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Streamlined TMT labeling workflow for proteomics and metaproteomics

ABSTRACT: This study established a streamlined TMT labeling workflow that is readily compatible with high throughput proteomics and metaproteomics sample preparation. The method was then evaluated using two different sample types, microbiome and intestinal Caco-2 cell line, with TMT11plex mix reagents.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Metagenome

TISSUE(S): Cell Culture

SUBMITTER: Xu Zhang

LAB HEAD: Xu Zhang

PROVIDER: PXD034791 | Pride | 2023-02-07

REPOSITORIES: Pride

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			Action	DRS
	20220309_Human_Sprot.fasta	Fasta
	20220513_DryTMT_Caco2_D100-1.raw	Raw
	20220513_DryTMT_Caco2_D100-2.raw	Raw
	20220513_DryTMT_Caco2_D100-3.raw	Raw
	20220513_DryTMT_Caco2_D100-4.raw	Raw

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Publications

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

Creskey Marybeth M Li Leyuan L Ning Zhibin Z Fekete Emily Ef EE Mayne Janice J Walker Krystal K Ampaw Anna A Ben Robert R Zhang Xu X Figeys Daniel D

Proteomics 20230113 21-22

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 sui ...[more]

PMID: 36528842

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Streamlined TMT labeling workflow for proteomics and metaproteomics

Project description:This study established a streamlined TMT labeling workflow that is readily compatible with high throughput proteomics and metaproteomics sample preparation. The method was then evaluated using two different sample types, microbiome and intestinal Caco-2 cell line, with TMT11plex mix reagents.

2023-02-07 | PXD034936 | Pride

Microbiota biobanking using ice recrystalization inhibitor

Project description:This study use different ice recrystalization inhibitors (IRIs) for better storing live microbiota. We evaluated whether the addition of IRIs can improve the cultivibility of microbiome and maintain their resposnes to prebiotic kestose. Frozen or fresh microbiota were cultured with or without kestose for 24 hours, and microbiota samples were collected for metaproteomics analysis.

2023-02-07 | PXD034979 | Pride

Effects of 5 drugs on the human gut microbiome.

Project description:We processed a human stool sample and cultured the processed microbiome in vitro under anaerobic condition for 48 hours using the rapidAIM assay. The microbiome was treated with either azathioprine, ciprofolxacin, diclofenac, nicatidine, paracetamol or control DMSO. Each drug was used at three different concentrations. We compared these results to those acquired using MS1-only data.

2022-07-29 | PXD024845 | Pride

Evaluate gut microbiome biobanking

Project description:A rapid ex vivo microbiome assay and metaproteomics approach was used for rapid evaluation of the cultivability of bio-banked live microbiota, which shows minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

2022-02-16 | PXD028857 | Pride

Effect of Edurans secretome of human microbiome

Project description:A rapid ex vivo microbiome assay and metaproteomics approach was used for evaluating the impacts of E. durans derived secretome on a human gut microbiome cultured in vitro.

2024-01-31 | PXD040751 | Pride

Multi-Level Analysis of Gut Microbiome Extracellular Vesicles-Host Interaction Reveals a Connection to Gut-Brain Axis Signaling

Project description:Microbiota-released extracellular vesicles (MEVs) have emerged as a key player in intercellular signaling. However, their involvement in the gut-brain axis has been poorly investigated. In this study, we aimed to investigate the cargo capacity of MEVs for bioactive metabolites and their interactions with the host. Metabolomics analysis identified various neuro-related compounds encapsulated within the 28 MEVs, such as arachidonyl-dopamine, gabapentin, glutamate, and N-acylethanolamines. 29 Metaproteomics unveiled an enrichment of enzymes involved in neuronal metabolism, primarily in the glutamine/glutamate/GABA pathway. The detected neuro-related proteins and metabolites were correlated with Bacteroides spp. A GABA-producing Bacteroides isolate, B. finegoldii, released EVs with a high GABA content (4 µM) as opposed to a low GABA-producing isolate, Phocaeicola massiliensis. MEVs exhibited a dose-dependent paracellular transport and were endocytosed by Caco-2 and hCMEC/D3 cells. RNA-Seq analyses showed that MEVs stimulate several immune pathways while suppressing cell apoptosis process. The in vivo biodistribution confirmed the presence of MEVs in the brain, liver, stomach, and spleen. Overall, our results highlight the ability of MEVs to cross the intestinal and blood-brain barriers to deliver their cargoes to distant organs, including the brain, where it may modulate the organ functionalities. MEVs could be an integral part of microbiome-host communications, with potential implication for the gut-brain axis.

2024-11-05 | PXD045162 | Pride

Functional effects of sweeteners on ex vivo human gut microbiome

Project description:We systematically investigated the responses of five human gut microbiomes to 21 common sweeteners, using an approach combining high-throughput ex vivo microbiome culturing and metaproteomics to quantify functional changes in different taxa. Hierarchical clustering based on metaproteomic responses of individual microbiomes resulted in two clusters. The first cluster was composed of non-caloric artificial sweeteners (NAS) and two sugar alcohols with shorter carbon backbones (4-5 carbon atoms), and the second cluster was composed of sugar alcohols with longer carbon backbones. The metaproteomic functional responses of the second cluster were similar to the prebiotic fructooligosaccharides and kestose, indicating that these sugar alcohol-type sweeteners have potential prebiotic functions.

2022-06-23 | PXD030458 | Pride

In-vitro human gut microbiota culture with added sugars

Project description:Three human gut microbiome samples from different individuals were cultured in an optimized culture medium with or without the presence of different sugars (10 mM glucose, 20 mM fructose, 10 mM glucose + 20 mM fructose, or 10 mM kestose). Samples were cultured in technical triplicates, and were taken at 0 hr, 1hr, 5 hr, 12 hr and 24 hr of culturing for optical density and metaproteomic analyses. Cultured microbiota cells were subjected to metaproteomics analysis using LC-MS/MS and a TMT approach.

2024-06-16 | PXD037925 | Pride

Effects of BAFASAL on human microbiome

Project description:This project aims to examinw the effects of an anti-Salmonella bacteriophage preparation, BAFASAL, on ex vivo human gut microbiome composition and function using a multi-omics approach

2022-02-17 | PXD027172 | Pride

Deciphering Differential Gut Bacterial Drug Metabolism with Activity-Based Protein Profiling

Project description:Gut bacterial β-glucuronidases (GUS) promote the toxic side effects of therapeutics by reactivating drugs from their inactive glucuronide conjugates. It is increasingly clear that the interindividual variability of bacterial GUS-producing species in the gut microbiota contributes to differential drug responses. Indeed, the anticancer drug irinotecan exhibits variable clinical toxicity outcomes that have been linked to interindividual differences in the composition of the gut microbiota. However, identification of the specific GUS enzymes responsible for drug metabolism in the context of the complexity of the human fecal microbiota has not been achieved. Here we pinpoint the specific bacterial GUS enzymes that reactivate SN-38, the active metabolite of irinotecan, from complex human fecal microbiota samples with activity-based protein profiling (ABPP). We identify and quantify gut bacterial GUS enzymes from human feces with ABPP-enabled proteomics and then integrate this information with ex vivo kinetics to reveal the specific GUS enzymes responsible for the reactivation of SN-38. The same ABPP approach also reveals the molecular basis for differential gut bacterial GUS inhibition between human fecal samples. Taken together, this work provides an unprecedented pipeline to identify the specific bacterial GUS enzymes responsible for drug-induced GI toxicity from the complexity of human feces, which may serve as highly precise biomarkers of clinical outcomes for irinotecan and other therapeutics.

2019-12-03 | PXD014864 | Pride

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