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Sequencing proteins with transverse ionic transport in nanochannels.

ABSTRACT: De novo protein sequencing is essential for understanding cellular processes that govern the function of living organisms and all sequence modifications that occur after a protein has been constructed from its corresponding DNA code. By obtaining the order of the amino acids that compose a given protein one can then determine both its secondary and tertiary structures through structure prediction, which is used to create models for protein aggregation diseases such as Alzheimer's Disease. Here, we propose a new technique for de novo protein sequencing that involves translocating a polypeptide through a synthetic nanochannel and measuring the ionic current of each amino acid through an intersecting perpendicular nanochannel. We find that the distribution of ionic currents for each of the 20 proteinogenic amino acids encoded by eukaryotic genes is statistically distinct, showing this technique's potential for de novo protein sequencing.

SUBMITTER: Boynton P

PROVIDER: S-EPMC4853742 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Sequencing proteins with transverse ionic transport in nanochannels.

Boynton Paul P Di Ventra Massimiliano M

Scientific reports 20160503

De novo protein sequencing is essential for understanding cellular processes that govern the function of living organisms and all sequence modifications that occur after a protein has been constructed from its corresponding DNA code. By obtaining the order of the amino acids that compose a given protein one can then determine both its secondary and tertiary structures through structure prediction, which is used to create models for protein aggregation diseases such as Alzheimer's Disease. Here, ...[more]

PMID: 27140520

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Project description:The electrokinetic behavior of molecules in nanochannels (<100?nm in length) have generated interest due to the unique transport properties observed that are not seen in microscale channels. These nanoscale dependent transport properties include transverse electromigration arising from partial electrical double layer overlap, enhanced solute/wall interactions due to the small channel diameter, and field-dependent intermittent motion produced by surface roughness. In this study, the electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) were investigated, including the effects of electric field strength, surface effects, and composition of the carrier electrolyte (ionic concentration and pH). The dNMPs were labeled with a fluorescent reporter (ATTO 532) to allow tracking of the electrokinetic transport of the dNMPs through a thermoplastic nanochannel fabricated via nanoimprinting (110?nm?×?110?nm, width?×?depth, and 100??m in length). We discovered that the transport properties in plastic nanochannels of the dye-labeled dNMPs produced differences in their apparent mobilities that were not seen using microscale columns. We built histograms for each dNMP from their apparent mobilities under different operating conditions and fit the histograms to Gaussian functions from which the separation resolution could be deduced as a metric to gage the ability to identify the molecule based on their apparent mobility. We found that the resolution ranged from 0.73 to 2.13?at pH?=?8.3. Changing the carrier electrolyte pH?>?10 significantly improved separation resolution (0.80-4.84) and reduced the standard deviation in the Gaussian fit to the apparent mobilities. At low buffer concentrations, decreases in separation resolution and increased standard deviations in Gaussian fits to the apparent mobilities of dNMPs were observed due to the increased thickness of the electric double layer leading to a partial parabolic flow profile. The results secured for the dNMPs in thermoplastic nanochannels revealed a high identification efficiency (>99%) in most cases for the dNMPs due to differences in their apparent mobilities when using nanochannels, which could not be achieved using microscale columns.

Dataset Information

Sequencing proteins with transverse ionic transport in nanochannels.

Publications

Sequencing proteins with transverse ionic transport in nanochannels.

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