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Label-Dependent Strategies

Creative Biolabs is a leading service provider that focuses on ribosomal proteomics research. Based on our advanced platforms and extensive experience, now we can provide a series of label-dependent strategies for ribosomal proteome identification.

Strategies for Ribosomal Proteome Identification

The research of ribosomal proteomics consists of four basic procedures, including ribosome isolation, ribosomal proteome separation, ribosomal proteome identification, and candidate protein validation. In each link, various strategies have been developed to improve the reliability and efficiency of research.

The radical-free and highly reducing (RFHR) 2-DE method is an optimal alternative for separating ribosomal proteins (RPs) that has been successfully performed in several ribosomal proteomic studies. However, ribosomal proteome identification performed only by RFHR 2-DE presents various disadvantages, such as accuracy and scale limitations. In this case, Creative Biolabs describes a range of MS-based label-dependent strategies for ribosomal proteome identification.

Fig. 1 Strategies of clinical cancer proteomics. (Macklin, A., Khan, S. & Kislinger, T., 2020)Fig. 1 Overview of clinical cancer proteomics strategies.¹

Description of Label-dependent Strategies

With the continuous improvement of mass spectrometry (MS) technology, a series of label-dependent strategies have been developed rapidly for ribosomal proteomics research. For example, stable isotope labeling by amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantitation (iTRAQ), and isotope-coded affinity tag (ICAT) are the most frequently used methods.

  • SILAC

SILAC is a metabolic labeling method in which a medium containing isotopomeric essential amino acids is incorporated into all proteins from the culture medium after multiple cell passages. Following cell harvest and protein collection, trypsinization and LC-MS/MS analysis can be used. Finally, the abundance of proteins can be obtained by determining the peak areas of different labeled peptides.

  • iTRAQ

iTRAQ is a powerful tool for multi-sample proteomic analysis. By adding isobaric tags to the amine groups of tryptic peptides, iTRAQ can analyze up to 8 samples simultaneously. Unlike SILAC, the iTRAQ strategy uses iTRAQ reagents to directly label tryptic peptides with subsequent liquid chromatography (LC) separation and two-step MS analysis. In addition, iTRAQ also demonstrated robust labeling efficiency and high quantitative sensitivity

  • ICAT

The ICAT method uses carbon isotopes (12C and 13C) for labeling and adds acid cleavage sites to remove affinity tags prior to MS analysis. Note that ICAT can only label and quantify cysteine-containing proteins, and many key proteins in the ribosomal proteome may not contain cysteine, so the use of ICAT is largely limited.

Creative Biolabs has been a long-term expert in the field of ribosome research. Based on our extensive experience and advanced platforms, we are confident in offering a series of ribosome-related services, including ribosome separation and extraction services, ribosome analysis services, as well as ribosomal marker antibody development services. If you are interested in our products or services, please do not hesitate to contact us for more detailed information.

Reference

  1. Macklin, Andrew, Shahbaz Khan, and Thomas Kislinger. "Recent advances in mass spectrometry based clinical proteomics: applications to cancer research." Clinical proteomics 17 (2020): 1-25.
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