Sample multiplexing for targeted pathway proteomics: application to aging mice

Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted TMT-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 first 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 (API) 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 comparable, if not better, sensitivity as deep fractionation and requires minimal total sample input (~10 µg). As a proof-of-principle experiment, we selected 4 pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (9 tissues from 5 old and 5 young mice) to explore effects of aging. Tissue-specific aging are 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.