Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics
Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have bee...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-06-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/11/13/2047 |
| _version_ | 1797480519824310272 |
|---|---|
| author | Jiaran Li Jifeng Wang Yumeng Yan Na Li Xiaoqing Qing Ailikemu Tuerxun Xiaojing Guo Xiulan Chen Fuquan Yang |
| author_facet | Jiaran Li Jifeng Wang Yumeng Yan Na Li Xiaoqing Qing Ailikemu Tuerxun Xiaojing Guo Xiulan Chen Fuquan Yang |
| author_sort | Jiaran Li |
| collection | DOAJ |
| description | Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO<sub>2</sub>-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO<sub>2</sub>-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO<sub>2</sub>-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO<sub>2</sub>-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics. |
| first_indexed | 2024-03-09T22:01:13Z |
| format | Article |
| id | doaj.art-0e8c318293bf40e8af626c2bb273dc6c |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-09T22:01:13Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-0e8c318293bf40e8af626c2bb273dc6c2023-11-23T19:48:41ZengMDPI AGCells2073-44092022-06-011113204710.3390/cells11132047Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for PhosphoproteomicsJiaran Li0Jifeng Wang1Yumeng Yan2Na Li3Xiaoqing Qing4Ailikemu Tuerxun5Xiaojing Guo6Xiulan Chen7Fuquan Yang8Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaKey Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, ChinaProtein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO<sub>2</sub>-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO<sub>2</sub>-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO<sub>2</sub>-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO<sub>2</sub>-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.https://www.mdpi.com/2073-4409/11/13/2047phosphoproteomicsTiO<sub>2</sub>phosphopeptide enrichmentphosphopeptide fractionationnon-phosphopeptide excluderdeamidation |
| spellingShingle | Jiaran Li Jifeng Wang Yumeng Yan Na Li Xiaoqing Qing Ailikemu Tuerxun Xiaojing Guo Xiulan Chen Fuquan Yang Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics Cells phosphoproteomics TiO<sub>2</sub> phosphopeptide enrichment phosphopeptide fractionation non-phosphopeptide excluder deamidation |
| title | Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics |
| title_full | Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics |
| title_fullStr | Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics |
| title_full_unstemmed | Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics |
| title_short | Comprehensive Evaluation of Different TiO<sub>2</sub>-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics |
| title_sort | comprehensive evaluation of different tio sub 2 sub based phosphopeptide enrichment and fractionation methods for phosphoproteomics |
| topic | phosphoproteomics TiO<sub>2</sub> phosphopeptide enrichment phosphopeptide fractionation non-phosphopeptide excluder deamidation |
| url | https://www.mdpi.com/2073-4409/11/13/2047 |
| work_keys_str_mv | AT jiaranli comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT jifengwang comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT yumengyan comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT nali comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT xiaoqingqing comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT ailikemutuerxun comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT xiaojingguo comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT xiulanchen comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics AT fuquanyang comprehensiveevaluationofdifferenttiosub2subbasedphosphopeptideenrichmentandfractionationmethodsforphosphoproteomics |