Tryptic digestion of human serum for proteomic mass spectrometry automated by centrifugal microfluidics

Klatt, Jan-Niklas; Depke, Maren; Goswami, Neha; Paust, Nils; Zengerle, Roland; Schmidt, Frank; Hutzenlaub, Tobias

doi:10.1039/d0lc00530d

Cited by 12 publications

(13 citation statements)

References 42 publications

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“…In addition to these significant advances enabling the in-depth characterization of the human plasma and serum proteome, other technological developments have focused on improving the reproducibility, throughput, and sample preparation prior to LC-MS/MS analyses. To circumvent lengthy, error-prone, and variable manual handling, sophisticated chips or microfluidic devices integrating biochemical steps of preanalytical protocols have been developed. − Some of these microfluidic devices integrate plasma preparation, depletion of abundant proteins, digestion, and peptide desalting for a fully automated processing of blood samples . Others developed highly parallelized preanalytical workflows based on automation using the 96-well plate format. − In line with such progress and to avoid bias in the distribution of samples before sample processing, dedicated applications have been developed to randomize samples, supporting the reliability of the entire workflow.…”

Section: Recent Advancesmentioning

confidence: 99%

Advances and Utility of the Human Plasma Proteome

et al. 2021

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The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.

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Section: Recent Advancesmentioning

confidence: 99%

Advances and Utility of the Human Plasma Proteome

et al. 2021

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“…37 To further increase differentiation among the positional isoforms, future work will focus on reducing instrumental noise 38 and on protein engineering to optimize the conformational degrees of freedom of peptides in the pore. Thus, in a combination of parallel high-resolution electrophysiology 15,39 with standardized and automated enzymatic generation of peptide fragments, 40 whole-molecule sensing with the aerolysin nanopore has the potential of being implemented in a practical technology to rapidly detect PTMs with positional resolution without requiring strand sequencing of proteins.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape

et al. 2022

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The chemical nature and precise position of posttranslational modifications (PTMs) in proteins or peptides are crucial for various severe diseases, such as cancer. State-of-theart PTM diagnosis is based on elaborate and costly massspectrometry or immunoassay-based approaches, which are limited in selectivity and specificity. Here, we demonstrate the use of a protein nanopore to differentiate peptides�derived from human histone H4 protein�of identical mass according to the positions of acetylated and methylated lysine residues. Unlike sequencing by stepwise threading, our method detects PTMs and their positions by sensing the shape of a fully entrapped peptide, thus eliminating the need for controlled translocation. Molecular dynamics simulations show that the sensitivity to molecular shape derives from a highly nonuniform electric field along the pore. This molecular shape-sensing principle offers a path to versatile, label-free, and highthroughput characterizations of protein isoforms.

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“…To further increase differentiation among the positional isoforms, future work will focus on reducing instrumental noise 45 and on protein engineering to reducing the conformational degree of freedom of peptides in the pore. Thus, in a combination of parallel high-resolution electrophysiology 15,18 with standardized and automated enzymatic generation of peptide fragments 46 , whole molecule sensing with the aerolysin nanopore has the potential of being implemented in a practical technology to rapidly detect PTMs with positional resolution without requiring strand sequencing of proteins.…”

Section: D Shift Of Maxima With Respect To That For Unmodified H4f Plotted Against the Mass Added By Monomethylation; E: Histogram Of I/imentioning

confidence: 99%

Resolving isomeric posttranslational modifications using a nanopore

Ensslen

Sarthak

Aksimentiev

et al. 2021

Preprint

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Posttranslational modifications (PTMs) of proteins are crucial for cellular function but pose analytical problems, especially in distinguishing chemically identical PTMs at different nearby locations within the same protein. Current methods, such as liquid chromatography-tandem mass spectrometry, are technically tantamount to de novo protein sequencing1. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems2–4. Here, we demonstrate the use of an engineered biological nanopore to differentiate positional isomers resulting from acetylation or methylation of histone protein H4, an important PTM target5,6. In contrast to strand sequencing, we differentiate positional isomers by recording ionic current modulations resulting from the stochastic entrapment of entire peptides in the pore’s sensing zone, with all residues simultaneously contributing to the electrical signal. Molecular dynamics simulations show that, in this whole-molecule sensing mode, the non-uniform distribution of the electric potential within the nanopore makes the added resistance contributed by a PTM dependent on its precise location on the peptide. Optimization of the pore’s sensitivity in combination with parallel recording and automated and standardized protein fragmentation may thus provide a simple, label-free, high-throughput analytical platform for identification and quantification of PTMs.

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Tryptic digestion of human serum for proteomic mass spectrometry automated by centrifugal microfluidics

Abstract:
Tryptic digestion of human serum automated by centrifugal microfluidics.

Cited by 12 publications

References 42 publications

Advances and Utility of the Human Plasma Proteome

Advances and Utility of the Human Plasma Proteome

Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape

Resolving isomeric posttranslational modifications using a nanopore

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Tryptic digestion of human serum for proteomic mass spectrometry automated by centrifugal microfluidics

Abstract: Tryptic digestion of human serum automated by centrifugal microfluidics.

Cited by 12 publications

References 42 publications

Advances and Utility of the Human Plasma Proteome

Advances and Utility of the Human Plasma Proteome

Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape

Resolving isomeric posttranslational modifications using a nanopore

Contact Info

Product

Resources

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Abstract:
Tryptic digestion of human serum automated by centrifugal microfluidics.