Xu Zhang

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in –omic study of complex samples. While TMT‐based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample‐to‐sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre‐aliquoted dry TMT reagents in a 96 well plate or 12‐tube strip. As little as 50 μg dry TMT per channel was used to label 6–12 μg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre‐aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.