Makarov, A. | bioRxiv Science

bioRxiv2019

Resolving heterogeneous high-mass macromolecular machineries by Orbitrap-based single particle charge detection mass spectrometry

Here we show that single particle charge-detection mass spectrometry (CD-MS) can be performed on a ubiquitous Orbitrap mass analyser and applied to the analysis of high-mass (megadalton) heterogeneous biomolecular assemblies. We demonstrate that single particle high-mass ions can survive in the Orbitrap for seconds, whereby their measured signal amplitudes scale linearly with charge state over the entire m/z range. Orbitrap based single particle CD-MS can be used to resolve mixed ion populations, accurately predict charge states, and consequently also the mass of the ions. We successfully applied CD-MS to challenging natural and biotherapeutic protein assemblies, such as IgM oligomers, designed protein nano-cages, ribosome particles and intact, empty- and genome-loaded Adeno-associated virus particles. Single particle CD-MS combined with native MS on existing Orbitrap platforms will greatly expand its application, especially in the mass analysis of megadalton heterogeneous biomolecular assemblies.

biophysics↗

bioRxiv2018

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

biochemistry↗