Application of Skyline for Analysis of Protein-Protein Interactions In Vivo.

Arman Kulyyassov

doi:10.3390/molecules26237170

Abstract

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

Highlights

Wide practical application of liquid chromatography in combination with mass spectrometry has been observed recently in proteomics [1,2] and metabolomics [3,4] as a routine method for the qualitative and quantitative analysis of biological samples
The principle of the proximity utilizing biotinylation (PUB) method is based on using enzyme/substrate pair reactions [25,26,27,28], where two proteins to be tested for their interaction in vivo are co-expressed in mammalian cells, one as fused to the biotin acceptor peptide (BAP), and the other fused to an enzyme BirA, which is an Escherichia coli protein biotin ligase [29]
When the two proteins are in proximity to each other, for example, when an interaction of X and Y occurs in vivo, a more efficient biotinylation of the BAP is to be expected (Figure 1A)

Summary

Introduction

Wide practical application of liquid chromatography in combination with mass spectrometry has been observed recently in proteomics [1,2] and metabolomics [3,4] as a routine method for the qualitative and quantitative analysis of biological samples. Another important task is to obtain information about changes in the expression of marker proteins under different physiological conditions of the cell [7] Examples of this include: differences in protein composition in a healthy/cancer cell or differences under the influence of external factors such as temperature, chemical agents, or radiation provide valuable information about metabolic and signaling pathways, mechanisms of stress response. In all these cases, the results are obtained as chromatograms in the multiple reaction monitoring (MRM) method, where many peptides derived from target proteins can be identified by retention time and mass spectra of fragment ions (or MS/MS spectra), and the relative amount of each peptide between samples can be determined by comparison of the peak areas [8,9,10]. These protein–protein interactions play an important role in almost all vital processes in cells, such as DNA replication, gene transcription and translation, signal transduction, cell-cycle control and proliferation, and cell–cell communication [12]

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Conclusion