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Detecting unexpected modifications from phosphoproteomic data generated by Orbitrap Astral mass spectrometer

ABSTRACT: We performed a phosphoproteomic analysis using tryptic phosphopeptides derived from HEK293T cells. The sample preparation workflow consisted of titanium dioxide (TiO₂)-based phosphopeptide enrichment, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Orbitrap Astral mass spectrometer.

ORGANISM(S): Homo Sapiens

SUBMITTER: Chao Liu

PROVIDER: PXD067374 | iProX | Fri Aug 15 00:00:00 GMT+01:00 2025

REPOSITORIES: iProX

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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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Detecting unexpected modifications from phosphoproteomic data generated by Orbitrap Astral mass spectrometer

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