A Tandem Mass Spectrometer for Improved Transmission and Analysis of Large Macromolecular Assemblies

TLDR

Large macromolecular assemblies generate signals above m/z 3 000–4 000, exceeding the limit of commercial quadrupoles and making them difficult to analyze by tandem‑MS. The authors designed and applied a quadrupole time‑of‑flight tandem mass spectrometer specifically optimized for transmitting and analyzing large macromolecular assemblies. The instrument uses a reduced‑frequency quadrupole as a narrow‑band filter, a gas‑filled collision cell for CID, and a high‑resolution TOF analyzer to isolate and fragment single charge states of assemblies such as transthyretin–thyroxine–retinol complexes. By precisely controlling the pressure gradient, lowering the quadrupole frequency to 300 kHz, and employing the narrow‑band filter, the system achieves a mass range beyond m/z 90 000, a resolution above 3 000 at m/z 44 500, and successful isolation and subunit identification of ions up to m/z 22 000.

Abstract

We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3,000-4,000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22,000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90,000, in theory to more than m/z 150,000. We also demonstrate that the resolution of the instrument is greater than 3,000 at m/z 44,500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5,340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.