Proteomics

Introduction

Proteomics aims at the comprehensive identification and quantification of all proteins in a biological system, to reveal the roles of proteins in biological, physiological, and pathological processes. Proteomics is widely applied in many research areas, such as the exploration of biological mechanisms, the discovery of biomarkers, and in drug design. As proteomics research progressed, it has become increasingly clear that quantitative information is critical to the systematic study of biological processes and the discovery of clinical markers for detection, diagnosis, and assessment of treatment outcome. So the quantitative proteomics is the basis of proteomics. In the past few years, several mass spectrometry (MS)-based quantitative proteomic methods have been developed. All these methods share the use of labeling by stable isotope-labeled compound to generate the mass signatures that identify the sample of origin and serve as the basis for accurate quantification. The strategy is also known as stable isotopes quantitative proteomics. Nowadays, the strategy has developed rapidly and provided important technical support for life science research.

Mechanism

Stable isotopes quantitative proteomics identify the equivalent peptides or peptide fragments by utilizing the specific increase in mass due to mass tags with stable isotope compound. The common workflow is to tag protein or peptide containing samples with stable isotopes reagents, one of which includes a heavy mass tag and the other a light mass or no tag. The labeled samples are analyzed by mass spectrometry after being mixed and fractionated. The peaks in the mass spectra reveal the ratio of the two different isotopic or mass tag variants. The ratio is then used to identify protein or peptide relative abundances.

labeling strategy

According to the approach of incorporating the isotopes, the quantitative proteomics methods based on labeling by stable isotope compound can be divided into chemical isotope labeling, enzymatic isotope labeling and metabolic isotope labeling ^[1-2].

Fig. 1 Schematic illustration of the strategies used to introduce stable isotope to proteins or peptides for quantitative proteomics.

Chemical isotope labeling: The method introduces isotopes compound through chemical labeling peptide chains or proteins in vitro, and is suitable for cell, body fluid, tissue and other samples analysis. The most commonly used of the methods include the isotope-coded affinity tag (ICAT) approach, nonselective isotope-coded protein labelling (ICPL), isobaric tags for relative and absolute quantitation, tandem mass tags (TMT), etc. Fig. 2 shows the structure of ICAT reagents, which are the first commercially available isotope reagents for the quantitative comparison of the relative expression level of targeted proteins extracted from two different samples. Fig. 3 shows some ICPL reagents with different numbers of isotope atoms.

Fig. 2 The structure of ICAT reagents.

Fig. 3 The structure of ICPL reagents.

Enzymatic isotope labeling: ¹⁸O labeling is currently the only method for enzymatic isotope labeling, which only requires H₂¹⁸O in the process of enzymolysis. The use of proteases can be serine proteases such as trypsin, chymotrypsin, Glu protease (Glu-C) and endopeptidease (Lys-C). Under the catalysis of these enzymes, two ¹⁸O atoms could be added to the C-terminal of the peptide to complete the process pf isotope labeling.

Metabolic isotope labeling: Metabolic labeling refers to the method of cell or organism labeling by adding medium containing stable isotope-labeled compounds in the process of cell or organism growth. The stable isotope labeling with amino acids in cell culture (SILAC) is the most widely used metabolic labeling method. The method is adding stable isotope-labeled amino acids (usually is ¹³C-labeled amino acids) such as lysine and arginine during cell culture. For instance, [¹³C₆] lysine was used to label S. cerevisiae proteins.

Fig. 4 Schematic representation of a stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic analysis ^[3].

What can we do?

Alfa Chemistry has many years of experience in the synthesis and development of stable isotope-labeled products. We can provide various stable isotope-labeled compounds for quantitative proteomics, including ICAT reagents, ICPL reagents, stable isotope-labeled amino acids and many others. And our professional technology teams that can also provide customers with high-quality stable isotope-labeled compounds design and customization services. No matter what design ideas you have, we will implement them together with you. Please contact us immediately to order or cooperate in research and development with high quality and reasonable price.

References

• Sheng, P. and Aebersold, R. Quantitative proteomics by stable isotope labeling and mass spectrometry[M]. Mass spectrometry data analysis in proteomics. Humana Press, 2007, 367: 209-218.
• Chahrour, O., et al. Stable isotope labelling methods in mass spectrometry-based quantitative proteomics[J]. Journal of pharmaceutical and biomedical analysis, 2015, 113: 2-20.
• Sethi, S., et al. Approaches for targeted proteomics and its potential applications in neuroscience[J]. Journal of biosciences, 2015, 40(3): 607-627.