Quantitative proteomics by metabolic labeling of model organisms

Abstract

In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Although in many of these studies using model organisms, the focus has primarily been on genetics and genomics approaches, it is important that methods become available to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dynamics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective means to accurately determine differential expression levels of proteins. Among these, metabolic incorporation of stable isotopes in vivo in whole organisms is one of the favored strategies. In this perspective, we will focus on methodologies to stable isotope label a variety of model organisms in vivo, ranging from relatively simple organisms such as bacteria and yeast to Caenorhabditis elegans, Drosophila, and Arabidopsis up to mammals such as rats and mice. We also summarize how this has opened up ways to investigate biological processes at the protein level in health and disease, revealing conservation and variation across the evolutionary tree of life.

Fig. 1.

Qualitative proteomics work flow. Proteins are extracted, digested, and separated by strong cation exchange. Each strong cation exchange fraction is then analyzed by nano-LC-MS/MS. Peptide fragment spectra are used in a database search to identify the peptide sequence and the corresponding protein.

Fig. 2.

Strategies for quantitative proteomics. Stable isotopes can be incorporated at different stages of the quantitative work flow and are indicated in black. The methods are metabolic labeling (left), protein labeling (middle), and peptide labeling (right). Relative expression levels are obtained by mass spectrometry where the signal of the unlabeled peptide is compared with that of the labeled peptide.

Fig. 3.

Overview of typical mass spectra that are obtained from different metabolic labeling strategies. In SILAC (A), labeled arginine can be converted to proline, indicated in red in the peptide sequence, resulting in peaks that are 6 Da higher than the labeled peptide. If such peaks are formed in heavy nitrogen labeling (B), they appear at the same position as the ¹⁵N-labeled peptide because they are isobaric. The red colored isotope at m/z 410.73 is a product of suboptimal ¹⁵N labeling. Minor enrichment of ¹³C in SMIRP leads to quantifiable peptide signals (C), whereas higher incorporation is used to determine protein synthesis and degradation (D).

Fig. 4.

Tree of metabolically labeled life. Branch lengths are not proportional to evolutionary distance.