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.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here, we present an ultra-fast scanning data-independent acquisition (DIA) strategy consisting on 2-Th precursor 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 that provides >200 Hz MS/MS scanning speed, high resolving power and sensitivity, as well as low ppm-mass accuracy. Narrow-window DIA enables profiling of up to 100 full yeast proteomes per day, or ∼10,000 human proteins in half-an-hour. Moreover, multi-shot acquisition of fractionated samples allows comprehensive coverage of human proteomes in ∼3h, showing comparable depth to next-generation RNA sequencing and with 10x higher throughput compared to current state-of-the-art MS. High quantitative precision and accuracy is demonstrated with high peptide coverage in a 3-species proteome mixture, quantifying 14,000+ proteins in a single run in half-an-hour. Teaser: Accurate and precise label-free quantification with comprehensive proteome coverage using narrow-window DIA

High-throughput proteomics is critical for understanding biological processes, enabling large-scale studies such as biomarker discovery and systems biology. However, current mass spectrometry technologies face limitations in speed, sensitivity, and scalability for analyzing large sample cohorts. The Thermo Scientific™ Orbitrap™ Astral™ Zoom mass spectrometer (MS) was developed to address these limitations by improving acquisition speed, ion utilization, and spectral processing, which are all essential for advancing proteome depth in high-throughput proteomics. The Orbitrap Astral Zoom MS achieves ultra-fast MS/MS scan rates of up to 270 Hz with enhanced ion utilization through pre-accumulation, enabling the identification of ∼100,000 unique peptides and >8,400 proteins in a single 300 samples-per-day (SPD) analysis of human cell lysate. The optimized system reduces analysis time by 40%, achieves near-complete proteome coverage (>12,000 proteins) in 2.7 hours, and enables ultra-high-throughput workflows, identifying >7,000 proteins in a 500 SPD method with exceptional reproducibility (Pairwise Pearson correlations >0.99). These advancements establish the Orbitrap Astral Zoom MS as a new benchmark in proteomics, significantly enhancing speed, sensitivity, and scalability, paving the way for routine large-scale proteome studies with applications in clinical research and systems biology. Teaser: High-Speed Human Proteome Analysis using the Orbitrap Astral Zoom Mass Spectrometer

High-throughput mass spectrometry-based proteomics has gained increasing interest for both academic and industrial applications. As implementation of faster gradients has facilitated higher sample throughput, mass spectrometers must adapt to shorter analysis times by enhancing scanning speed and sensitivity. For Orbitrap™ mass spectrometers, faster scan rates are constrained by the need for sufficient ion accumulation time, particularly given limitations on duty cycle at high repetition rate, and transient length, which determines analyzer sensitivity and resolving power. In this context, implementing alternative ion scheduling and better ion signal-processing strategies are needed to unleash the speed of these instruments. Here, we introduce a new scanning strategy termed pre-accumulation, which enables the storage of ions in the bent flatapole in parallel to the operation of the C-trap/IRM, leading to a significant improvement in ion beam utilization and enabling for the first time scanning speeds of >70 Hz on hybrid Orbitrap instruments. The combination of pre-accumulation and increased scan speeds notably enhances peptide and protein group identifications for short LC gradients and improves sensitivity for high-throughput applications. These benefits were further amplified when coupled with the full mass range phase-constrained spectrum deconvolution method (ΦSDM), especially for fast, lower-resolving Orbitrap measurements used with short LC gradients. Overall, we demonstrate that pre-accumulation of ions in the bent flatapole offers distinct advantages, particularly for conditions with reduced signal input. Since no hardware changes are required, this approach is highly attractive for Orbitrap mass spectrometers operated with fast MS/MS acquisition methods.

Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ∼50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employed a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which caused global proteome remodeling. Pulsed-SILAC experiments revealed that NatA-deficient strains consistently increased degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovered decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding. Teaser: A multidimensional proteomics approach reveals the effect of N-terminal acetylation on Saccharomyces cerevisiae cytosolic ribosomal proteins.

Formalin-fixed paraffin-embedded (FFPE) samples are essential for clinical research and spatial proteomics (SP) but are technically challenging to analyze by liquid-chromatography coupled mass-spectrometry (LC-MS) due to losses during protein extraction, evaporation during de crosslinking and low-throughput sample preparation methods. Here, we present a streamlined micro-FFPE proteomics sample preparation protocol for laser-capture micro-dissection (LMD) that enables scalable and high-throughput processing in 96-well format, completing the entire process from tissue lysis to tryptic peptide mixtures on LC-MS-ready Evotips in just 2-hours. Our method integrates whole-slide imaging, LMD, and ultra-sensitive narrow-window data-independent acquisition (nDIA)-MS and facilitates enhanced lysis, de crosslinking, and proteolytic digestion directly within Teflon-based EVO96 chips. By utilizing nano-wells and the hanging droplet concept, our approach significantly reduces surface adsorption, thereby improving processing efficiency for both higher- and very low-input FFPE samples. This enables quantification of >5,500 protein groups (PGs) by nDIA-MS from 50,000 µm² (∼250 cells) with robust performance maintained down to 1000 µm ² (∼5 cells) with ∼2,500 PGs identified. Application to a panel of breast cancer (BC) tissues precisely discerned BC subtypes and their tumor micro-environment (TME) based on differential MS-based protein abundance of biomarkers including TP53, HER2 and PGR. Individually excised subcellular regions (∼60 µm²) yielded ∼1,500 PGs, demonstrating reliable micro-scale spatial proteomics profiling approaching near-organelle resolution. Teaser: Streamlined micro-FFPE protocol enables scalable and high-throughput processing of LMD samples, from lysis to mass spectrometry in 2 hours.