Dataset Information

Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling.

ABSTRACT: Label-free protein characterization at surfaces is commonly achieved using digestion and/or matrix application prior to mass spectrometry. We report the assignment of undigested proteins at surfaces in situ using secondary ion mass spectrometry (SIMS). Ballistic fragmentation of proteins induced by a gas cluster ion beam (GCIB) leads to peptide cleavage producing fragments for subsequent OrbitrapTM analysis. In this work we annotate 16 example proteins (up to 272?kDa) by de novo peptide sequencing and illustrate the advantages of this approach by characterizing a protein monolayer biochip and the depth distribution of proteins in human skin.

SUBMITTER: Kotowska AM

PROVIDER: S-EPMC7672064 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling.

Kotowska Anna M AM Trindade Gustavo F GF Mendes Paula M PM Williams Philip M PM Aylott Jonathan W JW Shard Alexander G AG Alexander Morgan R MR Scurr David J DJ

Nature communications 20201117 1

Label-free protein characterization at surfaces is commonly achieved using digestion and/or matrix application prior to mass spectrometry. We report the assignment of undigested proteins at surfaces in situ using secondary ion mass spectrometry (SIMS). Ballistic fragmentation of proteins induced by a gas cluster ion beam (GCIB) leads to peptide cleavage producing fragments for subsequent Orbitrap<sup>TM</sup> analysis. In this work we annotate 16 example proteins (up to 272 kDa) by de novo pepti ...[more]

PMID: 33203841

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Project description:The Australian tobacco plant Nicotiana benthamiana is becoming increasingly popular as a platform for protein production and metabolic engineering. In this system, gene expression is achieved transiently by infiltrating N. benthamiana plants with suspensions of Agrobacterium tumefaciens carrying vectors with the target genes. To infiltrate larger numbers of plants, vacuum infiltration is the most efficient approach known, which is already used on industrial scale. Current laboratory-scale solutions for vacuum infiltration, however, either require expensive custom-tailored equipment or produce large amounts of biologically contaminated waste. To overcome these problems and lower the burden to establish vacuum infiltration in new laboratories, we present here 3D-printed plant holders for vacuum infiltration. We demonstrate that our plant holders are simple to use and enable a throughput of around 40 plants per hour. In addition, our 3D-printed plant holders are made from autoclavable material, which tolerate at least 12 autoclave cycles, helping to limit the production of contaminated waste and thus contributing to increased sustainability in research. In conclusion, our plant holders provide a simple, robust, safe and transparent platform for laboratory-scale vacuum infiltration that can be readily adopted by new laboratories interested in protein and metabolite production in Nicotiana benthamiana. Practical application Transient expression in Nicotiana benthamiana provides a popular and rapid system for producing proteins in a plant host. To infiltrate larger numbers of plants (typically >20), vacuum infiltration is the method of choice. However, no system has been described so far which is robust to use and can be used without expensive and complex equipment. Our autoclavable 3D-printed plant holders presented here will greatly reduce the efforts required to adopt the vacuum infiltration technique in new laboratories. They are easy to use and can be autoclaved at least 12 times, which contributes to waste reduction and sustainability in research laboratories. We anticipate that the 3D printing design provided here will drastically lower the bar for new groups to employ vacuum infiltration for producing proteins and metabolites in Nicotiana benthamiana.

Dataset Information

Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling.

Publications

Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling.

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