MSACL 2025 Abstract

Enhanced Sensitivity of a Modified Orbitrap Astral Mass Spectrometer for Deeper Proteome Coverage in Single-cell Proteomics Applications

Scott Peterman (1), Tabiwang N. Arrey (2), Julia Kraegenbring (2), Anjali Seth(3), Bernard Delanghe(2), Eduard Denisov(2), Johannes Petzoldt(2), Hamish Stewart(2), Eugen Damoc(2)
(1) Thermo Fisher Scientific, San Jose, California, (2) Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany, (3) Cellenion, Lyon, France

Scott Peterman, PhD (Presenter) Thermo Fisher Scientific

Relevant Financial Disclosures (within past 24 months, reported on Jul 16, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Single cell proteomics using liquid chromatography-mass spectrometry is transforming our understanding of cellular heterogeneity and function by enabling detailed analysis of protein expression in individual cells. However, to properly characterize these functions and heterogeneity, many single cells need to be analyzed. This raises challenges related to sample throughput and depth of proteome coverage. While throughput can be increased by multiplexing, e.g using TMT, achieving depth of coverage requires high sensitivity. Methods that provide the highest proteome coverage and throughput would be of great interest to single cell proteomics applications. Here we evaluated the depth of proteome coverage and throughput that can be achieved for low-input and single cell samples using a modified Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer.

METHODS:
For the dilution series, Pierce™ HeLa digest (20 µg) was reconstituted by adding 200 µL of 0.015% DDM/0.1% TFA, sonicated for 5 minutes, then diluted in 0.015% DDM/0.1% TFA to 5 ng/µL by adding the stock solution to 95 µL of 0.015% DDM solution in a low-binding sample well plate and vortexed for a few seconds. Diluted bulk HeLa digest (at different concentrations) and pre-digested single cells were separated using different gradients on the Thermo Scientific™ Vanquish™ Neo UHPLC system. Eluting peptides were measured on a modified Orbitrap Astral MS equipped with a Thermo Scientific™ FAIMS Pro™ interface. Data was processed with Thermo Scientific™ Proteome Discoverer™ software using Chimerys™ search algorithm and Spectronaut™ 19 software.

RESULTS:
The modified Orbitrap Astral MS was operated in DIA mode using the low input application mode. To evaluate its performance, 250 pg from the bulk HeLa digest was separated and analyzed. Triplicate raw data files were processed together without using a library, in Spectronaut 19. Compared to the standard Orbitrap Astral MS, we observed an increase in protein groups identifications of over 5% from 5,986 to 6,308 and over 6% in peptides identifications from 50,935 to 54,181. Next, we performed a dilution series from 50 pg to 10 ng by injecting the appropriate amounts from the 5 ng/µL stock solution.

DISCUSSION:
For mass spectrometers utilizing ion accumulation, the efficiency and time devoted to accumulation becomes the most critical element for achieving ultimate sensitivity. By introducing parallel ion packet management, trapping instruments can increase data acquisition speed while spending longer accumulation times. The original Oribtrap Astral MS parallelized five different ion packets and dual mass analysis of precursors and product ions to deliver high protein sample coverage while maximizing data quality. The new instrument enhancements applied to the Orbitrap Astral MS added an additional accumulation stage in the source region to initiate the accumulation step for an extra 1-3 milliseconds. While this seems minor, we observed an increase in identified proteins and peptides across all concentrations, with an average gain of 5% in protein groups and 10% in peptides. Among the identified protein groups, more than 80% had less than 20% CVs.Furthermore, we also analyzed a small number of single cells and observed an average increase in protein groups identifications of 5.5 % (6,041 compared to 5,724) and 4% for peptide groups (36,156 to 34,993) when using the modified Orbitrap Astral MS and the low input application mode compared to the standard Orbitrap Astral MS.