Unveiling proteomic insights: Orbitrap Astral's performance in multiplexed TMT analyses of brain Organoids in Alzheimer's disease research

New mass spectrometry technologies are crucial for driving scientific progress. In the last years, the development of fast, highly sensitive and resolution mass spectrometers allowing deep proteome coverage and accurate protein quantification has significantly expanded the capabilities and applications of this analytical technique.

The Orbitrap Astral state-of-the-art mass spectrometer delivers unprecedent performance in proteomics analyses. While the Orbitrap Astral has demonstrated impressive performance in label-free proteomics workflows, its applicability in multiplexed experiments, such as tandem mass tag (TMT) analyses, has not been extensively explored yet. To address this question, we have compared here the performance of the Orbitrap Astral to other leading Orbitrap-based mass spectrometers, the Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid, in TMT-based quantification experiments. To this end, we have investigated the protein content of brain organoids derived from healthy and Alzheimer's disease (AD) patients, which mimic the healthy and pathological brain context, respectively. A 16-plex TMT experiment was performed to comprehensively characterize the proteome of these organoids, representing two healthy brain models and two AD models (overexpressing APP or with a mutation in PSEN1), harvested at two time points (5 weeks and 2 months). AD is the most common type of dementia worldwide, and an in-depth characterization of the pathological changes during disease progression is crucial to elucidate novel proteins and biological processes that may serve as prognostic or diagnostic biomarkers, as well as potential therapeutic targets. The comparison of four different DDA-based TMT LC-MS/MS methods using the previously described three mass spectrometers aimed to identify the best approach for sensitive and accurate analysis of these organoids TMT samples.

After protein extraction, reduction, and alkylation, proteins were trypsin digested and TMT labeled. Labeled peptides were pooled together, and off-line fractionated into 12 fractions using a high-pH reverse phase column. Individual fractions were then analyzed by mass spectrometry on the three mass spectrometers in a 2-hours gradient. In addition, a 30-minutes gradient was also investigated with the Orbitrap Astral. Then, raw data were analyzed with the MaxQuant (version 2.4.2) and with the R program for the identification of dysregulated proteins in the disease.

The results obtained indicate that the Astral mass analyzer can deliver comparable or even superior depth of proteome coverage and quantification accuracy compared to the established Orbitrap platforms. A higher number of peptide and protein identifications were obtained with the Orbitrap Astral mass spectrometer, with significant improvements observed when using a 2-hour gradient instead of a 30-minute gradient.