Abstract
Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also preventing sensor overflow, as may be caused by highly abundant proteins in cell lysates or clinical samples. Here, we scale a bulk analysis method for protein separation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), to the single-molecule level using single-photon sensitive widefield imaging. Single-molecule sensing of the electrokinetically moving proteins is achieved by in situ polymerization of the PAGE in a low-profile fluidic channel having a depth of only ~ 0.6 µm. The polyacrylamide gel restricts the Brownian kinetics of the proteins, while the low-profile channel ensures that they remain in focus during imaging, allowing video-rate monitoring of single-protein migration. Calibration of the device involves separating a set of Atto647N-covalently labeled recombinant proteins in the size range of 14–70 kDa, yielding an exponential dependence of the proteins’ molecular weights on the measured mobilities, as expected. Subsequently, we demonstrate the ability of our fluidic device to separate and image thousands of proteins directly extracted from a human cancer cell line. Using single-particle image analysis methods, we created detailed profiles of the separation kinetics of lysine and cysteine -labeled proteins. Downstream coupling of the device to single-protein identification sensors may provide superior protein classification and improve our ability to analyze complex biological and medical protein samples.
Highlights
Accurate identification of both abundant and rare proteins hinges on the development of singleprotein sensing methods
In analogy to the profound impact that single-cell transcriptomics has made in contemporary scientific research, it is broadly anticipated that single-cell proteomics will carry a comparable or larger influence on all aspects of life sciences and medical r esearch[1,2,3,4,5,6,7]
One strategy that may considerably improve the ability to classify and correctly identify proteins involves the controlled separation of the proteins by their molecular weight, prior to their molecule-by-molecule identification. May this strategy remove highly abundant proteins, such as albumin[19], in favor of the more clinically-relevant proteins, it potentially improves the accuracy of the identification algorithms, due to the smaller repertoires of proteins that need to be matched in each molecular weight band
Summary
Accurate identification of both abundant and rare proteins hinges on the development of singleprotein sensing methods. We scale a bulk analysis method for protein separation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), to the single-molecule level using single-photon sensitive widefield imaging. Proteins cannot be replicated in an enzymatic reaction (i.e., polymerase chain reaction) This limitation, combined with the knowledge that many proteins in a cell’s proteome appear as a single or just a few c opies[8], motivates the development of single-molecule sensing techniques. Unlike in bulk SDS-PAGE in which the fluorescent signal is derived from ensembles containing tens of thousands of proteins, the device should enable real-time monitoring of singleprotein motion through the fluidic channels. Using single particle image analysis methods, we create detailed profiles of the separation kinetics of lysine and cysteine -labeled proteins spanning tens to hundreds of kDa
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