Non-mechanical disruption of<i>Bacillus subtilis spores</i>allows sensitive and deep characterization of the minimal proteome for resuming a cellular lifestyle
Abstract In response to extreme conditions,Bacillus subtilisgenerates highly resilient spores characterized by a unique multilayered structure. This confers resistance against various chemicals and enzymes yet adding complexity to the analysis of the spore proteome. As the first step in bottom-up proteomics, sample preparation poses a significant challenge. We assessed how an optimized protocol for sample preparation by easy extraction and digestion (SPEED) performed compared to previously established methods “One-pot” (OP) and single-pot, solid phase-enhanced sample-preparation (SP3) for the proteomic analysis ofB. subtiliscell and spore samples. We found that SPEED outperformed both OP and SP3 in terms of peptides and proteins identified, moreover SPEED highly reproducibly quantified over 1000 proteins in limited input samples as low as 1 OD600ofB. subtiliscells and spores. SPEED was applied to analyze spore samples of different purity by applying sequential purification following harvesting of spores. Comparison of the differential abundance of proteins revealed clusters likely partially stemming from remaining vegetative cells in less purified spore samples. We show that ranking of absolute protein abundance in cellular and spore samples further enables us to rationally differentiate integral spore proteins from vegetative remnants. This is of importance in applications and organisms where highly homogenous spore samples are difficult to obtain. A deep proteomic analysis of spore and vegetative cell samples with the new approach led to the identification of 2447 proteins, 2273 of which were further quantified and compared betweenB. subtilisspores and cells. Our findings indicate that pathways related to peptidoglycan biosynthesis, glycolysis, carbon metabolism, and biosynthesis of secondary metabolites are shared between cells and spores. This corroborates and extends earlier work stressing that despite marked differences in their physiological states, spores preserve vegetative cell (core) proteins, essential for revival under conditions conducive to growth.Abstract Figure <jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="584050v2_ufig1" position="float" orientation="portrait" /></jats:fig>.
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
bioRxiv.org - (2024) vom: 30. März Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Huang, Yixuan [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2024.03.08.584050 |
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| PPN (Katalog-ID): |
XBI042851890 |
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| 245 | 1 | 0 | |a Non-mechanical disruption of<i>Bacillus subtilis spores</i>allows sensitive and deep characterization of the minimal proteome for resuming a cellular lifestyle |
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| 520 | |a Abstract In response to extreme conditions,Bacillus subtilisgenerates highly resilient spores characterized by a unique multilayered structure. This confers resistance against various chemicals and enzymes yet adding complexity to the analysis of the spore proteome. As the first step in bottom-up proteomics, sample preparation poses a significant challenge. We assessed how an optimized protocol for sample preparation by easy extraction and digestion (SPEED) performed compared to previously established methods “One-pot” (OP) and single-pot, solid phase-enhanced sample-preparation (SP3) for the proteomic analysis ofB. subtiliscell and spore samples. We found that SPEED outperformed both OP and SP3 in terms of peptides and proteins identified, moreover SPEED highly reproducibly quantified over 1000 proteins in limited input samples as low as 1 OD600ofB. subtiliscells and spores. SPEED was applied to analyze spore samples of different purity by applying sequential purification following harvesting of spores. Comparison of the differential abundance of proteins revealed clusters likely partially stemming from remaining vegetative cells in less purified spore samples. We show that ranking of absolute protein abundance in cellular and spore samples further enables us to rationally differentiate integral spore proteins from vegetative remnants. This is of importance in applications and organisms where highly homogenous spore samples are difficult to obtain. A deep proteomic analysis of spore and vegetative cell samples with the new approach led to the identification of 2447 proteins, 2273 of which were further quantified and compared betweenB. subtilisspores and cells. Our findings indicate that pathways related to peptidoglycan biosynthesis, glycolysis, carbon metabolism, and biosynthesis of secondary metabolites are shared between cells and spores. This corroborates and extends earlier work stressing that despite marked differences in their physiological states, spores preserve vegetative cell (core) proteins, essential for revival under conditions conducive to growth.Abstract Figure <jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="584050v2_ufig1" position="float" orientation="portrait" /></jats:fig> | ||
| 650 | 4 | |a Biology |7 (dpeaa)DE-84 | |
| 650 | 4 | |a 570 |7 (dpeaa)DE-84 | |
| 700 | 1 | |a de Koster, Alphonse |4 aut | |
| 700 | 1 | |a Tu, Zhiwei |4 aut | |
| 700 | 1 | |a Gao, Xiaowei |4 aut | |
| 700 | 1 | |a Roseboom, Winfried |4 aut | |
| 700 | 1 | |a Brul, Stanley |4 aut | |
| 700 | 1 | |a Setlow, Peter |4 aut | |
| 700 | 1 | |a Kramer, Gertjan |0 (orcid)0000-0002-8541-0337 |4 aut | |
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