Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

Abstract

The complexity of human physiology arises from well-orchestrated interactions between trillions of single cells in the body. While single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of cell diversity, gene expression alone does not fully characterize cell phenotypes. Additional molecular dimensions, such as proteins, are needed to define cellular states accurately. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool for comprehensive protein analysis, including single-cell applications. However, challenges remain in terms of throughput and proteomic depth, in order to maximize the biological impact of single-cell proteomics by Mass Spectrometry (scp-MS) workflows. This study leverages a novel high-resolution, accurate mass (HRAM) instrument platform, consisting of both an Orbitrap and an innovative HRAM Asymmetric Track Lossless (Astral) analyzer. The Astral analyzer offers high sensitivity and resolution through lossless ion transfer and a unique flight track design. We evaluate the performance of the Thermo Scientific Orbitrap Astral MS using Data-Independent Acquisition (DIA) and assess proteome depth and quantitative precision for ultra-low input samples. Optimal DIA method parameters for single-cell proteomics are identified, and we demonstrate the ability of the instrument to study cell cycle dynamics in Human Embryonic Kidney (HEK293) cells, and cancer cell heterogeneity in a primary Acute Myeloid Leukemia (AML) culture model.

High-Throughput Nanoflow Proteomics Using a Dual-Column Electrospray Source

“…Recent advances in liquid chromatography (LC) and especially mass spectrometry (MS) allow researchers to dig deeper in the proteome of increasingly smaller samples. − MS infrastructure is now able to scan analytes eluting from the LC system with incredible speed and sensitivity, using up to nearly 100% of ions that are entering the MS system. , This is especially valuable for single cell and (single cell) laser capture microdissected (LCM) tissue proteomics applications, as well as low input clinical proteomics. Such applications, as well as clinical proteomics samples in general often consist of large sample cohorts, benefiting the most of high throughput.…”

High-throughput nano-flow proteomics using a dual-column electrospray source

“…Recent advances in liquid chromatography (LC) and especially mass spectrometry (MS) allow researchers to dig deeper in the proteome of increasingly smaller samples 1,2,3,4 . MS infrastructure is now able to scan analytes eluting from the LC system with incredible speed and sensitivity, using up to nearly a 100% of ions that are entering the MS system 5,6 .…”

Refining Spatial Proteomics by Mass Spectrometry: An Efficient Workflow Tailored for Archival Tissue

“…Yet, a prevailing limiting factor for the use of FFPE for proteomics has been the need for substantial sample quantities to acquire deep protein coverage, generally limiting FFPE proteomics by Mass Spectrometry (FFPE-MS) to bulk tissue analysis (3). This is highly problematic in the context of spatial proteomics, as it obscures the cell heterogeneity of the various cell populations within tissues such as cancer, which is defined by being exceptionally heterogeneous (4-8). In addition, there are limitations of available tissue associated with rare diseases, which can be too scarce or inadequately abundant to support comprehensive retrospective proteomic analyses.…”

“…The accelerated developments within the field of Liquid Chromatography-Mass Spectrometry (LC-MS) have recently enabled a level of sensitivity capable of analyzing ultra-low sample input (8,9), which consequently sparked a rapid emergence of methods transitioning from bulk or high-load input to low-load and near single-cell analysis (10-15). However, proteome coverage and ease of implementation on large-scale cohorts is still limited by sample preparation, which is especially true for FFPE tissue.…”