title: Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation.
abstract: Comprehensive analysis of post-translational modifications (PTMs) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. We report that the immuno-purification of lysine-acetylated peptides is hampered by the copurification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay.
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Martinez-Val
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Garcia
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Ximénez-Embún
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Ailyn
authors.initials: A
authors.lastname: Martínez Teresa-Calleja
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Nuria
authors.initials: N
authors.lastname: Ibarz
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Ruppen
authors.affiliation: ProteoRed-ISCIII, Proteomics Unit, Spanish National Cancer Research Centre (CNIO) , 28029 Madrid, Spain.
authors.forename: Javier
authors.identifier: 0000-0003-3288-3496
authors.initials: J
authors.lastname: Munoz
mesh_terms: D000107:Acetylation; D000906:Antibodies / Q000737:chemistry; D016477:Artifacts; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase / Q000592:standards*; D003840:Deoxycholic Acid / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques / Q000592:standards*; D008239:Lysine / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry; D014508:Urea / Q000737:chemistry*
chemical_list: D000906:Antibodies; D020543:Proteome; D003840:Deoxycholic Acid; D014508:Urea; D008239:Lysine
keywords: antibody; carbamylation; immuno-purification; lysine acetylation; mass spectrometry; sodium deoxycholate; urea