An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis

Abstract

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 increased cost due to the use of labeling reagents. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile organic 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 effectively labels 6-12 μg peptides, yielding efficient TMT labeling efficiency (~99%) in both microbiome and mammalian cell line samples. This streamlined workflow decreases reagent loss and reduces inter-sample variations. 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 streamlined and robust quantitative proteomics and metaproteomics in high throughput applications.

Pairing Metagenomics and Metaproteomics to Characterize Ecological Niches and Metabolic Essentiality of gut microbiomes

“…Samples were cultured in technical triplicates, and were taken at 0, 1, 5, 12, and 24 hours of culturing for optical density and metaproteomic analyses. 11-plex tandem mass tag (TMT11plex) was used for metaproteomic quantification 65 for a total of 189 samples. To reflect the effect of introduced sugars on protein expression levels, we used log2 of the ratio between normalized protein abundances/intensities (see Methods for details) in the treatment and that in the control group (i.e.…”

“…Proteins were digested with trypsin desalted 38 for LC-MS/MS analysis using an Orbitrap Exploris 480 mass spectrometer. For the cultured microbiomes, an automated process extracted and purified proteins, which were then digested, desalted, and quantified using TMT11plex 39 , ensuring mixed representation in labeling to avoid bias. Samples underwent a 2-hour LC gradient and were analyzed by mass spectrometry.…”

“…6a). Samples were cultured in technical triplicates, and protein abundances were quantified at 0, 1, 5, 12, and 24 hours using 11-plex tandem mass tag (TMT11plex) 39 for a total of 189 samples. We analyzed the Bray-Curtis dissimilarity of metaproteomes over time, finding that more complex sugars induce more pronounced alterations in protein profiles (Supplementary Fig.…”

RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics

“…Therefore, we updated the protocol to overcome this limitation. New feature and components of the protocol include 1) optimization of the protein extraction and purification protocol; 2) automation of the protein digestion and desalting protocol; 3) introduction of TMT multiplexing technique for labeling and quantitation of peptides and proteins allowing for the analysis of up to 10 samples in one LC-MS/MS run [21]; and 4) a TMT-based statistical analysis streamline for clustering functional responses. We estimate that, for an experiment containing 320 samples (four 96-well plates), this updated workflow requires only six days for culturing and sample processing, and it shortens LC-MS/MS sample analysis time from approximately 20 days to 3 days.…”

“…The use of TMT labeling also significantly reduce LC-MS/MS time and cost [20]. To enable high-throughput sample analysis, we have developed a streamlined TMT labeling workflow using pre-aliquoted dry TMT in 96-well plates, which achieved comparable labeling efficiency and improved inter-sample consistency for quantitation [21]. This streamlined TMT labeling workflow is fully compatible with automation of the metaproteomics sample preparation steps which altogether can significantly increase the robustness of liquid handling compared to manual operation, speed up the experimental workflow and further increase the throughput [22].…”