Isn’t it great when you can do two or even three tasks at once? I often check my emails on my smartphone as I shop for groceries, whilst listening to my favorite podcasts and learning something new.
But why stop at doing three things simultaneously? I often imagine how productive I’d be if I could do 10 jobs at the same time!
Well, the latest isobaric chemical tag reagents are helping proteomics researchers do just that.
TMTs are… terrific multi-taskers
Many quantitative proteomics approaches involve comparing changes in protein expression between multiple states to obtain a complete picture of how factors such as time, a disease or the environment affect the proteome. So the ability to perform these experiments quickly, accurately and with a large range of analysis variables is a big advantage.
Isobaric chemical tags are ideal for this approach as they can be used to identify and quantify relative changes in complex protein samples across several different experimental conditions in a single analysis. Thermo Scientific Tandem Mass Tag (TMT) reagents, such as the TMT 10plex tags, greatly increase the multiplexing potential of this approach – allowing simultaneous analysis of up to 10 different samples.
TMTs consist of a signature reporter group containing 13C and 15N isotopes connected to an amine reactive group by a spacer arm. By shifting the location of the heavy isotopes between the reporter group and the spacer, the total mass and chemical structure of each tag can be kept the same (isobaric), whilst ensuring each reporter ion is unique.
Like what you are learning?
These tags can covalently bind to the N-terminus or lysine side chains of peptides produced from protein digestions. The separate digests can be pooled together, allowing a range of samples to be studied simultaneously by tandem mass spectrometry (MS/MS). Upon fragmentation, each version of the tag produces a unique reporter ion, allowing peptides from multiple digestions to be investigated in one go, meaning proteomics researchers can study a wider range of conditions more conveniently, and have greater confidence in replicate experiments.
Other TMT reagents, such as those in the TMTzero, TMTduplex and TMTsixplex kits, share the same reagent structure. This is useful when optimizing studies, as it allows consistent and efficient transition from method development to multiplex quantitation.
TMTs let you… try more things
The wide range of reporter ions offered by TMT reagents aren’t just allowing scientists to generate results more quickly, they’re helping them unravel the intricacies of protein interactions in cells, tissues and organisms. Optimized for use with high resolution MS/MS platforms, when used together these multiplexing tools are helping researchers move beyond basic protein identification to multidimensional characterization of the proteome.
Hyperplexed localization of organelle proteins (hyperLOPIT), for example, is a new approach which combines density gradient centrifugation (to separate cellular organelles) with TMT10plex labeling to determine the spatial resolution of proteins within a cell. Used in conjunction with the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer, the technique has recently been used to provide data on the cellular distribution of more than 5,000 proteins. This information could be used, for instance, to reveal how organelles are organized and shed light on functional networks within the cell.
Multiplexed proteomics is revealing new insight into the molecular mechanisms underpinning complex biological processes and diseases. And with flexible isobaric tags, coupled with the latest MS/MS technology, this approach is becoming more convenient, more efficient and more powerful.
If only all my shopping trips could be that good!
- TMT workflow page
- Advances in Accurate Proteomics Quantitation using Isobaric Tags
- The Magic of Multiplexing in LC/MS
- Protein Quantitation Part 1: Mass Spectrometry Sample Prep for New and Experienced Users
- Protein Quantitation Part 2: LC-MS – Practical Guidelines for Robust, Accurate, and Meaningful Quantitative Proteomics
- Sample Multiplexing with 10-plex Tandem Mass Tags (TMT) Opens 10 Doors at Once, Providing a New Perspective on any Biology