Expert Advice: Unlocking efficiency in proteomics with TMT labeling

While genomics and transcriptomics have seen rapid advancements in high throughput analysis, achieving multiplex capabilities in mass spectrometry-based proteomics has remained challenging. The ability to analyze multiple samples in a single experiment not only saves researchers time but also accelerates discovery, especially in large-scale studies involving numerous samples.

The first step before conducting any experiments is to talk to somebody who is an expert in the field to understand what mass spectrometry can and cannot do.
- Shawn Li, Western University

Tandem mass tag (TMT) labeling helps bridge this gap in proteomics. The TMT technique involves labeling peptides from different samples with distinct chemical tags. These tags share the same mass but possess different isotopic compositions. This design allows researchers to differentiate peptides originating from different samples, enabling the simultaneous identification and quantification of multiple samples within a single mass spectrometry run. By adopting TMT labeling in mass spectrometry analysis, researchers can efficiently compare protein expression levels across multiple samples or experimental conditions.

Shawn Li, a biochemist from Western University, and his team are leveraging TMT mass spectrometry to unravel the molecular and epigenetic mechanisms underlying cancer development. By deciphering complex proteomic profiles associated with various cancer types, Li aims to identify novel protein- and peptide-based diagnostic and therapeutic agents for cancer.

What led you to use TMT labeling for your research?

My training involved a mix of organic chemistry and biochemistry, with a focus on protein biochemistry and structural biology. About 15 years ago, I started working with mass spectrometry. The reason for this shift was that when I was studying protein modifications like phosphorylation and methylation, I found it challenging to find antibodies specifically targeting these modified proteins, which was frustrating. I started exploring mass spectrometry as an alternative because I could identify where the modifications are on a protein without relying on antibodies. In the early days, I used techniques like multiple reaction monitoring and selected reaction monitoring to zoom in on specific proteins. About a few years ago, TMT labeling became quite popular, and we started to play around it.

What are the advantages of the TMT labeling technique?

It is a pretty cool technique as it allows scientists to multiplex samples. Today we can analyze 18 samples, which overcomes the throughput issue associated with mass spectrometry. Also, if scientists are limited by the quantity of samples, TMT labeling can be useful. For example, when dealing with micrograms of samples, it’s challenging to analyze them individually at such small volumes. However, by combining them, a lot more material becomes available for analysis. Another benefit of TMT is detecting trace amounts of post-translational protein or peptide modifications. For example, it can be very difficult to look at protein tyrosine phosphorylation, which accounts for only five percent of the total phosphorylated species in cells. This can be a limiting factor for a lot of studies, especially working with clinical samples. TMT is a good approach to overcome that, where combining small samples can increase the volume by ten or even 20 times.

How do you ensure labeling efficiency in TMT experiments?

Having high quality protein or peptides is essential, which is why we pre-purify our samples to ensure their quality. One thing to note is that TMT reagents are sensitive to moisture. We don’t advise people to reconstitute TMT reagents and use them repeatedly, as the labeling efficiency can go down very quickly. To maintain effectiveness, it’s best to use freshly prepared samples and TMT reagents just once. Additionally, we always do quality control by measuring the proportion of labeled peptides using a mass spectrometer. We aim to have 95 percent of peptides labeled before progressing to the next step.

What is the biggest challenge in TMT experiments for obtaining accurate results?

One of the most difficult issues is batch-to-batch variability in TMT experiments. To overcome this issue, it’s essential to conduct all experiments together. Performing TMT labeling on one batch one day and another batch the next day almost guarantees batch-to-batch discrepancies no matter how well we prepare the samples. Secondly, it’s important to reserve a mass spectrometry channel for data normalization. This channel typically involves a mixture of all samples, or at least representative ones. Labeling this mixture alongside other samples on the same day allows us to normalize the data from batch to batch.

How do you enhance the detection of low-abundance proteins?

In investigating phosphorylated proteins using TMT labeling, we incorporate a boost sample, which is a similar sample to what we analyze, such as a cell line, into our analysis. We treat the boost sample with phosphatase inhibitors to preserve the phosphorylation state of the proteins, which amplifies the phosphorylation signal in mass spectrometry analysis. Consequently, this augmentation enhances the mass spectrometer’s capability to detect phosphorylated peptides across all samples. This approach can significantly improve the detection limit and increase the number of molecules we can identify. By combining this approach with peptide enrichment, we can further enhance the detection limit.

What advice would you give to researchers for conducting a successful TMT experiment?

While mass spectrometry can answer certain questions, it cannot answer all of them. The first step before conducting any experiments is to talk to somebody who is an expert in the field to understand what mass spectrometry can and cannot do. Mastering TMT labeling isn’t too challenging, but it does require some learning. To perform a single experiment, it’s a good idea to collaborate with someone experienced in the technique. Otherwise, it’s essential to have a two-way discussion with a mass spectrometry expert about experimental design, sample requirements, and data analysis.

Where do you see the future of mass spectrometry-based proteomics heading?

The throughput of mass spectrometry analysis doesn’t match up to the efficiency of transcriptomic mapping or DNA sequencing yet, but the multiplexing capability will certainly improve. Future advancements will also rely on enhancing detection sensitivity, reducing turnaround time, and automating sample processing. Additionally, there is a trend toward integrating mass spectrometry with genomic analysis at the single cell level or in a spatially defined manner. Using multiomics is promising for analyzing clinical samples, and many labs are starting to adopt this approach. The next challenge will be combining these techniques onto a single platform.

This interview has been condensed and edited for clarity.