Quantitative Proteomics: Comparing the Big Three – iTRAQ, TMT, and SILAC

In a previous post, we covered quantitative proteomics and how it has emerged as an indispensable tool in modern biological research. The methodology helps scientists to understand the complexities of protein interactions, their relative abundances in samples, and how they change under different physiological or pathological conditions.

At the core of quantitative proteomics is a set of methods known as isotope labeling.

These techniques involve incorporating stable isotopes into proteins or peptides, which serve as markers to compare protein levels between different samples. Isotope labeling also allows for a more detailed and nuanced view of the proteome, to provide a dynamic picture of protein expression under varying conditions.

Among the different isotope labeling methods, there are techniques which have gained particular attention for their efficiency and wide-ranging applicability: iTRAQ (Isobaric Tags for Relative and Absolute Quantitation), TMT (Tandem Mass Tags), and SILAC (Stable Isotope Labeling by Amino acids in Cell culture).

Each of these methods has unique advantages, and they have already been used successfully in driving forward the field of quantitative proteomics. However, the same techniques also come with their own challenges, with each type best suited to specific research applications.

In this article, we are going to examine how these different methods compare and contrast, and when they should be used by scientists.

Method 1 – SILAC Methodology

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a metabolic labeling technique used for quantitative proteomics. In SILAC, cells are grown in culture media containing either ‘light’ or ‘heavy’ isotopes of essential amino acids, usually lysine and arginine.

The ‘light’ and ‘heavy’ proteins can then be mixed together and analyzed using mass spectrometry. The relative abundance of proteins in the sample is determined by comparing the peak intensities of the ‘light’ and ‘heavy’ peptide pairs in the mass spectra.

an overview of silac methodology
Image: www.researchgate.net

Advantages & Challenges of SILAC

The main advantage of the SILAC method is its simplicity and robustness.

As a metabolic labeling method, it avoids the potential for chemical artifacts associated with chemical labeling techniques. It also allows for accurate quantification, as the ‘light’ and ‘heavy’ peptides are chemically identical and therefore behave identically during separation and analysis.

Additionally, SILAC can be multiplexed, allowing for the comparison of multiple conditions in a single experiment.

SILAC has been further developed to label mammals including mice, fish, flies and worms and is called SILAM (stable isotope labeling in mammals).

While SILAC offers numerous benefits, it also has its limitations, SILAC requires a significant investment of time, as cells must be grown in the labeling media for several generations to ensure complete incorporation of the labeled amino acids.

Method 2 – iTRAQ Methodology

an overview of itraq methodology
Image: www.researchgate.net

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) is a chemical labeling technique methodology involving isobaric tags that bind to the N-terminus and side chain amines of amino acids.

These tags are ‘isobaric’ – identical in mass – but they fragment during tandem mass spectrometry (MS/MS) to produce reporter ions with different masses. The intensity of these reporter ions allows for the relative quantitation of proteins from different samples in the same experiment. This approach provides a way to compare the abundance of proteins across multiple samples simultaneously.

The Advantages and Limitations of iTRAQ

The major advantage of the iTRAQ method is its multiplexing capability, allowing multiple samples to be labeled with distinct tags and analyzed concurrently. This is an advantage because it reduces the variability caused by multiple separate runs, thus enhancing data reliability and consistency.

iTRAQ can also provide an overview of protein changes, hence making it an ideal method for large-scale proteomic studies. It is also beneficial for studying post-translational modifications, as the method labels all peptides, irrespective of their modification status.

A major challenge of iTRAQ is a phenomenon called ‘ratio compression’, where the quantitative ratios of the labeled peptides are underestimated due to co-isolation and co-fragmentation of unlabeled and labeled peptides. Because of its reliance on MS/MS for quantification, peptides that are not selected for fragmentation are not quantified.

iTRAQ is also more expensive than other techniques, which can limit its application in some settings.

Method 3 – Tandem Mass Tags Methodology

an overview of tandem mass tag
Image: ACS Publications

Tandem Mass Tags (TMT) is another labeling technique used in quantitative proteomics that works similarly to iTRAQ, involving the use of isobaric chemical tags that bind to the N-terminus and side chain amines of amino acids. Here, these tags consist of a mass reporter region, a mass normalization region, and a peptide reactive group.

While the total mass of the tags remains consistent across all samples, each tag differs in the mass of its reporter and normalization regions, which creates unique mass-to-charge ratios that can be detected during MS/MS analysis. The intensity of these reporter ions reflects the relative abundance of the respective peptides in the samples.

Advantages & Limitations of TMT

The main advantages of the TMT method is its expanded multiplexing capability, with the latest TMT kits allowing for the simultaneous analysis of up to 16 different samples. This reduces run-to-run variability, increases throughput, and improves the statistical power of the analysis.

Like iTRAQ, TMT is also applicable to global proteomics and targeted analysis of post-translational modifications. Additionally, TMT tags have been designed to be more resistant to fragmentation, which can result in a more accurate quantification.

Despite its advantages, TMT also has its challenges. As with iTRAQ, ratio compression due to co-isolation and co-fragmentation of peptides can be an issue, potentially leading to inaccurate quantification. TMT is also more expensive than other quantitative proteomics techniques, like label-free quantitation, which may limit its application in some cases.

Furthermore, the complexity and large amount of data generated by TMT analysis require sophisticated bioinformatics tools and resources for accurate interpretation and validation of results.

Comparing iTRAQ, TMT, and SILAC

Each of the three methods mentioned in this article employs a unique approach to protein quantification, but all rely on the use of mass spectrometry.

SILAC is a metabolic labeling method – particularly well-suited for studies involving cell cultures – focusing on dynamic processes such as protein turnover. This method provides accurate quantification and avoids chemical artifacts.

iTRAQ and TMT are isobaric labeling methods, where samples are chemically tagged, combined, and then analyzed together, making them suitable for global proteomics and targeted analysis of post-translational modifications in complex biological samples. iTRAQ and TMT offer the advantage of multiplexing many samples in one run, which reduces run-to-run variation and increases throughput. However, they also suffer from ratio compression due to co-isolation and co-fragmentation of peptides.

Selection Guide Based on Various Research Needs

The choice between SILAC, iTRAQ and TMT largely depends on the research question, sample type, and resources available.

If the study involves cell cultures and aims to investigate dynamic processes, SILAC would be the method of choice. However, if the study involves complex biological samples or requires the analysis of many samples simultaneously, iTRAQ or TMT would be more suitable.

Researchers also need to consider the cost, time, and computational resources required for each method before making a decision.

Optimizing Your Research With Silantes

Each of these quantitative proteomic methods has unique strengths and limitations.

SILAC, with its remarkable accuracy and avoidance of chemical artifacts, proves invaluable when investigating dynamic processes in cell cultures, delivering reliable and precise quantification in suitable settings.

On the other hand, iTRAQ and TMT, with their robust multiplexing capabilities, offer a wide range for comparative analysis of samples. They can provide detailed insights, especially in complex biological samples, making them ideal for large-scale proteomic studies.

At Silantes, we are dedicated to helping you optimize your research. Whether you need help choosing reagents, optimizing sample preparation, or troubleshooting method development, our team of scientists are experts in quantitative proteomics and can guide you through every step of your experimentation.

Do you need assistance or want to learn more? Contact our customer service team.


What is the difference between SILAC and iTRAQ?

SILAC is a metabolic labeling technique which involves growing cells in media containing heavy isotopes of essential amino acids, such as arginine and lysine, which then get incorporated into proteins during synthesis. On the other hand, iTRAQ is a chemical labeling technique. In iTRAQ, the proteins are digested into peptides which are then chemically labeled with isobaric tags.

What is the difference between iTRAQ and TMT?

iTRAQ allows for the simultaneous comparison of up to eight different samples, while TMT offers more multiplexing capacity, allowing the comparison of up to 16 samples. TMT may provide higher quantitative accuracy and precision compared to iTRAQ, but the choice between the two often depends on the specific requirements of the study.

What is the difference between SILAC and TMT?

The main difference is the stage at which labeling occurs. SILAC is a metabolic labeling method, where cells are grown in media containing heavy isotopes of certain amino acids, which then become part of the proteins during synthesis. TMT is a chemical labeling method, in which peptides are labeled with different isobaric tags.

What are the advantages of SILAC Labeling?

SILAC is a metabolic labeling method, which ensures the labeled amino acids are incorporated into all proteins, and provides highly accurate and reproducible quantification of proteins. It allows for the direct comparison of multiple samples in the same experiment, reducing variability.

What are the advantages of iTRAQ Labeling?

iTRAQ provides complete proteome coverage, as it labels all peptides in a sample. The labeling is performed after protein digestion and enables both relative and absolute quantification of proteins.

What are the advantages of TMT Labeling?

Like iTRAQ, TMT allows simultaneous comparison of multiple samples in a single experiment, reducing variation and increasing throughput. TMT labels all peptides in a sample, providing comprehensive proteome coverage.