Expanding the mass range for UVPD-based native top-down mass spectrometry.

ABSTRACT: Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles.

SUBMITTER: Greisch JF 

PROVIDER: S-EPMC6764275 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature