We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Orbitrap mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer is capable of producing high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

Matrix matched calibration curved on the Orbitrap Astral Mass Spectrometer from Thermo Fisher Scientific. We compare to data on the Orbitrap Fusion Lumos. For plasma experiments, DIA data was acquired in normal and enriched plasma.

Both SILAC labeled and unlabeled HeLa cell lysates were prepared using protein aggregation capture. Briefly, cell pellets were lysed with probe sonication in a lysis buffer containing 2% SDS. A Pierce BCA assay (Thermo Fisher Scientific) was used to estimate protein concentration and dilute lysates to a final concentration of 2 µg/µL in 1% SDS. Following reduction in 20 mM dithiothreitol and alkylation in 40 mM iodoacetamide, both samples were diluted to 70% acetonitrile and bound to MagReSyn Hydroxyl particles (ReSyn Biosciences) at a ratio of 100:1 (beads to protein). Subsequent washing was performed on a magnetic rack, with three washes in 95% acetonitrile followed by two washes in ethanol. Following the final wash, trypsin (in 50 mM ammonium bicarbonate) was added at an enzyme to protein ratio of 1:33. Samples were digested at 47˚ C for 4 hours, eluted from the beads and dried down via vacuum centrifugation. The digests were resuspended at a concentration of 200 ng/µL in 0.1% formic acid, Pierce peptide retention time calibration mix (PRTC) was spiked in to a final concentration of 5 pmol/µL, and digests were combined to form the following dilutions (X% unlabeled in (100-X)% heavy-labeled): 0, 0.5, 1, 5, 10, 25, 50, 70, 100%. An enriched membrane particle fraction was prepared from plasma using Mag-Net, a magnetic bead-based protocol developed by Wu et al. (2023). Plasma particle enrichment and protein aggregation capture steps were performed on a KingFisher Flex (Thermo Fisher Scientific). Briefly, HALT cocktail (protease and phosphatase inhibitors, Thermo Scientific) was added to 100 µL plasma and then mixed 1:1 by volume with Binding Buffer (BB, 100 mM Bis-Tris Propane, pH 6.3, 150 mM NaCl). MagReSyn® strong anion exchange beads (ReSyn Biosciences) were first equilibrated 2 times in Equilibration/Wash Buffer (WB, 50 mM Bis Tris Propane, pH 6.5, 150 mM NaCl) with gentle agitation and then combined in a 1:4 ratio (volume beads:volume starting plasma) with the plasma:BB sample for 45 minutes at room temperature. The beads were washed with WB 3 times 5 minutes with gentle agitation. The enriched membrane particles on the beads were then solubilized and reduced in 50 mM Tris, pH 8.5/1% SDS/10 mM Tris (2-carboxyethyl) phosphine (TCEP) with 800 ng enolase standard added as a process control. Following reduction, the plate was removed from the Kingfisher Flex. Samples were alkylated with 15 mM iodoaceamide in the dark for 30 minutes and then quenched with 10 mM DTT for 15 minutes. Total unfractionated plasma (1 µL) was prepared in parallel (reduction, alkylation, and quenched) and added to the Kingfisher plate as a control for enrichment. The samples were processed using protein aggregation capture with minor modifications. Briefly, the samples were adjusted to 70% acetonitrile, mixed, and then incubated for 10 minutes at room temperature to precipitate proteins onto the bead surface. The beads were washed 3 times in 95% acetonitrile and 2 times in 70% ethanol for 2.5 minutes each on magnet. Samples were digested for 1 hour at 47 ˚C in 100 mM ammonium bicarbonate with Pierce porcine trypsin (Thermo Scientific) at a ratio of 20:1 trypsin to protein. The digestion was quenched in 0.5% formic acid and spiked with Pierce Retention Time Calibrant peptide cocktail (Thermo Scientific) to a final concentration of 50 fmol/µL. Peptide digests were lyophilized and stored at -80°C.