Christian Hock

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Anion slow photoelectron velocity-map imaging (SEVI) spectroscopy is a high-resolution variant of photoelectron spectroscopy used to study the electronic and geometric structure of atoms, molecules, and clusters. To benefit from the high resolution of SEVI when it is applied to molecular species, it is essential to reduce the internal temperature of the ions as much as possible. Here, we describe an experimental setup that combines a radio-frequency ion trap to store and cool ions with the high-resolution SEVI spectrometer. For C(5)(-), we demonstrate ion temperatures down to 10 ± 2 K after extraction from the trap, as measured by the relative populations of the two anion spin-orbit states. Vibrational hot bands and sequence bands are completely suppressed, and peak widths as narrow as 4 cm(-1) are seen due to cooling of the rotational degrees of freedom.

We present an experimental study of the thermodynamics of free, size-selected water cluster anions consisting of 48 and 118 molecules. The measured caloric curves of the clusters are bulklike at low temperatures but show a well-defined, particle-size specific transition at $93\ifmmode\pm\else\textpm\fi{}3\text{ }\text{ }\mathrm{K}$ for $({\mathrm{H}}_{2}\mathrm{O}{)}_{48}^{\ensuremath{-}}$ and $118\ifmmode\pm\else\textpm\fi{}3\text{ }\text{ }\mathrm{K}$ for $({\mathrm{H}}_{2}\mathrm{O}{)}_{118}^{\ensuremath{-}}$. At the transition temperature the heat capacity strongly increases, which marks the onset of melting.