Progress and pitfalls of using isobaric mass tags for proteome profiling

ABSTRACT

Introduction: Quantitative proteomics using mass spectrometry is performed via label-free or label-based approaches. Labeling strategies rely on the incorporation of stable heavy isotopes by metabolic, enzymatic, or chemical routes. Isobaric labeling uses chemical labels of identical masses but of different fragmentation behaviors to allow the relative quantitative comparison of peptide/protein abundances between biological samples.

Areas covered: We have carried out a systematic review on the use of isobaric mass tags in proteomic research since their inception in 2003. We focused on their quantitative performances, their multiplexing evolution, as well as their broad use for relative quantification of proteins in pre-clinical models and clinical studies. Current limitations, primarily linked to the quantitative ratio distortion, as well as state-of-the-art and emerging solutions to improve their quantitative readouts are discussed.

Expert opinion: The isobaric mass tag technology offers a unique opportunity to compare multiple protein samples simultaneously, allowing higher sample throughput and internal relative quantification for improved trueness and precision. Large studies can be performed when shared reference samples are introduced in multiple experiments. The technology is well suited for proteome profiling in the context of proteomic discovery studies.

KEYWORDS:

• Proteomics
• quantification
• tagging

Article highlights

• Protein quantification using mass spectrometry (MS) can be achieved via label-free or label-based approaches.

• Label-based approaches rely on the incorporation of heavy stable isotopes. Labeling with isobaric chemical mass tags is one option for label-based relative quantification of proteins.

• Multiplexed comparison of two to sixteen samples is currently achievable with isobaric labeling in routine use, providing adequate precision and trueness. Higher order multiplexing options have been proposed.

• Increasing multiplexing capabilities allow a number of new applications in pre-clinical and clinical studies. Isobaric mass tags have been successfully employed in large-scale studies dealing with several hundred (up to a thousand) of samples.

• While quantitative performances of isobaric mass tags suffer from the well-known co-fragmentation issue (i.e. peptide precursor of interest isolated together with other peptides) that interferes with accurate quantification, solutions such as an additional round of fragmentation (i.e. MS/MS/MS or MS³) or further peptide separation/isolation (e.g. with ion mobility) can significantly improve performance.

• We consider that quantitative precision is a must while the trueness might be less of an issue in proteomic discovery applications.

• Data completeness is achieved within samples from a single experiment using isobaric mass tags. Missing values arise in multiple experiment comparisons due to the stochastic nature of the data-dependent mode of MS used for data acquisition. This limitation can be partly alleviated by replication of the liquid chromatography-MS analysis, sample fractionation, or refined data processing.

Declaration of interest

L. Dayon and M. Affolter are employees of the Société des Produits Nestlé SA. L. Dayon is a former employee of Proteome Sciences plc (2010–2011) and a former postdoctoral researcher funded by Proteome Sciences plc (2007–2009).

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was funded by Nestlé Research, Lausanne, Switzerland.