Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore

Zhang, Ming; Tang, Chao; Wang, Zichun; Chen, Shanchuan; Zhang, Dan; Li, Kaiju; Sun, Ke; Zhao, Changjian; Wang, Yu; Xu, Mengying; Dai, Lunzhi; Lu, Guangwen; Shi, Hubing; Ren, Haiyan; Chen, Lu; Geng, Jia

doi:10.1038/s41592-024-02208-7

Cited by 8 publications

(8 citation statements)

References 56 publications

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“…28 Another exciting option is nanopore technology, which was first commercialized for DNA and RNA sequencing, and is now emerging as a promising tool for peptide sequencing. Recent achievements include the detection of all 20 proteinogenic amino acids, real-time analyses of post-translational modifications (PTMs), 29 and peptide sequencing by sequentially cleaving amino acids from the Cterminus. 30 Although this technology currently struggles with large-scale sequencing under complex conditions, it has undoubtedly holds promise as a rising star.…”

Section: A Need For High Throughput Proteomicsmentioning

confidence: 99%

The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

Jiang,

DeBord,

Vitrac

et al. 2024

J. Proteome Res.

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The coevolution of liquid chromatography (LC) with mass spectrometry (MS) has shaped contemporary proteomics. LC hyphenated to MS now enables quantification of more than 10,000 proteins in a single injection, a number that likely represents most proteins in specific human cells or tissues. Separations by ion mobility spectrometry (IMS) have recently emerged to complement LC and further improve the depth of proteomics. Given the theoretical advantages in speed and robustness of IMS in comparison to LC, we envision that ongoing improvements to IMS paired with MS may eventually make LC obsolete, especially when combined with targeted or simplified analyses, such as rapid clinical proteomics analysis of defined biomarker panels. In this perspective, we describe the need for faster analysis that might drive this transition, the current state of direct infusion proteomics, and discuss some technical challenges that must be overcome to fully complete the transition to entirely gas phase proteomics.

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Section: A Need For High Throughput Proteomicsmentioning

confidence: 99%

The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

Jiang,

DeBord,

Vitrac

et al. 2024

J. Proteome Res.

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“…Step 2. In the sorted list count the number of duplicates down the string and output copy count for protein Complexity of mixture sequencing and quantification 1) In Procedure AssembleSequencesFromConjugateSequenceMatrix there are M 20 distinct paths of length 20 from the first column to the 20 th column of CSM[M] [20]. In the worst case all of them may be examined, so the worst-case behavior is O(M 20 ).…”

Section: Procedures Quantifysamplefrommproteinsequencesmentioning

confidence: 99%

“…For a recent review of nanopore-based methods, see [12]. Specific studies can be found in [13][14][15][16][17][18][19][20].…”

Section: Protein Sequencing In the Bulk And At The Single Molecule Levelmentioning

confidence: 99%

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Whole protein sequencing and quantification without proteolysis, terminal residue cleavage, or purification: A computational model

Sampath

2024

Preprint

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Sequencing and quantification of whole proteins in a sample without separation, terminal residue cleavage, or proteolysis are modeled computationally. Similar to recent work on DNA sequencing (PNAS 113, 5233-5238, 2016), a high-volume conjugate is attached to every instance of amino acid (AA) type AAi, 1 ≤ i ≤ 20, in an unfolded whole protein, which is then translocated through a nanopore. From the volume excluded by 2L residues in a pore of length L (a proxy for the blockade current), a partial sequence containing AAi is obtained. Translocation is assumed to be unidirectional, with residues exiting the pore at a roughly constant rate of ~1/μs (Nature Biotechnology 41, 1130-1139, 2023). The blockade signal is sampled at intervals of 1 μs and digitized with a step precision of 70 nm3; the positions of the AAis are obtained from the positions of well-defined quantum jumps in the signal. This procedure is applied to all 20 standard AA types, the resulting 20 partial sequences are merged to obtain the whole protein sequence. The complexity of subsequence computation is O(N) for a protein with N residues. The method is illustrated with a sample protein from the human proteome (Uniprot id UP000005640_9606). A mixture containing M unknown protein molecules (which may include multiple copies) is sequenced by mapping their partial sequences to a graph GM with 20M vertexes; GM contains M disjoint cliques of degree 20. Unlike in general clique identification (computationally an NP-hard problem) the M cliques can be obtained through a connected graph components algorithm in polynomial time. Quantification is done by sorting the M sequences on the sequence strings and counting duplicates down the sequence. The complexity of sequencing and quantification of a mixture of M proteins is O(MN). The possibility of translating this procedure into practice and related implementation issues are discussed.

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“…The other emerging technology from genomics is nanopore sequencing, in which the passing of a linearised amino acid chain through a protein-based nanopore induces specific measurable electrical current changes depending on each particular amino acid [ 214 , 215 ]. There is recent evidence to suggest that PTM can also be identified and localised with this technology [ 216 , 217 ].…”

Section: Recognising and Addressing Critical Issuesmentioning

confidence: 99%

Proteomics—The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience

Coorssen,

Padula

2024

Proteomes

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With growing recognition and acknowledgement of the genuine complexity of proteomes, we are finally entering the post-proteogenomic era. Routine assessment of proteomes as inferred correlates of gene sequences (i.e., canonical ‘proteins’) cannot provide the necessary critical analysis of systems-level biology that is needed to understand underlying molecular mechanisms and pathways or identify the most selective biomarkers and therapeutic targets. These critical requirements demand the analysis of proteomes at the level of proteoforms/protein species, the actual active molecular players. Currently, only highly refined integrated or integrative top-down proteomics (iTDP) enables the analytical depth necessary to provide routine, comprehensive, and quantitative proteome assessments across the widest range of proteoforms inherent to native systems. Here we provide a broad perspective of the field, taking in historical and current realities, to establish a more balanced understanding of where the field has come from (in particular during the ten years since Proteomes was launched), current issues, and how things likely need to proceed if necessary deep proteome analyses are to succeed. We base this in our firm belief that the best proteomic analyses reflect, as closely as possible, the native sample at the moment of sampling. We also seek to emphasise that this and future analytical approaches are likely best based on the broad recognition and exploitation of the complementarity of currently successful approaches. This also emphasises the need to continuously evaluate and further optimize established approaches, to avoid complacency in thinking and expectations but also to promote the critical and careful development and introduction of new approaches, most notably those that address proteoforms. Above all, we wish to emphasise that a rigorous focus on analytical quality must override current thinking that largely values analytical speed; the latter would certainly be nice, if only proteoforms could thus be effectively, routinely, and quantitatively assessed. Alas, proteomes are composed of proteoforms, not molecular species that can be amplified or that directly mirror genes (i.e., ‘canonical’). The problem is hard, and we must accept and address it as such, but the payoff in playing this longer game of rigorous deep proteome analyses is the promise of far more selective biomarkers, drug targets, and truly personalised or even individualised medicine.

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Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore

Cited by 8 publications

References 56 publications

The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

Whole protein sequencing and quantification without proteolysis, terminal residue cleavage, or purification: A computational model

Proteomics—The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience

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