Aizikov, K. | bioRxiv Science

bioRxiv2021

Frequency chasing of individual megadalton ions in an Orbitrap analyzer improves precision of analysis in single molecule mass spectrometry

To enhance the performance of charge detection mass spectrometry, we investigated the behavior of macromolecular single ions on their paths towards and within the Orbitrap analyzer. We discovered that ions in mass beyond one megadalton reach a plateau of stability and can be successfully trapped for seconds, travelling a path length of multiple kilometers, thereby enabling precise mass analysis with an effective resolution of greater than 100,000 at m/z 35,000. Through monitoring the frequency of individual ions, we show that these high mass ions, rather than being lost from the trap, can gradually lose residual solvent molecules and, in rare cases, a single elementary charge. Our observations highlight the importance of efficient desolvation for optimal charge detection mass spectrometry and inspired us to implement multiple improved data acquisition strategies. We demonstrate that the frequency drift of single ions due to desolvation and charge stripping can be corrected, which improves the effective ion sampling 23-fold and gives a two-fold improvement in mass precision and resolution, as demonstrated in the analysis of various viral particles.

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↗