scholarly journals Evaluation of FRET X for Single-Molecule Protein Fingerprinting

2021 ◽  
Author(s):  
Carlos de Lannoy ◽  
Mike Filius ◽  
Raman van Wee ◽  
Chirlmin Joo ◽  
Dick de Ridder
Keyword(s):  
Single Molecule ◽  
Protein Fingerprinting

scholarly journals Single-Molecule Protein Fingerprinting with Nanopores

2017 ◽  
Vol 112 (3) ◽  
pp. 330a ◽  
Author(s):  
Laura Restrepo-Perez ◽  
Misha Soskine ◽  
Shalini John ◽  
Aleksei Aksimentiev ◽  
Giovanni Maglia ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Fingerprinting

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 Evaluation of FRET X for Single-Molecule Protein Fingerprinting

iScience ◽  
2021 ◽  
pp. 103239
Author(s):  
Carlos Victor de Lannoy ◽  
Mike Filius ◽  
Raman van Wee ◽  
Chirlmin Joo ◽  
Dick de Ridder
Keyword(s):  
Single Molecule ◽  
Protein Fingerprinting

scholarly journals Protein identification by nanopore peptide profiling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Florian Leonardus Rudolfus Lucas ◽  
Roderick Corstiaan Abraham Versloot ◽  
Liubov Yakovlieva ◽  
Marthe T. C. Walvoort ◽  
Giovanni Maglia
Keyword(s):  
Mass Spectrometry ◽  
Nucleic Acid ◽  
Single Molecule ◽  
Protein Identification ◽  
Low Cost ◽  
Protein Fingerprinting ◽  
Nucleic Acid Analysis

AbstractNanopores are single-molecule sensors used in nucleic acid analysis, whereas their applicability towards full protein identification has yet to be demonstrated. Here, we show that an engineered Fragaceatoxin C nanopore is capable of identifying individual proteins by measuring peptide spectra that are produced from hydrolyzed proteins. Using model proteins, we show that the spectra resulting from nanopore experiments and mass spectrometry share similar profiles, hence allowing protein fingerprinting. The intensity of individual peaks provides information on the concentration of individual peptides, indicating that this approach is quantitative. Our work shows the potential of a low-cost, portable nanopore-based analyzer for protein identification.

scholarly journals In silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection

iScience ◽  
2021 ◽  
pp. 103202
Author(s):  
Carlos de Lannoy ◽  
Florian Leonardus Rudolfus Lucas ◽  
Giovanni Maglia ◽  
Dick de Ridder
Keyword(s):  
Single Molecule ◽  
In Silico ◽  
Protein Fingerprinting ◽  
Fingerprinting Method

scholarly journals Chop-n-drop: in silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection

2021 ◽  
Author(s):  
Carlos de Lannoy ◽  
Giovanni Maglia ◽  
Dick de Ridder
Keyword(s):  
Single Molecule ◽  
Protein Identification ◽  
Cost Effective ◽  
Biological Research ◽  
Fragmentation Pattern ◽  
Experimental Proof ◽  
Complex Samples ◽  
Single Molecule Level ◽  
Protein Fingerprinting ◽  
Fragmentation Patterns

The identification of proteins at the single-molecule level would open exciting new venues in biological research and disease diagnostics. Previously we proposed a nanopore-based method for protein identification called chop-n-drop fingerprinting, in which the fragmentation pattern induced and measured by a proteasome-nanopore construct is used to identify single proteins. However whether such fragmentation patterns are sufficiently characteristic of proteins to identify them in complex samples remained unclear. In the simulation study presented here, we show that 97.9% of human proteome constituents are uniquely identified under close to ideal measuring circumstances, using a simple alignment-based classification method. We show that our method is robust against experimental error, as 78.8% can still be identified if the resolution is twice as low as currently attainable and 10% of proteasome restriction sites and protein fragments are randomly ignored. Based on these results and our experimental proof-of-concept, we argue that chop-n-drop fingerprinting has the potential to make cost-effective single-molecule protein identification feasible in the near future.

scholarly journals Single-Molecule Protein Fingerprinting using Nanopores

2019 ◽  
Vol 116 (3) ◽  
pp. 316a
Author(s):  
Sonja Schmid ◽  
Laura Restrepo ◽  
Gang Huang ◽  
Chirlmin Joo ◽  
Giovanni Maglia ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Fingerprinting

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

Single molecule optical diffraction: A tool for understanding the structure of triple stranded left-hand helical cellulose

1989 ◽  
Vol 47 ◽  
pp. 1024-1025
Author(s):  
George C. Ruben ◽  
William Krakow
Keyword(s):  
Single Molecule ◽  
Helical Structure ◽  
Optical Diffraction ◽  
Maximum Diameter ◽  
Primary Cell Wall ◽  
Cellulose Synthesis ◽  
Left Hand ◽  
Left Handed ◽  
Freeze Dried ◽  
Diffraction Patterns

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and comparedIn this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.

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