Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies
The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Desp...
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| Format: | Article |
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MDPI AG
2021-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/4/1747 |
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| author | Barbara Sivakova Jan Jurcik Veronika Lukacova Tomas Selicky Ingrid Cipakova Peter Barath Lubos Cipak |
| author_facet | Barbara Sivakova Jan Jurcik Veronika Lukacova Tomas Selicky Ingrid Cipakova Peter Barath Lubos Cipak |
| author_sort | Barbara Sivakova |
| collection | DOAJ |
| description | The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast <i>Schizosaccharomyces pombe</i>. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the <i>S. pombe</i> phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast <i>S. pombe</i>. |
| first_indexed | 2024-03-09T04:56:48Z |
| format | Article |
| id | doaj.art-0b3b57c8af364fe493908331b4cdd66c |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T04:56:48Z |
| publishDate | 2021-02-01 |
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| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-0b3b57c8af364fe493908331b4cdd66c2023-12-03T13:04:32ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01224174710.3390/ijms22041747Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation StrategiesBarbara Sivakova0Jan Jurcik1Veronika Lukacova2Tomas Selicky3Ingrid Cipakova4Peter Barath5Lubos Cipak6Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 845 38 Bratislava, SlovakiaBiomedical Research Center, Cancer Research Institute, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, SlovakiaMedirex Group Academy, n.o., Jana Bottu 2, 917 01 Trnava, SlovakiaBiomedical Research Center, Cancer Research Institute, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, SlovakiaBiomedical Research Center, Cancer Research Institute, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, SlovakiaInstitute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 845 38 Bratislava, SlovakiaBiomedical Research Center, Cancer Research Institute, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, SlovakiaThe phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast <i>Schizosaccharomyces pombe</i>. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the <i>S. pombe</i> phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast <i>S. pombe</i>.https://www.mdpi.com/1422-0067/22/4/1747LFQ phosphoproteomicsSAXPGC<i>Schizosaccharomyces pombe</i> |
| spellingShingle | Barbara Sivakova Jan Jurcik Veronika Lukacova Tomas Selicky Ingrid Cipakova Peter Barath Lubos Cipak Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies International Journal of Molecular Sciences LFQ phosphoproteomics SAX PGC <i>Schizosaccharomyces pombe</i> |
| title | Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies |
| title_full | Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies |
| title_fullStr | Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies |
| title_full_unstemmed | Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies |
| title_short | Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies |
| title_sort | label free quantitative phosphoproteomics of the fission yeast i schizosaccharomyces pombe i using strong anion exchange and porous graphitic carbon based fractionation strategies |
| topic | LFQ phosphoproteomics SAX PGC <i>Schizosaccharomyces pombe</i> |
| url | https://www.mdpi.com/1422-0067/22/4/1747 |
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