Phospho-Proteomics Method Optimization and Application to Stimulated Jurkat Cells
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arXiv
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| Format: | Preprint |
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2025
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| _version_ | 1866911267634020352 |
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| author | Ndungu, Teresia Zecha, Jana Cazares, Lisa Hess, Sonja |
| author_facet | Ndungu, Teresia Zecha, Jana Cazares, Lisa Hess, Sonja |
| contents | In clinical proteomics, available input is often limited. In addition, phospho-proteomics is of particular interest since the dysregulation of these post-translational modifications (PTMs) has been implicated in various diseases such as cancer. We therefore assessed the feasibility of low input phospho-proteomics via phospho-bulk titration and low-input starting material. We found that there was identification of more phospho-peptides through phospho-bulk titration because of sample loss during preparation of low input starting material. Additionally, we explored various lysis buffers and boiling times for efficiency of decrosslinking formalin-fixed cells since cells and tissues are often fixed for preservation and sorting via FACS. We found that boiling in 0.05M Tris pH 7.6 with 5% SDS for 60 min yielded the highest number of phospho-peptides. Lastly, we applied Evotips Pure and phospho-bulk titration to treated Jurkat cells and identified 7 phospho-sites involved in T-cell stimulation. |
| format | Preprint |
| id |
arxiv_https___arxiv_org_abs_2511_02932 |
| institution | arXiv |
| publishDate | 2025 |
| record_format | arxiv |
| spellingShingle | Phospho-Proteomics Method Optimization and Application to Stimulated Jurkat Cells Ndungu, Teresia Zecha, Jana Cazares, Lisa Hess, Sonja Quantitative Methods Genomics In clinical proteomics, available input is often limited. In addition, phospho-proteomics is of particular interest since the dysregulation of these post-translational modifications (PTMs) has been implicated in various diseases such as cancer. We therefore assessed the feasibility of low input phospho-proteomics via phospho-bulk titration and low-input starting material. We found that there was identification of more phospho-peptides through phospho-bulk titration because of sample loss during preparation of low input starting material. Additionally, we explored various lysis buffers and boiling times for efficiency of decrosslinking formalin-fixed cells since cells and tissues are often fixed for preservation and sorting via FACS. We found that boiling in 0.05M Tris pH 7.6 with 5% SDS for 60 min yielded the highest number of phospho-peptides. Lastly, we applied Evotips Pure and phospho-bulk titration to treated Jurkat cells and identified 7 phospho-sites involved in T-cell stimulation. |
| title | Phospho-Proteomics Method Optimization and Application to Stimulated Jurkat Cells |
| topic | Quantitative Methods Genomics |
| url | https://arxiv.org/abs/2511.02932 |