No abstract

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

We demonstrate single-particle charge detection mass spectrometry on an Orbitrap for the analysis of megadalton biomolecular assemblies. We establish that the signal amplitudes of individual ions scale linearly with their charge, which can be used to resolve mixed ion populations, determine charge states and thus also determine the masses of individual ions. This enables the ultrasensitive analysis of heterogeneous protein assemblies including immunoglobulin oligomers, ribosomes, proteinaceous nanocontainers and genome-packed adeno-associated viruses.Native mass spectrometry (MS) is a powerful tool, enabling mass analysis of intact macromolecular assemblies in the megadalton range 1 . The exact mass of the intact macromolecular complex is then used to infer its composition and the stoichiometry of subunits, post-translational modifications and ligands bound to the complex 2 . Various mass analyzers, including quadrupole time-of-flight, Fourier-transform ion cyclotron resonance and, most recently, Orbitraps, have all been adapted for native MS experiments 3 .Notably, masses are not measured directly in most MS approaches, but need to be inferred from the mass-to-charge (m/z) ratios of the detected ions. As pioneered by Mann and Fenn 4 , the charge states from a population of multiply charged ions generated by electrospray ionization (ESI) can be determined from the m/z values by matching consecutive peaks in the charge state distribution to calculate accurate masses. A general limitation in native MS studies then stems from the fact that the charge state, and thus also the mass, can only be accurately measured when multiple charge states of the same molecular species can be resolved and assigned. This hampers the analysis of larger heterogeneous protein assemblies, such as highly glycosylated proteins, amyloid fibrils, genomepacked viruses and membrane protein complexes decorated with multiple lipid molecules.Even small variabilities in the monomeric building blocks can result in wide distributions of masses in their larger assemblies. In combination with the often poor desolvation, these broadened mass distributions result in overlapping signals between consecutive charge states, leading to inaccurate mass assignments. A possible solution to these problems is to measure one particle (or ion) at a time, thereby avoiding the convolution of signals that stem from insufficient resolving power 5,6 . When such single-particle detection approaches can be combined with an independent measure of the