MacCoss - AstralBenchmarking

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data Independent Acquisition
Data License: CC BY 4.0 | ProteomeXchange: PXD042704 | doi:
  • Organism: Homo sapiens
  • Instrument: Orbitrap Fusion Lumos,Orbitrap Astral
  • SpikeIn: No
  • Keywords: plasma, DIA, Astral, Orbitrap, limit of quantification
  • Lab head: Michael MacCoss Submitter: Lilian Heil
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.
Experiment Description
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.
Sample Description
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.
Created on 6/2/23, 3:27 PM

Here, we are evaluating the performance of Astral compared to the Orbitrap for DIA quantitation. To do this, we have two main experiments:

  1. Matrix-matched calibration curve to evaluate quantitative precision and accuracy in a HeLa cell lysate
  2. Plasma proteome measurements to showcase the potential of Astral and a new extracellular vesicle enrichment strategy to reach new depths of coverage in a single shot


mzML files are not included on this page due to space considerations and the fact that they are functionally the same as raw files. mzML files can be generated using the ProteoWizard Docker image available on Docker Hub at “ proteowizard/pwiz-skyline-i-agree-to-the-vendor-licenses:3.0.22335-b595b19”. 

To re-generate the files in MSConvert, the following command can be used: msconvert *.raw --simAsSpectra --ignoreUnknownInstrumentError --filter "peakPicking true"


Below is a summary of the run types.

File prefixSampleAnalyzerNormalized AGC TargetMaximum injection timeIsolation WindowMass rangeColumnGradient length (min)
20230403_OLEP08_EV_1ug_MB_30min_AS_10ms_4Th_ EV plasma Astral 500% 10 ms 4 Th 400-1000 110 cm 30
20230403_OLEP08_EV_1ug_MB_30min_AS_3p5ms_2Th_ EV plasma Astral 500% 3.5 ms 2 Th 400-1000 110 cm 30
20230403_OLEP08_TP_1ug_MB_30min_AS_10ms_4Th_ Total plasma Astral 500% 10 ms 4 Th 400-1000 110 cm 30
20230403_OLEP08_TP_1ug_MB_30min_AS_3p5ms_2Th_ Total plasma Astral 500% 3.5 ms 2 Th 400-1000 110 cm 30
20230404_OLEP08_EV_1ug_MB_60min_AS_15ms_4Th_ EV plasma Astral 500% 15 ms 4 Th 400-1000 110 cm 60
20230404_OLEP08_EV_1ug_MB_60min_AS_dDIA_15ms_2Th_ EV plasma Astral 500% 15 ms 2 Th 300 Th dDIA 110 cm 60
20230404_OLEP08_TP_1ug_MB_30min_AS_dDIA_20ms_4Th_ Total plasma Astral 500% 20 ms 4 Th 300 Th dDIA 110 cm 30
20230404_OLEP08_TP_1ug_MB_60min_AS_15ms_4Th_ Total plasma Astral 500% 15 ms 4 Th 400-1000 110 cm 60
20230404_OLEP08_TP_1ug_MB_60min_AS_dDIA_15ms_2Th_ Total plasma Astral 500% 15 ms 2 Th 300 Th dDIA 110 cm 60
20230405_OLEP08_EV_1ug_MB_30min_AS_dDIA_20ms_4Th_ EV plasma Astral 500% 20 ms 4 Th 300 Th dDIA 110 cm 30
20230405_OLEP08_EV_1ug_MB_60min_AS_23ms_2Th_ChrLib_ EV plasma Astral 500% 23 ms 2 Th 100 Th GPF 110 cm 60
20230405_OLEP08_EV_1ug_MB_60min_OT_23ms_2Th_ChrLib_ EV plasma Orbitrap 800% 23 ms 2 Th 100 Th GPF 110 cm 60
20230406_OLEP08_MMCC_1ug_MB_24min_AS_10ms_4Th_ MMCC Astral 500% 10 ms 4 Th 400-1000 110 cm 24
20230406_OLEP08_MMCC_1ug_MB_24min_AS_3p5ms_2Th_ MMCC Astral 500% 3.5 ms 2 Th 400-1000 110 cm 24
20230406_OLEP08_MMCC_1ug_MB_24min_AS_dDIA_10ms_2Th_ MMCC Astral 500% 10 ms 2 Th 300 Th dDIA 110 cm 24
20230406_OLEP08_MMCC_1ug_MB_24min_OT_75Th_23ms_2Th_ MMCC Orbitrap 800% 23 ms 2 Th 500-575 110 cm 24
20230410_OLEP08_HeLa_1ug_MB_24min_AS_23ms_2Th_ChrLib_ 200 ng HeLa  Astral 500% 23 ms 2 Th 100 Th GPF 110 cm 24

MMCC: Matrix-matched calibration curve of HeLa into SILAC labeled HeLa. Consists of 9 dilution points injected in triplicate.

EV plasma: 1 ug EV enriched plasma

Total plasma: 1 ug of total plasma

GPF: 6 gas phase fraction runs, each covering 100 Da range

dDIA: dynamic DIA

Clustergrammer Heatmap
Download 11:30:394,45331,37331,373137,8076 11:30:394,40533,03533,035142,4076 11:30:394,95240,67140,671199,2726 11:30:395,16344,66844,668217,5596 11:30:393,37118,83018,83079,2726 11:30:393,69224,47424,474103,3606 11:30:394,27030,14030,140147,0166 11:30:393,73125,70425,704107,9326 11:30:394,70437,53437,534182,8896 11:30:394,77743,88243,882214,1206 11:30:394,12410,63610,63651,79427 11:30:397,70161,17361,173299,12227 11:30:398,17379,47079,470387,16227 11:30:396,88559,29659,296290,42627 11:30:398,10174,26874,268360,08627