The nanopore mass spectrometer

Bush, Joseph; Maulbetsch, William; Lepoitevin, Mathilde; Wiener, Benjamin; Skanata, Mirna Mihovilovic; Moon, Wooyoung; Pruitt, C. D.; Stein, Derek

doi:10.1063/1.4986043

Cited by 23 publications

(34 citation statements)

References 40 publications

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“…The use of ever-smaller electrospray ion source apertures has led to substantial improvements in the sensitivity of MS 31,32 . Mass spectrometers with a nanopore ion source have been developed for the purpose of sequencing single proteins 33 (Fig. 2d).…”

Section: Box 2 | Mass Spectrometry-based Single-cell Proteomicsmentioning

confidence: 99%

The emerging landscape of single-molecule protein sequencing technologies

et al. 2021

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Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

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Section: Box 2 | Mass Spectrometry-based Single-cell Proteomicsmentioning

confidence: 99%

The emerging landscape of single-molecule protein sequencing technologies

et al. 2021

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“…The ions pass through focusing ion optics, a quadrupole mass filter and an ion bender (Extrel CMS) before being measured by a continuous dynode single-ion detector (DeTech). [17]. The background pressure inside the instrument is typically 10 −7 torr, and the mean free path of an amino acid ( > 100 m) is orders of magnitude larger than the size of the instrument.…”

Section: Figmentioning

confidence: 99%

“…Figure 1c shows an overview of our custom mass spectrometer [17]. Ions are emitted from the source by applying a voltage between a Ag/AgCl electrode inside the nanocapillary and a ring-shaped extraction electrode located about 5 mm in front of the nanopore tip.…”

mentioning

confidence: 99%

Nanopore ion sources deliver single amino acid and peptide ions directly into high vacuum

Drachman

Lepoitevin

Szapary

et al. 2021

Preprint

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A technology for sequencing single proteins would expand our understanding of biology and improve the detection and treatment of disease. Approaches based on fluorosequencing, nanopores, and tunneling spectroscopy are under development and show promise. However, only mass spectrometry (MS) has demonstrated an ability to identify amino acids with minimal degeneracy. We envision sequencing a protein by fragmenting it and delivering its constituent amino acids into a mass spectrometer in sequential order, but existing ion sources employ a background gas that scrambles the spatial ordering of ions and degrades their transmission. Here we report an ion source comprising a glass capillary with a sub-100 nm diameter pore that emits amino acid ions from aqueous solution directly into vacuum. Emitted ions travel collision-less trajectories before striking a single-ion detector. We measured unsolvated ions of 16 different amino acids as well as glutathione and two of its post-translationally modified variants.

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“…If at least one AMP-sized blockade is observed Identity of AA = AA corresponding to charged tRNA (8) If no AMP-sized blockade is observed the contents of trans are transferred back to the reservoir (Figure 1). A different tRNA and related AARS are added in up to N (20 ≤ N ≤ 61) cycles until AA is identified.…”

Section: Current Blockade As a Proxy For Analyte Volumementioning

confidence: 99%

“…In [7] it is reported that 13 of the 20 standard amino acids can be identified based solely on the volume excluded by an amino acid as it translocates through a nanopore. In [8,9] standard mass spectrometry methods are used to sequence monomers that are ionized after they are photo-cleaved from a polymer as it emerges from a nanopore.…”

Section: Introductionmentioning

confidence: 99%

Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

Sampath¹

2020

Preprint

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Label-free identification of amino acids (AAs) based on superspecific transfer RNAs (tRNAs) and amino-acyl tRNA synthetase (AARS) is explored. Molecules of a specific AA, a tRNA and its cognate AARS, and adenosine triphosphate (ATP) from a reservoir are confined to a micro-sized cavity by hydraulic pressure to counter the effects of diffusion. In this confined space a cognate tRNA in the cavity gets charged with AA and adenosine monophosphate (AMP) is released. The products are transferred to an electrolytic cell with a nanopore where AA, AMP, and ATP cause current blockades of different sizes. If an AMP-sized blockade occurs it means that a tRNA molecule has been charged with AA, this identifies AA; otherwise the procedure is repeated with a different tRNA and AARS until AA is identified. Unlike in most other nanopore-based methods, there is no need for precise measurement of current blockade levels. The efficacy of the procedure is assessed by simulating the movement of particles in a reservoir-cavity structure and analyzing the blockades that occur in the nanopore. In the former the probability of reactants escaping the cavity is found to be near 0. In the latter the probability of an error in identifying an AMP blockade is found to be less than 10% in the worst case (which occurs with the largest volume AA, namely Tryptophan). Detailed information and data are provided in a Supplementary File.

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The nanopore mass spectrometer

Cited by 23 publications

References 40 publications

The emerging landscape of single-molecule protein sequencing technologies

The emerging landscape of single-molecule protein sequencing technologies

Nanopore ion sources deliver single amino acid and peptide ions directly into high vacuum

Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

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