1. Nanodiamonds for Membrane Proteins
Minh D. Pham, Ting-Chun Wen, Hung-Cheng Li, Huan-Cheng Chang, Chau-Chung Han
Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan,
Membrane Proteins
What have nanodiamonds offered?
Conclusions
Despite the important roles play in the cells and its great attraction to scientists, membrane proteins are still extremely challenging to study. This is because of their intrinsic hydrophobicity, low abundance, and highly dynamics expression levels in cells. In addition, how to reduce the negative effects of extracting and solubilizing reagents on membrane protein characterization is another big issue . Novel methods, tools, or approaches are thus urgently desired to facilitate studies of the protein class.
Facile, speedy enrichment of membrane proteins from highly contaminated and diluted buffers.
ND surface-enhanced proteolytic cleavage/digestion of membrane proteins:.
Figure 8 .Flow chart of the ND-based platform for gel elctrophoreis and mass spectrometric analysis of membrane proteins.
Enrichment of pMMO[1] 1% 2%
input
SDS
SDS/50%MeOH 0%
Enrichment of E.coli’s
membrane Proteins [1]
We have established a novel platform based on nanodiamond particles for (1) enrichment of hydrophobic membrane proteins either from water-soluble proteins or highly contaminated and diluted membrane proteins solutions, (2) fractionation of membrane proteins solubilized in detergent micelles or chaotropic reagent buffers, and (3) enhancement of chemical modifications, proteolytic cleavage/digestion of membrane proteins. It is demonstrated that the platform is highly effective for studying membrane proteins with gel electrophoresis, MALDI-MS, and high-throughput shot-gun proteomics.
Compared with other methods, our platform is more versatile, faster, cheaper, and easier to deploy.
Figure 3 .SDS-PAGE analysis of membrane proteins enriched from various buffers. It is demonstrated that a multi-subunit transmembrane metallo-enzyme (named particulate methane monooxygenase, or pMMO), and the total membrane proteins from E.coli cells or HeLa cells purposely admixed in various buffers (detergents, even sodium dodecyl sulfate (SDS), salts, and organic solvents) could be enriched and extracted easily from these solutions within 5 minutes.
Applications
Figure 6 .(a) SDS-PAGE analysis of tryptic digestion of a mouse myeloma membrane proteome in a time-scale experiment; (b) Flow chart of facilitating the characterization of membrane proteins by MALDI-MS and LC-MS/MS
5min
Reduce or eliminate the negative effects of extracting/solubilizing reagents on gel electrophoresis (GE), MALDI-TOF-MS analysis of membrane proteins: Thanks to their high affinity for the NDs, the adsorbed membrane proteins could sustain extensive washes for removal of salts, detergents, and other impurities to facilitate down-stream analysis.
Combination of sequential pH-tuned ND extraction triton detergent-based and two-phase separation [2] for enrichment and fractionation of hydrophobic membrane proteins from water-soluble proteins.:
Outlooks
Nanodiamonds
Applying the developed platform to study biological issues of significance (e.g brain membrane proteomes, biomarker discovery, and so on).
Functionalization of nanodiamonds with membrane proteins of biomedical significance.
Nanodiamonds as nucleation enhancing factor for membrane protein crystallization.
Nanodiamonds may be helpful in antigen design in the production of antibodies against membrane proteins.
Preparation of oxidized nanodiamond particles (NDs): 100nm - diamond particles were surface-functionalized with carboxyl and carbonyl groups in strong oxidative acids under microwave heating[1].
Properties of oxidized NDs used in this method: (i) compatible with all detergents, salts, organic solvents, and acid/base buffers, (ii) large specific surface area, and (iii) easy to handle, requiring the use of no specialized equipment other than a bench-top centrifuge.
Figure 7 .(a) SDS-PAGE analysis of 6 fractions obtained from sequential pH-tuned ND extraction (pH3, pH7, and pH11) for membrane protein fractionation. Fractionation was conducted with proteins in both Triton-X100 phase (D) and aqueous phase (A) after detergent two-phase enrichment of hydrophobic membrane proteins; (b) “Shot-gun proteomics evaluation of the enrichment and fractionation of the platform.
Acknowledgements: This work is supported by Academia Sinica and grants from the National Science Council of the Republic of China (NSC).
Figure 4 .MALDI-MS analysis of pMMO complex solubilzed in different buffers with and without the assistant of NDs (left panel).
ND Surface-enhanced chemical modifications of proteins”: (
Oxidation
Figure 2 .Surface of oxidized nanodiamond particles
ND-based enrichment, fractionation, and sample preparation of membrane protomes for high-throughput shot-gun proteomics.
References: [1] Minh D. Pham, Steve S.-F. Yu, Chau-Chung Han, Sunney I. Chan, Anal Chem 85, 6748–6755 (2013); [2] Thomas Arnold and Dirk Linke, BioTechniques 43, (2007).
Membrane proteome
Identified proteins (all)
Membrane proteins
Methods of analysis
E.Coli
406
63%
Single 2h-1D LC-MS/MS
Mouse Myeloma
2163
68%
Six protein fractions (2h-1D LC-MS/MS per fraction)
Figure 5 .Reduction and alkylation of di-sulfur bonds in proteins adhering to the surface of nanodiamond
Poster presented during ?, Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan, from February 24-26