Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho‐form distributions
Abstract The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or ‘phospho‐form’—giving biological systems profound capabilities for dynamic information processing. A central problem i...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Springer Nature
2011-04-01
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| Series: | Molecular Systems Biology |
| Online Access: | https://doi.org/10.1038/msb.2011.15 |
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| Summary: | Abstract The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or ‘phospho‐form’—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the ‘phospho‐form distribution’: the relative amount of each of the 2n phospho‐forms of a protein with n‐phosphorylation sites. We compared four potential methods—western blots with phospho‐specific antibodies, peptide‐based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein‐based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well‐studied mitogen‐activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16‐member phospho‐form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem. |
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| ISSN: | 1744-4292 |