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A novel ultra-high resolution multi-reflecting time-of-flight mass spectrometer in Y-MRT configuration for bottom-up proteomics

ABSTRACT: The first results of using a new type of ultrahigh-resolution mass analyzer based on a planar multi-pass time-of-flight mass spectrometer with periodic reflecting lenses (Y-MRT MS) for bottom-up whole-proteome analysis are presented. The instrument achieves a resolving power of 1,000,000 for peptides across an m/z range of 200-2000, with a high repetition rate of 300 Hz (averaged to 1 Hz for enhanced dynamic range). In preliminary experiments, a single 30-minute gradient HPLC separation of 1 µg proteolytic cell digest yielded over 3700 protein groups (4500 at 5% FDR) in DirectMS1 mode (without fragmentation). Combining three technical runs increased this to 5200 protein groups at 5% FDR. Peptide ion mass measurements demonstrated an accuracy of 100-150 ppb across the whole m/z range, with a dynamic range exceeding 10⁴. In DIA mode (SWATH-DIA, 20 Th window, 30-minute gradient), 4200 protein IDs were obtained at 1% FDR. These results highlight Y-MRT mass analyzer potential as a powerful addition to existing high resolution mass spectrometers for bottom-up proteomics. Further improvements in proteome coverage and analysis time are anticipated with optimized HPLC configurations and the integration of gas-phase ion mobility separation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Mark Ivanov

LAB HEAD: Mikhail. V. Gorshkov

PROVIDER: PXD065853 | Pride | 2025-11-05

REPOSITORIES: Pride

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			Action	DRS
	DIANN_report_DIA_30minLC_1000ng_rep1.pg_matrix.tsv	Tabular
	DIANN_report_DIA_30minLC_1000ng_rep2.pg_matrix.tsv	Tabular
	DIA_30minLC_1000ng_rep1.mzML	Mzml
	DIA_30minLC_1000ng_rep2.mzML	Mzml
	MS1_10minLC_scan05s_250ng_rep1.features_proteins.tsv	Tabular

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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.

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A novel ultra-high resolution multi-reflecting time-of-flight mass spectrometer in Y-MRT configuration for bottom-up proteomics

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