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Introduction
Proteomics is the large-scale study of the complete set of proteins, known as proteome, within a cell, tissue, or organism. Mapping and understanding the proteome is vital for researchers to better understand the expression, structure, function, and interaction of proteins for applications in disease diagnosis, drug discovery, and understanding fundamental biological processes. Mass spectrometry (MS) is a widely used technique in proteomics for identifying and characterizing molecules within a sample based on their mass-to-charge ratio.
Tandem Mass Tag (TMT) labeling is a popular chemical labeling method for multiplexing applications in quantitative proteomics to measure protein abundance across multiple samples simultaneously. TMTs are small, reactive amine tags that attach to peptides. TMT-labeled peptides fragment during MS measurement to release multiple reporter ions. As TMT are isobaric reagents, each fragmented reporter ion confers a separate identity, enabling the identification and quantification of different samples during a single MS run. Using TMT-labeling, researchers can simultaneously quantify up to 18 samples, significantly reducing time spent on extensive preparation stages.
Challenges
Chemical labeling reagents have specific handling and compatibility requirements to achieve optimal labeling efficiencies. When combined with the multi-step methods of proteomics sample preparation for mass spectrometry analysis, these protocols can be time-consuming, taking up to two full days to prepare adequate samples for MS. The sample preparation stages will also limit the accuracy of the MS results, as a single error within the two-day work window can hemorrhage result quality.
Labeling conditions must also be optimized for high efficiency without excessive reagent use, as TMT reagents are usually expensive, and the efficiency of labeling directly impacts the quantification and identification of peptides. However, using an insufficient quantity of the TMT reagent can lead to incomplete labeling, which adversely affects peptide identification and quantification. This limits both the reproducibility and accuracy of the data, compromising MS performance for a guarantee in labeling.
Solution
To overcome these limitations, PreOmics has developed the iST-NHS kit, which is fully compatible with chemical labeling reagents. It provides a streamlined and fast solution for robust and reproducible sample preparation compatible with chemical labeling such as iTRAQ or TMT, achieving excellent labeling efficiency and reproducibility required for robust quantification.
The chemical labeling is directly integrated within the intuitive four-step protocol (LYSE, DIGEST, LABEL, PURIFY) of the iST-NHS kit. It is fully compatible with 96-well plate formats, as well as automated liquid handling platforms, enabling high-throughput chemical labeling experiments. Using the iST-NHS kit, researchers can decrease MS downtime and guarantee reliable results.
Optimizing labeling ratios
To test the TMT-labeling efficiency, the iST-NHS kit was employed to prepare yeast samples, and the ratio of TMT reagent per g of peptide input material was compared. Most TMT manufacturers recommend an 8:1 TMT-to-peptide ratio to achieve optimal labeling efficiency, but it can sometimes compromise quantification accuracy if not carefully controlled. The iST-NHS technology provided a 98% labeling rate at a 4:1 ratio, decreasing the amount of TMT tags required for MS. Preparation with a 4:1 ratio also reduces reagent costs, making the iST-NHS kit a more affordable long-term alternative to standard methods. High reproducibility was also observed in the data obtained, marking the iST-NHS kit as a useful tool for highly accurate and reproducible chemical labeling experiments.
Practical benefits of the iST-NHS kit
The iST-NHS kit provides reproducible, high-quality MS preparation within several hours, decreasing the processing time from 2 days to below 4 hours. The iST-NHS also guarantees high labeling efficiency, favoring a 4:1 tag: peptide ratio instead of the traditional 8:1 ratio recommended by manufacturers, saving money on TMT tags.
This technology has already been used to reveal altered histone modification landscapes in heart failure patients. The study employing a multi-omics approach using samples prepared with iST-NHS labeled with TMT-16plex revealed that histone post-translational modifications are deposited due to metabolic changes, showing a 16-fold upregulation of the H3K27ac and significant upregulation of other active transcriptional markers like H3K36me3 and H3S28ph.
The iST-NHS kit is fully compatible with PreOmics’ propriety automation platform PreON, enabling the processing of up to 18 labeled samples using the TMTproTM 18-plex upgrade set. Combining iST-NHS with PreON automatic sample preparation can reduce hands-on time to 5 minutes with high labeling efficiency. This enables efficient high-throughput chemical labeling experiments, saving valuable time whilst guaranteeing large reproducibility and low variability in results.
References & Further Reading
1. Al-Amrani S, Al-Jabri Z, Al-Zaabi A, Alshekaili J, Al-Khabori M. Proteomics: Concepts and applications in human medicine. World J Biol Chem. 2021 Sep 27;12(5):57-69.
2. Guo Y, Yu D, Cupp-Sutton KA, Liu X, Wu S. Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics. Anal Chim Acta. 2022 Aug 15;1221:340037.
3. Hutchinson-Bunch C, Sanford JA, Hansen JR, Gritsenko MA, Rodland KD, Piehowski PD, Qian WJ, Adkins JN. Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH. ACS Omega. 2021 May 6;6(19):12660-12666.
4. Precise and robust automated proteomics sample prep with PreON and TMTpro-18 plex.
5. Application Note: iST-NHS Sample Preparation kit
6. Jong-woo Yoo et al. Histone PTM profiling of human heart failure biopsies reveals H3K27ac-mediated metabolic dysfunction.
