Abstract
Immobilized-metal-ion affinity chromatography (IMAC) is used extensively for phosphopeptide enrichment in phosphoproteomics. However, the effect of nucleic acids in protein samples on phosphopeptide enrichment by IMAC has not yet been well clarified. In this study, we demonstrate that IMAC beads possess a strong adsorption of nucleic acids, especially single-stranded or single-stranded-region-containing nucleic acids, leading to approximately 50% loss of phosphopeptides during the process of IMAC enrichment. Therefore, nucleic acids must be removed from protein samples prior to IMAC. Acetonitrile (ACN) precipitation, a simple and efficient procedure, was established to remove nucleic acids from the protein samples. We showed that ACN precipitation approximately doubled the phosphopeptide number identified by IMAC and mass spectrometry, indicating that nucleic acid removal significantly improves the identification of phosphopeptides.
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Abbreviations
- ACN:
-
Acetonitrile
- IMAC:
-
Immobilized-metal-ion affinity chromatography
- LC:
-
Liquid chromatography
- MALDI-TOF-MS:
-
Matrix-assisted desorption/ionization time-of-flight mass spectrometry
- MS/MS:
-
Tandem mass spectrum
- TFA:
-
Trifluoroacetic acid
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Acknowledgment
This study was partially supported by Natural Science Foundation of China Grant (30623009 and 30721063) and State Key Basic Research Program of China Grant (2007CB507404).
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Li, Y., Luo, Y., Wu, S. et al. Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry. Mol Biotechnol 43, 59–66 (2009). https://doi.org/10.1007/s12033-009-9176-6
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DOI: https://doi.org/10.1007/s12033-009-9176-6