time, attapulgite nanoparticles were employed to modify the inner wall of separation capillaries for capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS)-based top-down proteomics. The attapulgite nanoparticles and the inner wall of a fused silica capillary were first functionalized with ?-methacryloxypropyl trimethoxysilane. Then the modified nanoparticles and acrylamide were copolymerized in the fused silica capillary with the assistance of azobisisobutyronitrile and heat. The incorporation of high-surface-area nanoparticles in the linear polyacrylamide (LPA) coating resulted in significantly lower electroosmotic mobility compared with the typical LPA coating (3.48?×?10-5 vs. 9.03?×?10-5 cm2 V-1 S-1), most likely because more LPA molecules were immobilized on the inner wall of the separation capillary. The attapulgite nanoparticles functionalized separation capillaries have shown great stability and reproducibility across 43 discontinuous CZE-MS runs of a standard protein mixture. We applied the CZE-MS/MS system for top-down proteomics of Escherichia coli cells. In a proof-of-principle experiment, the CZE-MS/MS system achieved a 90-min separation window and a 1-?L sample loading volume, leading to nearly 300 proteoform and 135 protein identifications in a single run. Many post-translational modifications (PTMs) were identified, including methylation, acetylation, phosphorylation, biotinylation, succinylation, and disulfide bond.

SUBMITTER: Wang T 

PROVIDER: S-EPMC6557293 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature