Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Astral
ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Joshua J Coon
PROVIDER: MSV000093613 | MassIVE | Fri Dec 08 15:10:00 GMT 2023
REPOSITORIES: MassIVE
Similar Datasets
Project description:We highlight the leap in performance made possible with new instrumentation in the Thermo Scientific Orbitrap Astral™ mass spectrometer. Integrated alongside a high-resolution accurate mass (HRAM) Orbitrap analyzer, the instrument introduces a fast-switching quadrupole mass filter congruent with downstream speeds of ion detection, a dual-pressure ion processor cell which fragments ions in the high-pressure cell before they are moved to the low-pressure cell for further accumulation and orthogonal extraction to the Astral analyzer, and a high dynamic range detector. Altogether, ions can be manipulated in five stages of the MS at once (Orbitrap, ion routing multipole, two ion processor stages, Astral analyzer), allowing for a high degree of parallelization which enables MS/MS scan speeds of 200 Hz while HRAM MS1 full scans are synchronously acquired in the Orbitrap analyzer. To provide such fast scan speeds, the Astral analyzer is capable of single-ion detection sensitivity like a linear ion trap, all while enabling resolving powers of 80,000 at m/z 524. Here, in triplicate 7-min microflow active LC gradients on the Orbitrap Astral MS, we report 7,852 protein groups from 94,267 peptides on average. When employing 15-, 30-, and 60-min active, nano-LC gradients, triplicate experiments yield an average of 9,831, 10,411, and 10,645 unique protein groups from 195,612, 234,406, and 245,754 unique peptides, respectively. These nanoflow methods delivered a median sequence coverage of 38%, 42.4% and 44.4% across identified proteins for each respective gradient length. Our 30-min method delivered approximately 347 proteins per minute. Finally, we applied our 30-min active gradient method to analyze a CRISPR-Cas9 knockout (KO) of the mitochondrial gene MGME1 in human HAP1 cells. There, we quantified an average of 10,353 proteins across three technical replicates and find 449 significantly up- or down-regulated proteins relative to wild-type (WT) HAP1 proteins. Proteins from the same sample quantified using a 61-min active gradient Orbitrap Ascend DIA method show strong fold-change correlations to and gene ontology (GO) term agreement with our results.Building upon the immense progress of the mass spectrometry and proteomics communities of the past ten years, here we report alongside other recent works, that the one-hour human proteome is now within reach.
2024-03-29 | PXD049028 | Pride
Project description:We evaluated the performance of the Orbitrap Ascend in comparison to the Orbitrap Eclipse in RTS-SPS-MS3-based analysis of human proteomes using fractionated and unfractionated samples labeled with TMT and TMTpro reagents. We did this comparison in the context of different sample input amounts; we analyzed differences in quantitative precision; we compared the effectiveness in identifying protein abundance changes induced by treatment with covalent inhibitors of PIN1.
2023-10-13 | PXD043660 | Pride
Project description:Mass spectrometry is the premier tool for identifying and quantifying protein phosphorylation on a global scale. Analysis of phosphopeptides requires enrichment, and even after the samples remain highly complex and exhibit broad dynamic range of abundance. Achieving maximal depth of coverage for phosphoproteomics therefore typically necessitates offline liquid chromatography prefractionation, a time-consuming and laborious approach. Here, we incorporate a recently commercialized aerodynamic high-field asymmetric waveform ion mobility spectrometry (FAIMS) device into the phosphoproteomic workflow. We characterize the effects of phosphorylation on the FAIMS separation, describe optimized compensation voltage settings for unlabeled phosphopeptides, and demonstrate the advantages of FAIMS-enabled gas-phase fractionation. Standard FAIMS single-shot analyses identified around 15-20% additional phosphorylation sites than control experiments without FAIMS. In comparison to liquid chromatography prefractionation, FAIMS experiments yielded similar or superior results when analyzing up to four discrete gas-phase fractions. Although using FAIMS led to a modest reduction in the precision of quantitative measurements when using label-free approaches, the data collected with FAIMS yielded a 26% increase in total reproducible measurements. Overall, we conclude that the new FAIMS technology is a valuable addition to any phosphoproteomic workflow, with greater benefits emerging from longer analyses and higher amounts of material.
| S-EPMC8051555 | biostudies-literature
Project description:In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and we present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. Here we show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.
2022-03-14 | PXD029320 | Pride
Project description:The Orbitrap Ascend Tribrid mass spectrometer was systematically evaluated in the context of shotgun proteomics, and its performance was compared to that of the previous generation of Tribrid instrument - Orbitrap Eclipse
2023-03-10 | MSV000091467 | MassIVE
Project description:We modified a dual pressure linear ion trap Orbitrap to permit infrared multiphoton dissociation (IRMPD) in the higher energy collisional dissociation (HCD) cell for high resolution analysis. A number of parameters, including the pressures of the C-trap and HCD cell, the radio frequency (rf) amplitude applied to the C-trap, and the HCD DC offset, were evaluated to optimize IRMPD efficiency and maintain a high signal-to-noise ratio. IRMPD was utilized for characterization of phosphopeptides, supercharged peptides, and N-terminal modified peptides, as well as for top-down protein analysis. The high resolution and high mass accuracy capabilities of the Orbitrap analyzer facilitated confident assignment of product ions arising from IRMPD.
| S-EPMC3202985 | biostudies-literature
Project description: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. Narrowwindow 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.
2024-02-01 | PXD046372 | Pride
Project description: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. Narrowwindow 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.
2024-02-01 | PXD046444 | Pride
Project description: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. Narrowwindow 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.
2024-02-01 | PXD046357 | Pride
Project description: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. Narrowwindow 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.
2024-02-01 | PXD046417 | Pride