SYSTEMS, APPARATUSES AND METHODS FOR READING AN AMINO ACID SEQUENCE
Embodiments of the present disclosure relate to amino acid, modified amino acid, peptide and protein identification and sequencing, by means of, for example, electronic detection of individual amino acids or small peptides.
This is a divisional application of U.S. patent application Ser. No. 14/376,154, filed Aug. 1, 2014, which is the U.S. national stage of International Application No. PCT/US2013/024130, which was filed Jan. 31, 2013, which claims benefit under 35 USC 119(e) of U.S. provisional patent application nos. 61/593,552, filed Feb. 1, 2012, and entitled, “Apparatus And Method For Reading Amino Acid Sequence,” and 61/647,847, filed May 16, 2012 of the same title. Each of these disclosures is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT
This invention was made with government support under grant RO1HG006323 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE
Embodiments of the present disclosure relate to amino acid, peptide and protein, sequencing and identification, by means of, for example, electronic detection of individual amino acids or small peptides.BACKGROUND OF THE DISCLOSURE
The human proteome, which is encoded by just some 25,000 genes, consists of millions of proteins variants, due to RNA splicing, post translational modifications (PTM), somatic DNA rearrangements, and single nucleotide polymorphisms (SNP). Despite the widespread interest in whole genome sequencing, the genome is largely fixed. While sequencing of mRNA is more informative, giving an average picture of the levels of gene expression, it yields little of post-translational modifications that generate the millions of proteins from just thousands of genes. There is a need of a massively parallel method to read human proteomes with high throughput and low cost. In contrast to the human genome in which DNA exists as diploid, the proteome has a wide dynamic range, for example the abundance of proteins in human plasma spans more than 10 orders of magnitude. Some proteins are expressed in a low quantity. Proteomics has no tool equivalent to the polymerase chain reaction (PCR) for protein sample amplification. There is no cost effective way to faithfully reproduce a protein population from a source. Thus, protein analysis must be carried out by extracting materials from samples removed from humans in some quantity.
There have been remarkable advances in sample preparation, and sequencing techniques, most notably based on mass spectroscopy as a proteomic tool. However, mass-spectrometers are large, costly machines. Their size is dictated by the need for very high mass resolution to obtain accurate identification of the amino acid components (and even then, readout is complicated by isobaric amino acids). Accordingly, there is a need for an alternative method for identifying amino acids, particularly in small quantities, and ideally at the single-molecule level.
In a series of earlier disclosures, WO2009/117522A2, WO 2010/042514A1, WO 2009/117517, WO2008/124706A2, US2010/0084276A1, and US2012/0288948, each of which is incorporated herein by reference, a system was disclosed where nucleic acid bases could be read by using the electron tunneling current signals generated as the nucleobases pass through a tunnel gap functionalized with adaptor molecules. A demonstration of the ability of this system to read individual bases embedded in a polymer was given by Huang et al.1 This method is referred to as “Recognition Tunneling” (RT).2 It was earlier recognized in these previous disclosures that, because the method is purely physical, in that it did not rely on the reactions of a DNA polymerase or ligase, any chemical residue should be able to be recognized provided that it generates a distinctive tunneling current signal.
Yet another problem in determining the human proteome arises because of the stereochemistry of amino acids. Amino acids can exist in two forms (“enantiomers”) that are mirror images of each other. Nature has chosen the so called “L” (for left-handed) form in general, but the presence of “D” (for dextro or right handed) amino acids is an important biomarker for diseases such as ALS or schizophrenia. Since these enantiomers are isobaric, they cannot be sensed by mass spectroscopy, so large amounts of additional sample are needed for optical identification. Furthermore, control of optical isomers is one of the most difficult problems in chemical synthesis and separation. Since the isomers are chemically identical, it is very hard to extract pure samples of one isomer or the other. Thus, a method to read the relative concentrations of isomers from very small amounts of sample would represent a major advance. Present, optical techniques require large amounts of sample. In addition, it would be advantageous to provide a simple method for reading the identity of optical isomers at the single molecule level.SUMMARY OF THE DISCLOSURE
Accordingly, it is an object of some of the embodiments of the present disclosure to provide a method for sequencing and/or identifying amino acid molecules. It is another objective of some of the embodiments of the present disclosure to provide a method to read sequences of proteins and peptides. It is yet another objective of some embodiments to detect enantiomers at the single molecule level. Accordingly, it is an object of some of the embodiments of the present disclosure to provide a method for sequencing and/or identifying at least one protein, peptide, amino acid and/or modified amino acid.
These and other objectives are achieved by at least some of the embodiments of the present disclosure. In some embodiments, an apparatus is provided which includes a plurality of electrodes which are closely spaced, where at least one electrode (and, in some embodiments preferably more than one electrode) is functionalized with molecules that bind transiently to the analyte target. In some embodiments, the apparatus includes two such electrodes where each is functionalized with such molecules. In some embodiments, at least two of the electrodes enclose a space into which an analyte can be drawn, by diffusion, electrophoresis, dielectrophoresis and/or electro-osmosis (for example). Accordingly, in some embodiments, on binding an analyte to the recognition molecules, a series of current spikes signal the identity of the molecule upon a bias being applied between the electrodes. In some embodiments, the small analyte molecules are generated from a parent protein by placing the electrode(s) in close proximity to a bead containing proteases that digest the protein to be analyzed.
According to some of the embodiments of the present disclosure, a computer system is additionally included, as well as associated software for operating such a computer and operating embodiments for identifying and, in some embodiments, sequencing/identifying proteins anal/or peptides, analyzing amino acids, as detailed herein.
According to some embodiments, an apparatus for identifying anal/or sequencing at least one protein, peptide, amino acid and/or modified amino acid is provided, where the apparatus includes a plurality of electrodes, at least a first electrode of the plurality of the electrodes being functionalized with a molecular adaptor that is strongly bonded to at least the first electrode and that forms non-covalent bonds with an amino acid, voltage means (e.g., a voltage biasing device, which may comprise a power supply) for applying a voltage between the first electrode and a second electrode of the plurality of electrodes, where the first and second electrodes are arranged such that a gap is produced between them, and current data monitoring means (e.g., a current measuring device) for monitoring current passing between the first and second electrodes upon a solution containing at least one protein, peptide, amino acid and/or modified amino acid being passed in the gap established between the first and the second electrodes. In some embodiments, a database and processing means (e.g., a computer processor, which may also include a memory) may also be included where the database includes data associated with characteristic current signatures for a plurality of amino acids and amino acid derivatives, where the processing means has computer instructions operating thereon configured to compare the amino acid and the derivative signatures (which are contained in the database, for example) with the current data to determine the identity of the amino acid, peptide and/or protein. Moreover, the chirality of the amino acid may be determined from the current data.
In some embodiments, an assembly used in an apparatus for sequencing and/or identifying at least one protein, peptide, amino acid, and/or modified amino acid, may include a silicon substrate covered with a thin layer of dielectric between about 1 nm and about 100 nm in thickness, a window etched through the silicon substrate, a metal electrode layer deposited onto the dielectric layer which may comprise at least one of silver, gold, palladium or platinum between about 1 nm and about 10 nm in thickness, an adhesive layer of at least one of Ti or Cr of about 1 nm, a second layer of dielectric deposited onto the metal electrode layer comprising at least one of silicon nitride, silicon dioxide or hafnium oxide, the layer having a thickness of between about 1 nm and about 4 nm in thickness, a second metal electrode deposited on top of the second dielectric layer comprising at least one of silver, gold, palladium or platinum between about 1 nm and about 10 nm in thickness, a third layer of dielectric deposited on top of the apparatus and is formed to contact each pair of electrode planes at each apparatus, and a nanopore of between about 1 and 5 nm in diameter provided through the entire assembly.
In some embodiments, a method for sequencing/identifying at least one protein, peptide, amino acid, and/or modified amino acid is provided and may include one or more of the following steps: flowing a fluid containing at least one protein, peptide, amino acid, and/or modified amino acid in a gap provided between two closely spaced electrodes, where: at least one of the electrodes is functionalized with molecules that form transient, non-covalent, bonds with a target amino acid, and the target molecules diffuse freely into the gap from solution, and/or can be driven into the gap upon the gap spanning a nanopore through which fluid flows. Fluid flow may be accomplished via at least one of a pressure gradient, electroosmosis, dielectrophoresis and electrophoresis. Signals may be generated as the fluid flows through the gap which is representative of the at least one of protein, peptide and amino acid contained in the fluid. Additional steps may include determining the at least one of protein, peptide and amino acid based on the signals.
Determining the at least one of protein, peptide and amino acid may comprise determining at least one of: a width of signal spikes, a ratio of “on” to “off” time in a signal burst, a duration of a signal burst, a degree of clustering of the peaks, a flatness of the top of the spike of a signal, a frequency of the signal spikes, a rise and/or fall time of the signal spikes, and a shape of the signal spikes.
Some embodiments may include a computer system for sequencing/identifying at least one protein, peptide, amino acid, and/or modified amino acid which comprises a processor, where the processor includes computer instructions operating thereon for performing steps of a method for identifying and/or sequencing at least one protein, peptide, amino acid and/or modified according to any of the disclosed embodiments. Other related embodiments include a computer program for identifying and/or sequencing at least one protein, peptide, amino acid, and/or modified amino acid comprising computer instructions for performing such steps, as well as computer readable media (e.g., CD, DVD, flashdrive, and the like) containing such a program operable on a computer system.
In some embodiments, a molecular recognition apparatus is provided which may be configured to identify at least one portion of a target molecule. The apparatus may include a microfluidic channel containing first and second sensing electrodes, where the electrodes are separated from one another by a gap for receiving a target molecule, a first affinity element connected to the first electrode, and a second affinity element connected to the second electrode. When a portion of the target molecule is adjacent to the gap and completes a circuit between the electrodes, the device is configured to enable an electrical current to pass through the first electrode, the first affinity element, the portion of the target molecule, the second affinity element, and the second electrode.
The above-noted embodiments, and embodiments disclosed throughout this application, are merely examples of the many embodiments disclosed herein. Such embodiments further include embodiments which are similar to those set out above/below, but which may include less than the recited features, and others still, more than the recited features, thereof. Moreover, other embodiments include a combination of features of the embodiments exemplified above, below and throughout the application.
Some embodiments of the disclosure are further described in the following detailed description and included drawings.
In some embodiments of the present disclosure, RT relies on passing an analyte between two closely spaced electrodes, each of which is functionalized with molecules that form transient, non-covalent, bonds with the target analyte. The target molecules may diffuse freely into the gap from solution, and/or they can be driven into the gap if the gap spans a nanopore through which fluid flows. Fluid can be made to flow through the gap by means of, for example, at least one of a pressure gradient electrophoresis, dielectrophoresis and electroosmosis (e.g., if the walls of the nanopore carry a charge). If the analyte molecules are charged, they can also be driven through the gap by electrophoresis.
For the data presented here, for some embodiments, the buffer is a 1 mM phosphate buffer, having a pH=7.0. Provided that only (sub-micron)2 areas of one of the electrodes are exposed to electrolyte, electrochemical leakage currents in some embodiments are considerably smaller than tunnel current between the electrodes. The gap 3 may be defined by the current I and the voltage V (electrical bias being the voltage, being supplied by a power supply device, and structure I can be a monitoring device for monitoring current). For a voltage of 0.5V applied between the electrodes, a current of 6 pA is indicative of a gap of about 2.5 nm5 (for example). A bias of 0.5V and a set-point current of 4 pA were used to collect the data shown in
In some embodiments, when amino acids are added to the solution (“aa” in
While there is considerable overlap between the peak height distributions from these three exemplary amino acids, other parameters can be used to make assignments. Examples of such parameters may include:
- Width of the spikes;
- Ratio of “on” to “off” time in a signal burst;
- Duration of a burst of signal;
- Degree of clustering of the peaks;
- Flatness of the top of the spike;
- Frequency of the spikes; and
- Rise and fall times of the spikes
- In some embodiments, a combination of these parameters may be used.
In some embodiments, the shape of the spikes may be characterized by the size of frequency components in a wavelet analysis, and any number of components can be included in a principle component analysis, for example, conveniently carried out with Support Vector Machine (SVM) code. Accordingly, with adequate training of a recognition code, an assignment of signals from all 20 of the amino acid residues may be obtained.
In some embodiments, the system may be sensitive to chirality (i.e., optical isomerism) of individual molecules. Specifically, in some embodiments, the amino acid is trapped by bonding to the recognition molecules in the gap. Thus, the three dimensional details of its non-covalent bonding to the recognition molecules becomes important. To demonstrate this, exemplary data for L-asparagine and D-asparagine is illustrated in
In some embodiments, L-asparagine produces larger signals in these conditions.
The ability of the methods and systems according to some embodiments of the present disclosure to distinguish between two enantiomers at the single molecule level is illustrated in
A readout apparatus, according to some embodiments, which enables a scheme for sequencing and/or identifying one or more proteins and/or one or more peptides is illustrated in
Each chamber of the apparatus may be filled with an electrolyte such as KCl or NaClO3 (for example), in concentrations that range from about 1 mM to about 1M, and to which may be added Mg ions and/or ATP as required to activate enzymes 113 attached to one or more beads 112 which are fixed in turn to the walls of the channel 117 in close proximity to the nanopore 102. Alternatively, the enzyme is directly attached to the channel in close proximity to the nanopore. The enzymes may then be any one of the well-known proteases which include carboxypeptidase, aminopeptidase, trypsin, chymotrypsin, pepsin, papain or elastase (for example). Since each of these proteases is somewhat selective in their hydrolysis of peptide bonds, the beads may ideally contain a mixture of these enzymes. In some embodiments, the bead may be functionalized with proteosomes, assemblies that sequentially degrade proteins into their component amino acids. The isolated protein or peptide 114 to be identified/sequenced may also bound to the bead. In some embodiments, digestion of the protein may be initiated by binding the protein to the bead in the absence of Mg or other chemicals needed to initiate digestion, the bead placed in the channel, and then digestion is initiated by the addition of Mg or other chemicals (needed to initiate digestion). The resulting small fragments, small peptides, or optimally amino acids 115, may be released into the solution. A bias 118 may be applied between the cis and trans chambers by means of the reference electrodes 111, 110, for example. In the figure, this is shown with the negative electrode in the trans chamber 109. Accordingly, this draws positively charged amino acids and small peptides through the nanopore where they bind transiently to the recognition molecules 107 generating current spikes recorded by a transconductance amplifier 106. A bias 105 of between about 0.1V and about 1V is preferably applied between the electrodes 103, 104. The walls of an oxide layer (such as the surfaces of silicon, silicon nitride or silicon dioxide or hafnium oxide) in aqueous electrolyte are negatively charged 116 owing to the accumulation of OH groups on the surface. This negative charge causes an electro-osmotic flow of water towards the negative electrode 115. Thus, neutral amino acids that diffuse into the vicinity of the nanopore may be swept through it by the electro-osmotic flow of the water.
All the neutral and positively charged amino acid residues can be read by the same nanopore apparatus, according to some embodiments. Accordingly, the amino acid mixture will generate a characteristic set of tunneling signals that will allow the protein or peptide to be identified. To the extent that the digestion of the target proteins is sequential and synchronized, the train of signals could also be used to deduce protein sequence, while the sequence of small peptides may be read directly from the time series of signals generated as each amino acid in the chain passes through the tunnel gap.
Fabrication of the apparatus according to some embodiments is illustrated in more detail in
In this example, the two most aromatic amino acids, tyrosine and tryptophan, appear not to give signals in these conditions. Signals can be obtained by increasing the tunneling set point to 6 pA and 10 pA respectively, but the signals contain some spurious background from water. This can be removed using a SVM analysis.7 Thus, in some embodiments, there may be an advantage in having an adjustable tunnel gap (as described in an additional embodiment detailed below).
An SVM7, 8 trained on 18 amino acid data sets simultaneously, and 3000 peaks were sampled for each of 17 amino acids. The exception was isoleucine, where only 800 peaks were measured. 400 of the 3000 were used for training, and then the remaining 2,600 peaks were called using the resulting support vectors. The accuracy with which the peaks were called is listed in Table 1 below. Tyrosine and tryptophan were excluded from the analysis shown here, but also give distinctive signals.
Thus, individual amino acids may be read with high fidelity based on, in some circumstances, a single signal spike. A final accuracy of a call may depend on how many amino acids cluster near a correct one in parameter space. Should the missed calls be spread among many other amino acids, then the probability of a majority call being incorrect would be even smaller than suggested by the numbers in Table 1.
These data were taken with just one run each, and the SVM was trained on a single amino acid at a time. Run to run variations in the tunnel gap were not accounted for, and the problem of identifying multiple analytes from a mixed pool of signals was not addressed. These problems are addressed for a limited pool of amino acids below. Note that the ability to separate leucine and isoleucine is another example of how RT may be used to separate isobaric isomers which are not distinguished by mass spectroscopy in addition to the earlier case of L- and D-Apargine.
In some embodiments, an amino acid/protein sequence reader which eliminates the requirement for a nanopore is provided. The principle of this embodiment is illustrated in
In some embodiments, smaller volumes can be utilized, with reaction chambers down to the femtoliter range (for example), given the relatively small tunneling volume between electrodes. In the case of a 3.5 nanoliter reaction volume, for example, a final concentration of 10−4 moles/liter (which yields hundreds of counts per second in our detector) would require just 350 fM of starting protein. If the volume was reduced to 10 picoliters (a cell of 20 microns on each side), then less than a fM would be needed. In some embodiments, the reading volume and the reaction volume are substantially the same (and in some embodiments, the same) or similar. With appropriate treatment of the microfluidic channels, the only dilution may be caused by diffusion of the aliquot of molecules along the channels. In the case of a 3.5 nanoliter volume, a characteristic channel length is 150 microns. Given that the diffusion constant of a typical amino acid is 8×10−4 cm2/s, the aliquot requires nearly 30 s to diffuse over this distance. In the case of a 10 picoliter volume, this time is reduced to 0.5 s. Thus, in some embodiments, there is an ample amount of time to move the sample and record recognition signal peaks.
As shown in
In operation (for example), an aliquot to be measured is passed to the tunneling junction, measured by means of its characteristic tunneling signal, and then flushed out by passing clean buffer into the measurement cell via the channel 601/602. The cycle is then repeated as needed.
Another advantage of using a microfluidic channel with a scanning-tunneling micropscopy (STM) apparatus, according to some embodiments, is that an adjustable tunnel gap can be included for added versatility and ease of manufacture. Such embodiments also may allow identification of species that require a different tunneling gap. For example, in the case of tryrosine and tryptophan, a “null” read in the standard tunneling conditions (0.5V, 4 pA) can be followed rapidly by a sample taken at a smaller gap (6 or 10 pA current) to see if the aliquot of sample that produced no signal was tyrosine or tryptophan. In that way, all 20 amino acids can be identified.
A view of an exemplary nanoliter scale reaction system, according to some embodiments, is shown in
The overall assembly, according to some embodiments, is shown in
Prior to sequencing, both the probe 603 and the substrate 606 may be functionalized with an adaptor molecule as previously described. Buffered electrolyte may then be pushed from a reservoir on the microfluidic chip 901 to fill the reading channel 602 and the probe advanced to the desired tunneling current by means of the actuator 903, and subsequently controlled by the servo circuit. A set point at a bias of 0.5V between the probe and substrate is a current of 4 pA (for example).
The first Edman degradation may then be carried out, and control valves on the microfluidic chip may be set so as to release the first amino acid aliquot released from the peptide into the reading channel up to the point where it is preferably centered on the junction region between the probe apex 604 and the substrate 606. RT signals may then be acquired for a period of between about 0.1 to about is and recorded for subsequent analysis. Valves on the microfluidic chip may then be set to flush the sample out of the junction area. Then a next cycle of Edman chemistry may be carried out, releasing the next amino acid to the junction for the next read. This cycle may be repeated out to the limit of reliable cleaving by Edman chemistry, which may be (for example) up to about 50 amino acid residues. In some embodiments, mixtures of amino acids can be analyzed by this technique with each read producing hundreds or thousands of signal peaks, each one of which can be assigned to a particular amino acid, enabling analysis of the data well into a number of amino acids beyond 50 amino acid residues, based on the identity of the last reliably called residue. This is because contaminants from residues that may fail to have cleaved in earlier reactions will have been previously recorded, so their presence in a subsequent reaction can be recognized as an artifact of the chemistry.
Accordingly, in some embodiments, the same ability to make hundreds or even thousands of single molecule calls from even a few femtomoles of sample for each amino acid, which, corresponds to small amounts of post translationally modified amino acids, can be identified.
Analysis of the signals by means of a multiparameter characterization of each pulse is described in the publication by Chang et al.7 Wavelet and Fourier analysis may be used to characterize the shape of each pulse in the signal train and a SVM8 to assign the pulse using data obtained from known calibration samples. Using a systematic search of parameter combinations, the SVM may be trained to recognize new data with an accuracy that can exceed 90% based on just one pulse of the data train. Thus, many calls will be made for each aliquot of amino acids that pass into the reader channel.
In some embodiments, a fixed tunnel gap can generate RT signals from an amino acid.
The data analysis shown in Table 1 was restricted to just one run for each amino acid tested with the true positive rate quoted for the SVM trained on a subset of the data in that run so they do not reflect the important influence of variations in the atomic details of the tunnel junction form experiment to experiment. Here, it is shown that amino acids may be reproducibly identified across different measurements with different tunnel junctions set to the same set point current. In order to determine transferability of data, a study was conducted on a limited number of amino-acids. Since no two tunnel junctions are identical at the atomic level, that is, signals will likely be different form device to device, the following measurements correspond with four different junctions to find characteristics of the signals that are conserved from junction to junction. In addition, an investigation was conducted to determine whether the technique would discriminate between those molecules that are challenging for mass spectral analysis, without the aid of other techniques.
Accordingly, leucine and isoleucine (isobaric isomers), L-asparagine and D-asparagine (as an example of enantiomers), L-arginine (with a charged side chain) and glycine (with no side chain), were chosen for analysis. Data was generated according to the following constraints: (1) control runs (phosphate buffer alone with concentration in the range of 1.0 to 10.0 mM) were free of any features (indicating that the buffer solution was free of contaminants); (2) insulated STM probes had to show no electrochemical leakage down to below the measurement limit (<1pA)—electrochemical leakage is sensitive to the tip-to-surface distance and cannot be simply backed out of the signal. Thus, leakage often introduces an error into the tunneling current set point, resulting in variability of the tunnel gap from experiment to experiment.
Finally, data from four (4) runs (meeting the above criteria) for each sample was collected. A key collected criterion was to find one or more signal parameters that varied systematically from analyte to analyte, but that remained constant with repeated runs (using different junctions) on the same analyte. Examples of signals from L-leucine and L-isoleucine are shown in
The plots show scatter plots for peak widths and amplitudes (symbols) overlaid on the fitted probability distribution functions used by the SVM. The circled region shows that isoleucine can generate significantly larger peaks than leucine. This is a notable result, since the structural difference between these two amino acids is small and subtle (see insets in
The parameter sets were first subjected to a covariance analysis and parameters that were highly correlated were rejected (so do not convey new information). Other parameters were rejected whose distributions vary from data set to data set with the same analyte. This reduced the subset of useful parameters to 14. Using these parameters, the SVM was trained on small subsets of the data, and then analyzed all of the remaining data (pooled from all six amino acids). The results of which are illustrated below in Table 2 (parameter combinations not optimized).
Random calling of a peak would result in a 17% probability of a correct call. Even at this early stage, a single peak can call an amino acid with better than 0% true positive rate (with 90% discrimination between pairs such as leucine and isoleucine). Each trapped analyte generates many peaks.
Post translational modifications play an extremely important role and detecting them can require very high resolution mass spectroscopy. Here it is shown that a modified amino acid is readily identified from within a larger pool of analytes. Signals were obtained from sarcosine (methylglycine), again running at least four repeated experiments with different tunnel junctions (
Table 3 below illustrates a comparison of analytical methods for detection of amino acids and peptides.
While recognition tunneling has potential advantages in terms of sensitivity, lack of labeling and single molecule counting, the accuracy of the assignment of single molecule signals can be improved. In some embodiments, accuracy is better than the true positive rate shown in Table 3 since each trapped molecule generates multiple spikes (e.g., at least 10 or more). In some embodiments, one way to exploit the repeated data is to use, for example, a majority vote algorithm. For example, if the case of a pool of six (6) amino acids with a 0.5 true positive rate considered, with the remaining calls being distributed amongst the five (5) remaining miscalls with 0.1 probability each, then two (2) correct calls will occur one time in four (4) reads (1/(0.5)2). Two (2) incorrect calls of the same kind occur only one (1) in twenty (20) times (e.g., 1/(0.5×0.1) The remaining outcomes consist of ambiguous reads with two (2) different calls. The accuracy improves rapidly with the number of spikes sampled as shown in
Lower Concentrations. The collected data outlined in
With dielectophoresis, by applying alternating current (AC) fields to the tunnel gap, charged and dipolar molecules can be concentrated into the gap. Accordingly, in some embodiments, a one-dimensional STM (
With pressure-flow, microfluidic cells are provided configured to inject the sample in μl (for example) quantities adjacent to the tunnel gap, and includes a flow profile configured to maximize injection into the gap itself. In such embodiments, this may reduce the absolute amount of sample needed. An exemplary embodiment is show in
In some embodiments, the recognition tunneling, amino-acid analysis system may be integrated with a high-performance liquid chromatography (HPLC) system. As illustrated in
In some embodiments, the RP-HPLC includes a flow rate of about 0.1 mL/min with the microfluidic flow cell shown in
Thus far, data for amino acids and modified amino acids has been shown. Since the ends amino and carboxy ternmini might be expected to interact strongly with the recognition molecules, it is not obvious that peptide chains will generate signals.
Various implementations of the embodiments disclosed above (e.g., protein, amino acid and/or peptide sequencing), in particular at least some of the processes discussed, may be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and/or combinations thereof. These various implementations may include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.
Such computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, for example, and may be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language. As used herein, the term “machine-readable medium” refers to any computer program product, apparatus and/or device (e.g., magnetic discs, optical disks, memory, Programmable Logic Devices (PLDs)) used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and/or data to a programmable processor.
To provide for interaction with a user, the subject matter described herein may be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor and the like) for displaying information to the user and a keyboard and/or a pointing device (e.g., a mouse or a trackball) by which the user may provide input to the computer. For example, this program can be stored, executed and operated by the dispensing unit, remote control, PC, laptop, smart-phone, media player or personal data assistant (“PDA”). Other kinds of devices may be used to provide for interaction with a user as well; for example, feedback provided to the user may be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user may be received in any form, including acoustic, speech, or tactile input.
Certain embodiments of the subject matter described herein may be implemented in a computing system and/or devices that includes a back-end component (e.g., as a data server), or that includes a middleware component (e.g., an application server), or that includes a front-end component (e.g., a client computer having a graphical user interface or a Web browser through which a user may interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, or front-end components. The components of the system may be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include a local area network (“LAN”), a wide area network (“WAN”), and the Internet.
The computing system according to some such embodiments described above may include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.
For example, as shown in
Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented in the present application, are herein incorporated by reference in their entirety.
Although a few variations have been described in detail above, other modifications are possible. For example, any logic flow depicted in the accompanying figures and described herein does not require the particular order shown, or sequential order, to achieve desirable results. Other implementations may be within the scope of at least some of the following exemplary claims.
Example embodiments of the devices, systems and methods have been described herein. As noted elsewhere, these embodiments have been described for illustrative purposes only and are not limiting. Other embodiments are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the above-described embodiments but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, embodiments of the subject disclosure may include methods, systems and devices which may further include any and all elements from any other disclosed methods, systems, and devices, including any and all elements corresponding to protein/peptide/amino-acid sequencing. In other words, elements from one or another disclosed embodiments may be interchangeable with elements from other disclosed embodiments. In addition, one or more features/elements of disclosed embodiments may be removed and still result in patentable subject matter (and thus, resulting in yet more embodiments of the subject disclosure).REFERENCES
1. Huang, S.; He, J.; Chang, S.; Zhang, P.; Liang, F.; Li, S.; Tuchband, M.; Fuhrman, A.; Ros, R.; Lindsay, S. M., Identifying single bases in a DNA oligomer with electron tunneling. Nature Nanotechnology 2010, 5, 868-73.
2. Lindsay, S.; He, J.; Sankey, O; Hapala, P.; Jelinek, P.; Zhang, P.; Chang, S.; Huang, S., Recognition Tunneling. Nanotechnology 2010, 21, 262001-262013.
3. Liang, F.; Li, S.; Lindsay, S.; Zhang, P., Chemical and Hydrogen Bonding Properties of Imidazole-2-carboxamide, a reagent for DNA Sequencing by Recognition Tunnelling Chemistry 2011, submitted.
4. Lawson, J. W.; Bauschlicher, C. W., Transport in Molecular Junctions with different molecular contacts. Physical Review B 2006, 74, 125401.
5. Chang, S.; He, J.; Zhang, P.; Gyarfas, B.; Lindsay, S., Analysis of Interactions in a Molecular Tunnel Junction. J. Am Chem Soc 2011, 133, 14267-14269.
6. Hempel, J., An oritentation to Edman chemistry. In Modern Protein Chemistry, Howard, G. C.; Brown, W. E., Eds. CRC Press: 2002; pp 103-122.
7. Chang, S., S. Huang, H. Liu, P. Zhang, R. Akahori, S. Li, B. Gyarfas, J. Shumway, B. Ashcroft, J. He, and S. Lindsay, Chemical Recognition and Binding Kinetics in a Functionalized Tunnel Junction. Nanotechnology, 2012.
8. Chang, C.-C. and C.-J. Lin, LIBSVM: a library for support vector machines. ACM Transactions on Intelligent Systems and Technology, 2011: p. 2:27:1-27:27.
9. Chen, W., X. Yin, J. Q. Mu, and Y. Yin, Subfemtomole level protein sequencing by Edman degradation carried out in a microfluidic chip. Chem. Commun., 2007: p. 2488-2490.
10. Chen, W., J. Shen, X. Yin, and Y. Yin, Optimization of microfabricated nanoliter-scale solid phase extraction device for detection of gel-separated proteins in low abundance by matrix assisted laser desorption/ionization mass spectrometrry. Rapid Communications in Mass Spectrometry, 2007. 21: p. 35-43.
11. Tuchband, M., J. He, S. Huang, and S. Lindsay, Insulated gold scanning tunneling microscopy probes for recognition tunneling in an aqueous environment. Rev, Sci. Instrum., 2012. 83: p. 015102.
12. Louwagie, M.; Kieffer-Jaquinod, S.; Dupierris, V.; Coute, Y.; Bruley, C.; Garin, J.; Dupuis, A.; Jaquinod, M.; Brun, V., Introducing AAA-MS, a Rapid and Sensitive Method for Amino Acid Analysis Using Isotope Dilution and High-Resolution Mass Spectrometry. J. Proteome Res. 2012, 11, p. 3929-3936.
13. Rosnack, K. J.; Stroh, J. G., C-Terminal Sequencing of Peptides Using Electrospray Ionization Mas Spectrometry. Rapid Commun. Mass Spectrom. 1992, 6, 637-640.
14. Walker, J. M.; Winder, J. S., C-Terminal Sequence Analysis with Carboxypeptidase Y. In The Protein Protocols Handbook, Walker, J. M., Ed. Humana Press Inc.: 1996; pp 569-571;
15. Yan-Fei, G.; Hong-Xia, W., Development of C-terminal Sequencing Analysis of Protein and Peptide. Chin J Anal Chem 2007, 35, 1820-1826.
1. An apparatus for sequencing and/or identifying at least one of protein, peptide, amino acid and/or modified amino acid, the apparatus comprising:
- a plurality of electrodes;
- at least a first electrode of the plurality of the electrodes being functionalized with a molecule strongly bonded to at least the first electrode and that forms transient bonds with an amino acid;
- voltage bias means for applying a voltage between the first electrode and a second electrode of the plurality of electrodes, wherein the first and second electrodes are arranged such that a gap is produced between them; and
- current data monitoring means for monitoring current passing between the first and second electrodes upon a solution containing at least one of protein, peptide, amino acid, and/or modified amino acid being passed in the gap established between the first and the second electrodes.
2. The apparatus according to claim 1, further comprising means for recording and/or storing data corresponding to the monitored current.
3. The apparatus according to claim 1, further comprising a database and processing means, wherein the database includes data associated with characteristic current signatures for a plurality of proteins, peptides, amino acids, and/or modified amino acids, and/or derivatives thereof, and wherein the processing means compares such stored signatures with the collected current data to determine the identity of the protein, peptide, amino acid and/or modified amino acid.
4. The apparatus according to claim 1, wherein the chirality of an amino acid is determined from the current data.
5. The apparatus according to claim 1, further comprising one or more proteases for digesting a peptide, the proteases being arranged in proximity to the first and second electrodes.
6. The apparatus according to claim 5, wherein the proteases are provided on a bead.
7. The apparatus according to claim 1, further comprising one carboxypeptidase for digesting a peptide or protein from its C-terminus, the peptidase being arranged in proximity to the first and second electrodes.
8. The apparatus according to claim 1, further comprising one aminopeptidase for digesting a peptide or protein from its N-terminus, the peptidase being arranged in proximity to the first and second electrodes.
9. The apparatus according to claim 1, further comprising one or more proteins, peptides, amino acids and/or modified amino acids on a bead to be sequenced using Edman chemistry.
10. The apparatus according to claim 1, further comprising one or more proteases for digesting a protein and/or peptide, the proteases being arranged in proximity to the first and second electrodes.
11. The apparatus according to claim 1, wherein the distance between electrodes is adjustable.
12. The apparatus according to claim 10, wherein the proteases are provided on a bead.
13. A system for sequencing at least one of protein, peptide, and/or amino acids comprising the apparatus according to claim 1, and further comprising:
- one or more proteases arranged in proximity to at least one of the electrodes for digesting a protein and/or peptide, and
- injection means for injecting at least one reagent into the solution, wherein the activity of the proteases are synchronized via an injection of a reagent by the injection means that activates said proteases.
15. An apparatus for capturing and reading negatively charged amino acids and/or peptide fragments, the apparatus comprising:
- a cis chamber;
- a trans chamber;
- a first apparatus comprising the apparatus of claim 1;
- a second apparatus comprising the apparatus of claim 1, wherein the second apparatus is arranged substantially orthogonal to the first apparatus, and wherein the second apparatus separates the cis chamber from the trans chamber;
- a plurality of reference electrodes arranged among the chambers, wherein one of the reference electrodes is provided in the trans chamber and is positively biased with respect to the cis chamber, and wherein the gap provided by the second apparatus acts to capture negatively charged amino acid and/or peptide residues.