Nucleic acids in protein samples interfere with phosphopeptide identification by immobilized-metal-ion affinity
Yaojun Li1, Yuanming Luo, Shuzhen Wu
1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China.
Insights
Nucleic acids interfere with immobilized-metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. Removing nucleic acids using acetonitrile precipitation significantly enhances phosphopeptide identification in phosphoproteomics.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Immobilized-metal-ion affinity chromatography (IMAC) is a key technique for enriching phosphopeptides in phosphoproteomics.
- The impact of nucleic acids on IMAC-based phosphopeptide enrichment remains unclear.
Purpose of the Study:
- To investigate the effect of nucleic acids on phosphopeptide enrichment using IMAC.
- To develop a method for removing nucleic acids to improve phosphoproteomic analysis.
Main Methods:
- Demonstration of nucleic acid adsorption by IMAC beads.
- Development and application of acetonitrile (ACN) precipitation for nucleic acid removal.
- Comparison of phosphopeptide identification with and without nucleic acid removal using IMAC and mass spectrometry.
Main Results:
- IMAC beads exhibit strong adsorption of nucleic acids, particularly single-stranded forms.
- Nucleic acid adsorption leads to a significant loss (approximately 50%) of phosphopeptides during IMAC.
- ACN precipitation effectively removes nucleic acids from protein samples.
- ACN precipitation approximately doubled the number of identified phosphopeptides.
Conclusions:
- Nucleic acids interfere with IMAC-based phosphopeptide enrichment.
- Pre-treatment of protein samples to remove nucleic acids is crucial for maximizing phosphopeptide recovery.
- Acetonitrile precipitation is an effective method for removing nucleic acids, thereby improving phosphoproteomic identification.
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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