Nucleic acids in protein samples interfere with phosphopeptide identification by immobilized-metal-ion affinity chromatography and mass spectrometry.
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.
Li
Yaojun
Y
National 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.
Luo
Yuanming
Y
Wu
Shuzhen
S
Gao
Youhe
Y
Liu
Yanxin
Y
Zheng
Dexian
D
D000327:Adsorption; D016477:Artifacts; D002846:Chromatography, Affinity / Q000379:methods*; D045424:Complex Mixtures / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008670:Metals / Q000737:chemistry; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
D045424:Complex Mixtures; D008670:Metals; D009696:Nucleic Acids; D010748:Phosphopeptides; D011506:Proteins