Sample multiplexing for targeted pathway proteomics in aging mice
| Autor: | Haopeng Xiao, José Navarrete-Perea, Jeffrey Knott, Steven P. Gygi, Devin K. Schweppe, Mark P. Jedrychowski, Edward T. Chouchani, John C. Rogers, Qing Yu |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Proteomics
Aging tissue-specific aging Computer science Sample (statistics) Computational biology Tandem mass tag Orbitrap Mass spectrometry Multiplexing law.invention Mice law targeted pathway proteomics Animals Tomahto Multidisciplinary Systems Biology Biological Sciences Isobaric labeling Chemistry Proteome Physical Sciences isobaric labeling real-time instrument control |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Significance A user-friendly workflow, termed Tomahto, enables real-time targeted pathway proteomics assays using two-dimensional multiplexing technology. Hundreds of proteins of interest from a multitude of samples can be targeted and accurately quantified in a remarkably sensitive fashion. We highlight Tomahto’s ease of use, sensitivity, and accuracy and present proof-of-principle utility by targeting 260 metabolism- and inflammation-related proteins across 90 samples (nine tissues from five old and five young mice). Tissue-specific aging effects are presented with specific interest in metabolic and inflammatory pathways of white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community. Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over targeted sample multiplexing experiments, termed Tomahto, and present its implementation on the Orbitrap Tribrid mass spectrometer platform. Importantly, this software monitors via the external desktop computer to the data stream and inserts optimized MS2 and MS3 scans in real time based on an application programming interface with the mass spectrometer. Hundreds of proteins of interest from diverse biological samples can be targeted and accurately quantified in a sensitive and high-throughput fashion. It achieves sensitivity comparable to, if not better than, deep fractionation and requires minimal total sample input (∼10 µg). As a proof-of-principle experiment, we selected four pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (nine tissues from five old and five young mice) to explore effects of aging. Tissue-specific aging is presented here and we highlight the role of inflammation- and metabolism-related processes in white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community. |
| Databáze: | OpenAIRE |
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