Speaker
Description
The analysis of intact proteins and protein complexes presents unique challenges to mass spectrometry (MS), prompting a fundamental re-evaluation of principles previously validated for small molecules and peptides. As the initial limitations of fragmentation have largely been overcome in the past two decades—culminating in near-complete freedom of fragmentation in the latest instruments—severe spectral congestion has emerged as a major obstacle, persisting despite advances in modern liquid separation techniques.
Until recently, this issue could only be addressed by high-resolution MS, preferably featuring isotopic resolution and accurate mass. Orbitrap mass spectrometry has proven particularly effective in such analyses, but further progress has been hampered by inherently limited dynamic range for species that overlap in m/z space.
Nowadays, this limitation can be addressed using two distinct approaches: charge reduction and direct charge detection. This presentation explores the practical implementation of both techniques on the latest generation of Orbitrap instruments.
Charge reduction is achieved through high-speed ion–ion reactions, allowing proteins to shed a number of protons and shift to significantly higher and less congested m/z values. By stepping a narrow m/z isolation window in a data-independent manner, this method enables detection of up to ten times more proteoforms—not only for intact proteins, but also for protein complexes. It is also compatible with spectra produced by a broad range of fragmentation techniques.
For direct charge detection, Direct Mass Technology (DMT) allows the charge states of individual ions to be determined in parallel for hundreds to thousands of ions. While typically suited to low-intensity ion beams, DMT is shown to offer intriguing possibilities for top-down analysis as well.
In conclusion, recent rapid advances all aspects of mass spectrometry instrumentation and analysis are shown to open up exciting new possibilities for top-down proteomic analysis.
| User consent | yes |
|---|
