scholarly journals Single-Molecule Peptide Fingerprinting

2017 ◽  
Author(s):  
Jetty van Ginkel ◽  
Mike Filius ◽  
Malwina Szczepaniak ◽  
Pawel Tulinski ◽  
Anne S. Meyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Single Cell ◽  
Single Molecule ◽  
Medical Diagnostics ◽  
Cellular Systems ◽  
Protein Sequencing ◽  
Small Scale ◽  
Proof Of Concept ◽  
Essential Information ◽  
First Choice

ABSTRACTProteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study, such as single-cell analysis, challenging. Here we demonstrate a proof of concept of singlemolecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits uni-directional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.SIGNIFICANCEProtein sequencing remains a challenge for small samples. A sensitive sequencing technology will create the opportunity for single-cell proteomics and real-time screening for on-site medical diagnostics. In order to resolve protein identity, we previously developed a computational algorithm that analyzes the ordered sequence of only two types of amino acids within a protein species. Through modification of a biological nanomachine, here we developed single-molecule fluorescence technology to linearize protein molecules and to read signals from labeled amino acids in an ordered manner. This proof of concept of singlemolecule fingerprinting will open a new door to single-molecule protein sequencing and pave the road towards the development of a new, fast, and reliable diagnostic tool.

scholarly journals Single-molecule peptide fingerprinting

2018 ◽  
Vol 115 (13) ◽  
pp. 3338-3343 ◽  
Author(s):  
Jetty van Ginkel ◽  
Mike Filius ◽  
Malwina Szczepaniak ◽  
Pawel Tulinski ◽  
Anne S. Meyer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Living Organism ◽  
Cellular Systems ◽  
Small Scale ◽  
Proof Of Concept ◽  
Essential Information ◽  
Protein Substrates ◽  
Single Molecule Fret ◽  
Protein Fingerprinting ◽  
First Choice

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.

scholarly journals Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

Nano Research ◽  
2020 ◽  
Vol 14 (1) ◽  
pp. 328-333 ◽  
Author(s):  
Andrea Spitaleri ◽  
Denis Garoli ◽  
Moritz Schütte ◽  
Hans Lehrach ◽  
Walter Rocchia ◽  
...  
Keyword(s):  
Amino Acids ◽  
Single Molecule ◽  
Dynamic Range ◽  
Protein Sequencing ◽  
High Fidelity ◽  
Label Free ◽  
Therapeutic Approaches ◽  
Highly Sensitive ◽  
Dynamics Simulations ◽  
Structure Of Proteins

AbstractSingle molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the “shape” of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing.

scholarly journals Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

2022 ◽  
Author(s):  
Brian D Reed ◽  
Michael J Meyer ◽  
Valentin Abramzon ◽  
Omer Ad ◽  
Pat Adcock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Dynamic Range ◽  
Protein Sequencing ◽  
Peptide Sequence ◽  
Single Amino Acid ◽  
Functional Components

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

Protein Sequencing, One Molecule at a Time

2022 ◽  
Vol 51 (1) ◽  
Author(s):  
Brendan M. Floyd ◽  
Edward M. Marcotte
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Large Scale ◽  
Dynamic Range ◽  
Medical Diagnostics ◽  
Protein Quantification ◽  
Protein Sequencing ◽  
Protein Isoforms ◽  
Annual Review ◽  
Publication Date

Despite tremendous gains over the past decade, methods for characterizing proteins have generally lagged behind those for nucleic acids, which are characterized by extremely high sensitivity, dynamic range, and throughput. However, the ability to directly characterize proteins at nucleic acid levels would address critical biological challenges such as more sensitive medical diagnostics, deeper protein quantification, large-scale measurement, and discovery of alternate protein isoforms and modifications and would open new paths to single-cell proteomics. In response to this need, there has been a push to radically improve protein sequencing technologies by taking inspiration from high-throughput nucleic acid sequencing, with a particular focus on developing practical methods for single-molecule protein sequencing (SMPS). SMPS technologies fall generally into three categories: sequencing by degradation (e.g., mass spectrometry or fluorosequencing), sequencing by transit (e.g., nanopores or quantum tunneling), and sequencing by affinity (as in DNA hybridization–based approaches). We describe these diverse approaches, which range from those that are already experimentally well-supported to the merely speculative, in this nascent field striving to reformulate proteomics. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals A single-molecule electrical approach for amino acid detection and chirality recognition

2021 ◽  
Vol 7 (10) ◽  
pp. eabe4365
Author(s):  
Zihao Liu ◽  
Xingxing Li ◽  
Hiroshi Masai ◽  
Xinyi Huang ◽  
Susumu Tsuda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Theoretical Calculations ◽  
Protein Sequencing ◽  
Current Fluctuations ◽  
Point Contacts ◽  
Conductance Data ◽  
Single Molecule Junctions

One of the ultimate goals of analytic chemistry is to efficiently discriminate between amino acids. Here we demonstrate this ability using a single-molecule electrical methodology based on molecular nanocircuits formed from stable graphene-molecule-graphene single-molecule junctions. These molecular junctions are fabricated by covalently bonding a molecular machine featuring a permethylated-β-cyclodextrin between a pair of graphene point contacts. Using pH to vary the type and charge of the amino acids, we find distinct multimodal current fluctuations originating from the different host-guest interactions, consistent with theoretical calculations. These conductance data produce characteristic dwell times and shuttling rates for each amino acid, and allow accurate, statistical real-time, in situ measurements. Testing four amino acids and their enantiomers shows the ability to distinguish between them within a few microseconds, thus paving a facile and precise way to amino acid identification and even single-molecule protein sequencing.

scholarly journals A theoretical analysis of single molecule protein sequencing via weak binding spectra

2018 ◽  
Author(s):  
Samuel Rodriques ◽  
Adam Marblestone ◽  
Ed Boyden
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Protein Sequencing ◽  
Single Amino Acid ◽  
Weak Binding ◽  
Binding Spectra ◽  
Accuracy Determination

AbstractWe propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding [1] to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to extremely weak-binding probes. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.Author summaryWe simplify the problem of single molecule protein sequencing by proposing and analyzing an approach that makes use of low-affinity, low-specificity binding reagents. This decouples the problem of protein sequencing from the problem of generating a high-quality library of binding reagents against each of the amino acids.

scholarly journals Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

2021 ◽  
Author(s):  
Prem Kumar Viji Babu ◽  
Ursula Mirastschijski ◽  
Ganzanfer Belge ◽  
Manfred Radmacher
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Actin Filaments ◽  
Adherens Junctions ◽  
Bond Formation ◽  
Force Spectroscopy ◽  
Cellular Systems ◽  
Bond Rupture ◽  
Extracellular Region ◽  
Cell Force

AbstractCadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

Mapping global water projects: improving access to donor investment information on the web

Water Policy ◽  
2003 ◽  
Vol 5 (3) ◽  
pp. 203-212
Author(s):  
J. Lisa Jorgensona
Keyword(s):  
Water Resources ◽  
Web Sites ◽  
Large Scale ◽  
Technical Assistance ◽  
Water Withdrawal ◽  
Small Scale ◽  
Water Projects ◽  
Essential Information ◽  
Water Project ◽  
The Web

This paper discusses a series of discusses how web sites now report international water project information, and maps the combined donor investment in more than 6000 water projects, active since 1995. The maps show donor investment:  • has addressed water scarcity,  • has improved access to improvised water resources,  • correlates with growth in GDP,  • appears to show a correlation with growth in net private capital flow,  • does NOT appear to correlate with growth in GNI. Evaluation indicates problems in the combined water project portfolios for major donor organizations: •difficulties in grouping projects over differing Sector classifications, food security, or agriculture/irrigation is the most difficult.  • inability to map donor projects at the country or river basin level because 60% of the donor projects include no location data (town, province, watershed) in the title or abstracts available on the web sites.  • no means to identify donor projects with utilization of water resources from training or technical assistance.  • no information of the source of water (river, aquifer, rainwater catchment).  • an identifiable quantity of water (withdrawal amounts, or increased water efficiency) is not provided.  • differentiation between large scale verses small scale projects. Recommendation: Major donors need to look at how the web harvests and combines their information, and look at ways to agree on a standard template for project titles to include more essential information. The Japanese (JICA) and the Asian Development Bank provide good models.

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