We hope that this protocol encourages other research groups to consider incorporating this powerful methodology into their studies. In addition, we describe how to analyze TMT data to screen thousands of proteins in a short amount of time. The major benefits of this optimized protocol are that it reduces the costs of labeling, improves protein extraction, and consistently generates high-quality data. ![]() The sample preparation protocol consists of the following major steps: extraction, quantification, precipitation, digestion, and labeling. For detailed descriptions of the mass spectroscopy portion of this process please see Kirshenbaum et al. It can be beneficial to perform the sample preparation and labeling prior to submission to a core because the labeled tryptic peptides are more stable than raw frozen samples, not all cores have experience handling all sample types, and preparing samples in a laboratory can save time for cores, which often have long backlogs. Here, we describe a detailed method to perform multiplex TMT quantitative proteomics analyses using tissue samples or cell pellets. In so doing, we were able to demonstrate widespread improvement in the cardiac profiles of BTHS mice treated with gene therapy and identify novel proteins impacted by BTHS that revealed novel therapeutic pathways involved in cardiomyopathies. We recently used TMT to characterize the cardiac proteomic profile of a mouse model of Barth Syndrome (BTHS) 17. As a result, it is possible to measure peptide abundance in multiple conditions with biological replicates at the same time 14, 15, 16. As of this publication date, TMT kits have been developed that can simultaneously label 6, 10, 11, or 16 samples. TMT labeling is a powerful method due to its increased sensitivity to detect relative protein expression levels and posttranslational modifications 13. In 2003, a novel and robust proteomics technique involving tandem mass tag (TMT) isobaric labels was introduced to the field 12. ![]() The main limitation to the SILAC approach is primarily the reduced cell growth rate caused by isotope label incorporation, which can be particularly challenging in relatively sensitive cell lines modeling human diseases 11. The advantage of this technique is its efficiency and precise labeling 9. It consists of metabolic labeling of cells that are incubated in medium lacking a standard essential amino acid and supplemented with an isotope-labeled version of that specific amino acid 10. The stable isotope labeling by amino acids in cell culture (SILAC) method became the next popular approach to identify and quantify protein abundance in samples 9. General disadvantages to the 2D-PAGE approach are that it is time-consuming, does not work well for hydrophobic proteins, and there are limitations in the total number of proteins assessed due to low sensitivity 7, 8. For years, the combination of 2D-PAGE and subsequent tandem mass spectrometry performed on each gel component was the most common untargeted protein expression analysis technique performed and identified numerous previously unknown protein expression profiles 5, 6. The 2D method separates proteins based on charge (isoelectric focusing, IEF) and molecular mass (sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE) 4. The initial descriptions of such studies were published in 1975 and demonstrated the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for this purpose 1, 3. Proteomic analyses enable the investigation of mechanisms and cellular processes involved in disease development, therapeutic pathways, and healthy systems using techniques to perform relative quantitation of protein expression levels 2. The term “proteomics” was first defined as the large-scale characterization of the entire protein complement of a cell, tissue, or organism 1.
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