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. 2017;7(1):50-57.
doi: 10.5539/jmbr.v7n1p50.

Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

Fadi El-Rami  1   2 , Kristina Nelson  3 , Ping Xu  1   2
Affiliations

Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

Fadi El-Rami et al. J Mol Biol Res. 2017.

Abstract

Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining "proteomic signatures" as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis.

Keywords: Streptococcus sanguinis SK36; mass spectrometry; oral cavity; proteomics; soluble protein.

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Figures

Figure 1
Figure 1
Schematic presentation of the multi-step protocol for high resolution soluble proteome analysis
Figure 2
Figure 2
Coomassie Blue stained gel of soluble proteins extracted from Streptococcus sanguinis SK36. (from left) Lane 1: Protein ladder; Lanes 2–4: soluble proteins extracted from S. sanguinis SK36 in triplicate
Figure 3
Figure 3
The peptides and proteins identified for the samples (soluble proteins) are shown in the Scaffold file. Many proteins from Streptococcus were identified as upregulated (green). From right, the three lanes (1–3) are displays of protein from wild type S. sanguinis strain run in triplicate
See this image and copyright information in PMC

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