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. 2019 Oct 31;9(1):15729.
doi: 10.1038/s41598-019-52188-4.

Covalently Modified Carboxyl Side Chains on Cell Surface Leads to a Novel Method Toward Topology Analysis of Transmembrane Proteins

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Free PMC article

Covalently Modified Carboxyl Side Chains on Cell Surface Leads to a Novel Method Toward Topology Analysis of Transmembrane Proteins

Anna Müller et al. Sci Rep. .
Free PMC article

Abstract

The research on transmembrane proteins (TMPs) is quite widespread due to their biological importance. Unfortunately, only a little amount of structural data is available of TMPs. Since technical difficulties arise during their high-resolution structure determination, bioinformatics and other experimental approaches are widely used to characterize their low-resolution structure, namely topology. Experimental and computational methods alone are still limited to determine TMP topology, but their combination becomes significant for the production of reliable structural data. By applying amino acid specific membrane-impermeable labelling agents, it is possible to identify the accessible surface of TMPs. Depending on the residue-specific modifications, new extracellular topology data is gathered, allowing the identification of more extracellular segments for TMPs. A new method has been developed for the experimental analysis of TMPs: covalent modification of the carboxyl groups on the accessible cell surface, followed by the isolation and digestion of these proteins. The labelled peptide fragments and their exact modification sites are identified by nanoLC-MS/MS. The determined peptides are mapped to the primary sequences of TMPs and the labelled sites are utilised as extracellular constraints in topology predictions that contribute to the refined low-resolution structure data of these proteins.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The reaction flow of the applied labelling method. The carboxyl amino acid side chains of Bovine Serum Albumin model protein were activated by EDC and Sulfo-NHS, and then biotinylated by Biotinyl Cystamine. During the nanoLC-MS/MS sample preparation, the disulphide bridges were cleaved by DTT and alkylated by Iodoacetamide reagents.
Figure 2
Figure 2
SDA-PAGE and Western Blot analysis of the biotinylated BSA model protein. The protein successfully bound the biotin-residues in both experiments when the labelling agent was applied but the activation step resulted in a higher biotinylated BSA yield. Images were captured by a Bio-Rad ChemiDoc XRS+ Imaging system. The full-length images are presented in Supplementary Fig. 2.
Figure 3
Figure 3
The Intensities of the fragments of a successfully modified BSA peptide. During the applied nanoLC-MS/MS analysis, we were looking for the known fix covalent modifications on the carboxyl amino acid side chains of BSA model protein. Based on peptide sequencing, here we present the +116 Da modification on the highlighted aspartic acid (E). The image was created by Byonic 2.15.7 (Protein Metrics Inc., Cupertino, CA, USA).
Figure 4
Figure 4
The effect of different activating agent concentrations on cell surface biotinylation. EDC and Sulfo-NHS were applied in a 1:2 molar ratio. According to the fluorescent intensities emitted by CF488A anti-biotin antibody, we found that cell surface biotinylation did not increase when applying the activating agents over the concentrations 70 mM EDC and 140 mM Sulfo-NHS.
Figure 5
Figure 5
Examining the surface biotinylation of the cells by applying the observed maximal labelling concentrations. The fluorescent signal of the applied dyes and the HL60 cells were detected by confocal microscopy (A: Alexa Fluor 488 conjugated anti-biotin antibody fluorescence, B: Hoechst 33342 DNA dye fluorescence; C: Differential Interference Contrast; D: Merged picture). Scale bar: 20 µm. The images were created by Zeiss ZEN lite software (Carl Zeiss, Oberkochen, Germany).

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