2009
DOI: 10.1007/s12033-009-9176-6
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Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

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. T… Show more

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Cited by 6 publications
(3 citation statements)
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“…For sufficient IMAC phosphoprotein/phosphopeptide enrichment to occur, it is crucial to perform protein extraction properly in order to dispose of all traces of nucleic acids (Li et al 2009b). The contaminating nucleic acids would bind to the IMAC matrix and thus result in phosphopeptide losses.…”
Section: Immobilized Metal Affinity Chromatography (Imac)mentioning
confidence: 99%
“…For sufficient IMAC phosphoprotein/phosphopeptide enrichment to occur, it is crucial to perform protein extraction properly in order to dispose of all traces of nucleic acids (Li et al 2009b). The contaminating nucleic acids would bind to the IMAC matrix and thus result in phosphopeptide losses.…”
Section: Immobilized Metal Affinity Chromatography (Imac)mentioning
confidence: 99%
“…It is also important to remove all traces of nucleic acids during sample protein extraction. These can competitively bind to the IMAC matrix, thus reducing IMAC efficiency [ 71 ].…”
Section: General Workflow For Targeted Phosphoproteomicsmentioning
confidence: 99%
“…Therefore, it is recommended to avoid using urea, a commonly used denaturant, in phosphoprotemic studies due to its tendency to induce these modifications (deamidation and carbamylation) in peptides. Additionally, because interfering nucleic acids contain abundant phosphate groups that have strong affinities for IMAC beads, ACN precipitation has been employed for protein extraction to efficiently remove nucleic acids before the IMAC enrichment step . To improve phosphopeptide enrichment, the loading, washing, and elution buffers all need to be carefully adjusted.…”
Section: Advances In Enrichment Techniquesmentioning
confidence: 99%