The importance of adding EDTA for the nanopore analysis of proteins

Krasniqi, Besnik; Lee, Jeremy S.

doi:10.1039/c2mt20050c

Cited by 4 publications

(4 citation statements)

References 41 publications

(51 reference statements)

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“…In most cases, this process would generate bumping events. By analyzing the duration and current distributions of the bumping events, the a-HL achieved the detection of single molecule behavior of the analytes of a large size, such as illuminating the interactions between prion proteins and metal ions 52,53 and analyzing the aggregation states of peptides. [54][55][56] A previous study showed that the aggregation states of bamyloid 42 (Ab42) could be analyzed by monitoring the bumping events using an a-HL nanopore (Fig.…”

Section: Analysis Of the Bumping Events In Nanopore Detectionsmentioning

confidence: 99%

Single molecule analysis by biological nanopore sensors

Ying

Cao

Long

2014

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Recent advances in top-down mass spectrometry enabled identification of intact proteins, but this technology still faces challenges. For example, top-down mass spectrometry suffers from a lack of sensitivity since the ion counts for a single fragmentation event are often low. In contrast, nanopore technology is exquisitely sensitive to single intact molecules, but it has only been successfully applied to DNA sequencing, so far. Here, we explore the potential of sub-nanopores for single-molecule protein identification (SMPI) and describe an algorithm for identification of the electrical current blockade signal (nanospectrum) resulting from the translocation of a denaturated, linearly charged protein through a sub-nanopore. The analysis of identification p-values suggests that the current technology is already sufficient for matching nanospectra against small protein databases, e.g., protein identification in bacterial proteomes. Author summary Protein identification is the key step in many proteomics studies. Currently, the most popular technique for intact protein analysis is top-down mass spectrometry which recently enabled high-throughput identification of many proteins and their proteoforms. However , this approach requires large amounts of materials and is currently limited to short proteins, typically less than 30 kDa. On the other hand, nanopore sensors promise single molecule sensitivity in protein analysis, but an approach for the identification of a single protein from its blockade current (nanospectrum) has remained elusive, since the signal from the sensors relates to the amino acid sequence of the protein in a poorly understood way. In this work we describe the first algorithm for protein identification based on nanospectra associated with translocation of proteins through pores with sub-nanometer diameters. While identification accuracy currently does not allow reliable processing of complex protein samples yet, we believe, that the rapidly improving experimental protocols along with the new computational algorithms will transform into a viable protein identification approach in the near future. PLOS Computational Biology | https://doi.

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Section: Analysis Of the Bumping Events In Nanopore Detectionsmentioning

confidence: 99%

Single molecule analysis by biological nanopore sensors

Ying

Cao

Long

2014

Analyst

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“…Most reports show that proteins induce large blockade events when interacting with the α-hemolysin pore [4], [5], [7], [40], [41], [42], [43]. We have analyzed eight different proteins (unpublished data) using alpha-hemolysin and all induce large blockade events.…”

Section: Introductionmentioning

confidence: 99%

“…At the first glance, the profile of the events observed are similar to those obtained with small peptides and single-stranded DNA or RNA which can be mistakenly identified as translocation events. However, most proteins analyzed with nanopore sensing are larger than the smallest constriction of the α-hemolysin pore [2], [5], [7], [8], [10], [14], [15], [17], [43], [44]. Therefore, in order for proteins to translocate the pore, they must unfold.…”

Section: Introductionmentioning

confidence: 99%

RNase A Does Not Translocate the Alpha-Hemolysin Pore

Krasniqi

Lee

2014

PLoS ONE

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The application of nanopore sensing utilizing the α-hemolysin pore to probe proteins at single-molecule resolution has expanded rapidly. In some studies protein translocation through the α-hemolysin has been reported. However, there is no direct evidence, as yet, that proteins can translocate the α-hemolysin pore. The biggest challenge to obtaining direct evidence is the lack of a highly sensitive assay to detect very low numbers of protein molecules. Furthermore, if an activity based assay is applied then the proteins translocating by unfolding should refold back to an active confirmation for the assay technique to work. To overcome these challenges we selected a model enzyme, ribonuclease A, that readily refolds to an active conformation even after unfolding it with denaturants. In addition we have developed a highly sensitive reverse transcription polymerase chain reaction based activity assay for ribonuclease A. Initially, ribonuclease A, a protein with a positive net charge and dimensions larger than the smallest diameter of the pore, was subjected to nanopore analysis under different experimental conditions. Surprisingly, although the protein was added to the cis chamber (grounded) and a positive potential was applied, the interaction of ribonuclease A with α-hemolysin pore induced small and large blockade events in the presence and the absence of a reducing and/or denaturing agent. Upon measuring the zeta potential, it was found that the protein undergoes a charge reversal under the experimental conditions used for nanopore sensing. From the investigation of the effect of voltage on the interaction of ribonuclease A with the α-hemolysin pore, it was impossible to conclude if the events observed were translocations. However, upon testing for ribonuclease A activity on the trans chamber it was found that ribonuclease A does not translocate the α-hemolysin pore.

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“…Hence, nanopores can, in principle, provide precise information on a single-molecule level. Significant efforts have been devoted to nanopore DNA sequencing [2] and only recently possible proteomic applications have started being explored [3][4][5][6][7][8][9][10][11][12] using both solid-state and biological pores.…”

Section: Introductionmentioning

confidence: 99%

Protein translocation in narrow pores: Inferring bottlenecks from native structure topology

et al. 2013

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Coarse-grained simulations of protein translocation across narrow pores suggest that the transport is characterized by long stall events. The translocation bottlenecks and the associated free-energy barriers are found to be strictly related to the structural properties of the protein native structure. The ascending ramps of the free-energy profile systematically correspond to regions of the chain denser in long range native contacts formed with the untranslocated portion of the protein. These very regions are responsible for the stalls occurring during the protein transport along the nanopore. The decomposition of the free energy in internal energyand entropic terms shows that the dominant energetic contribution can be estimated on the base of the protein native structure only. Interestingly, the essential features of the dynamics are retained in a reduced phenomenological model of the process describing the evolution of a suitable collective variable in the associated free-energy landscape.

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The importance of adding EDTA for the nanopore analysis of proteins

Cited by 4 publications

References 41 publications

Single molecule analysis by biological nanopore sensors

Single molecule analysis by biological nanopore sensors

RNase A Does Not Translocate the Alpha-Hemolysin Pore

Protein translocation in narrow pores: Inferring bottlenecks from native structure topology

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