Mass spectrometry instrumentation continues to evolve rapidly, yet quantifying these advances beyond conventional peptide and protein detections remains challenging. Here, we evaluate a modified Orbitrap Astral Zoom mass spectrometer (MS) prototype and compare its performance to the standard Orbitrap Astral MS. Across a range of acquisition methods and sample inputs, the prototype instrument outperformed the standard Orbitrap Astral MS in precursor and protein identifications, ion accumulation efficiency, and reproducibility of measurements. To enable meaningful cross-platform comparisons, we implemented an ion calibration framework that converts signal intensity from arbitrary units to absolute ion counts (ions/sec). This benchmarking strategy showed that the prototype consistently sampled more ions per peptide, resulting in improved sensitivity and quantitative precision. To make these metrics broadly accessible, we added new metrics to the Skyline document grid to report ion counts directly from data-independent acquisition (DIA) data. Taken together, our results demonstrate the Orbitrap Astral Zoom prototype as a high-performance platform for DIA proteomics and establish a generalizable framework for ion-based evaluation of mass spectrometer performance.

HeLa samples preparation: HeLa cells were grown until 70% confluent and lysed in a buffer containing 2% SDS, 100 mM Tris-HCl (pH 8.5), and Thermo Fisher protease inhibitors, and the lysates were briefly sonicated using a Branson probe sonicator. The total protein concentration was quantified using the bicinchoninic acid (BCA) assay (BCA kit, Thermo Fisher Scientific) using bovine serum albumin standards. The lysate was diluted to 1 µg/µL, reduced with 20 mM dithiothreitol (DTT), and alkylated with 40 mM iodoacetamide (IAA). Protein aggregate capture was used for cleanup protocol19,20. Briefly, proteins were bound to ReSyn hydroxyl magnetic beads at 4 µL beads per 25 µg protein by adding acetonitrile to 70% final concentration. The beads were washed with three cycles of 95% ACN and two cycles of 70% ethanol. Residual ethanol was removed by centrifugation. The washed beads were resuspended in 50 mM ammonium bicarbonate containing trypsin (1:20 enzyme-to-protein ratio) and incubated at 47 °C for 3 hours. After digestion, peptides were eluted from the beads and dried using a speedVac vacuum centrifuge. The dried peptides were stored at -80˚C until analysis. Prior to LC-MS, the frozen peptides were reconstituted in 0.1% formic acid to a final concentration of 0.2 µg/µL. Plasma Preparation and extracellular vesicle (EV) Enrichment: Plasma membrane particle enrichment was performed on a Thermo Scientific KingFisher Flex instrument following the Mag-Net protocol, a magnetic bead approach developed by Wu et al. (2024)21. The same protocol was described in Heil et al (2023)2. To start, 100 µL of plasma was first mixed with protease and phosphatase inhibitors and then combined with an equal volume of Binding Buffer (BB) containing 100 mM Bis-Tris Propane, pH 6.3, 150 mM NaCl. MagReSyn strong anion exchange beads (ReSyn Biosciences) were pre-equilibrated twice in Equilibration/Wash Buffer (WB) containing 50 mM Bis-Tris Propane, pH 6.5, 150 mM NaCl). These beads were mixed with gentle agitation and then added to the plasma:BB mixture in a 1:4 bead-to-plasma ratio, followed by a 45-minute incubation at room temperature. Post-incubation, the beads were washed three times with WB for five minutes each using gentle agitation. Membrane particles bound to the MagReSyn beads were solubilized by a lysis buffer containing 1% SDS, 50mM Tris at pH 8.5, 10mM TCEP, and 800ng yeast enolase protein. Post reduction, 15mM iodoacetamide was added and incubated for 30 minutes in the dark and quenched with 10mM DTT for 15 minutes. For sample clean-up, protein aggregate capture (PAC) was performed by adding acetonitrile to a final concentration of 70% to precipitate the proteins at room temperature for 10 minutes19,20. The beads were washed three times in 95% acetonitrile and two washes in 70% ethanol for 2.5 minutes per wash, using the magnetic field on the KingFisher Flex to separate the beads. After clean-up, the proteins were digested at 47°C for 1 hour in a 20:1 ratio of trypsin-to-protein containing 100 mM ammonium bicarbonate. Digestion was halted by adding 0.5% formic acid, and an internal control, Pierce Retention Time Calibrant peptide cocktail (Thermo Fisher Scientific), was included at a final concentration of 50 fmol/uL. The peptides were dried using a speedVac vacuum centrifuge and frozen until analysis.