Application of Skyline for Analysis of Protein–Protein Interactions In Vivo

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.

Comparative transcriptome and proteome reveal the unique genes and proteins of female parasitic wasps, Lysiphlebia japonica Ashmead

“…Multiple MRM transitions were monitored using the unit resolution of Q1 and Q3 quadrupoles to maximize specificity. The raw data obtained were imported and processed using Skyline 28 . MRM peaks were manually checked to ensure correct peak detection, absence of interference, and accurate integration of peaks.…”

“…The raw data obtained were imported and processed using Skyline. 28 MRM peaks were manually checked to ensure correct peak detection, absence of interference, and accurate integration of peaks. MRM signals were detected for all leaps from endogenous peptides and all leaps from stable isotope-labeled peptides were fully coeluted.…”

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

“…Due to this DNA-dependent binding and interaction of these proteins on such motifs, this results in the labeling of the BAP target and high level of biotinylation detected by MRM mode [45] of LC-MS/MS and streptavidin Western blotting in comparison with controls, where we used GFP or Tap54beta fusions of BAP (Figure 3D). Quantification of MRM data was performed using Skyline software [46]. It is interesting to note that, the using of another widely applied method, such as Co-immunoprecipitation (Co-IP) in the search of Oct4 interactors did not reveal one of the best-studied partners of Oct4, namely Sox2 [31].…”

Uniprot Database - Universal Information Resource of Protein Sequences

“…The direct contact of the biotin acceptor peptide (BAP) and wild-type BirA results in site-specific biotinylation of the target molecule[55]. The level of BAP biotinylation can be detected using Western blotting or quantified using MRM mode of LC-MS/MS[56] followed by raw data processing with Skyline software[57]. Sequence Logo of HMG DNA-binding domain sequences of some mammalian species (human, cow, sheep, goat, pig, mouse), chicken, frog and fish.…”