title: Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.
abstract: Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research.
authors.affiliation: Institute of Toxicology, Russian Federal Agency for Biology and Medicine, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Gladilovich
authors.affiliation: Universität Leipzig, Faculty of Chemistry and Mineralogy, Germany.
authors.forename: Uta
authors.initials: U
authors.lastname: Greifenhagen
authors.affiliation: Saint Petersburg State University, Russia.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Sukhodolov
authors.affiliation: Saint Petersburg State University, Russia.
authors.forename: Artem
authors.initials: A
authors.lastname: Selyutin
authors.affiliation: Universität Leipzig, Faculty of Chemistry and Mineralogy, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Singer
authors.affiliation: Leibniz Institute of Plant Biochemistry, Proteome Analytics Research Group, Germany.
authors.forename: Domenika
authors.initials: D
authors.lastname: Thieme
authors.affiliation: Leibniz Institute of Plant Biochemistry, Proteome Analytics Research Group, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Majovsky
authors.affiliation: Institute of Analytical Instrumentation, Russian Academy of Science, Russia.
authors.forename: Alexey
authors.initials: A
authors.lastname: Shirkin
authors.affiliation: Leibniz Institute of Plant Biochemistry, Proteome Analytics Research Group, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hoehenwarter
authors.affiliation: Institute of Toxicology, Russian Federal Agency for Biology and Medicine, Russia.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Bonitenko
authors.affiliation: Institute of Toxicology, Russian Federal Agency for Biology and Medicine, Russia; Institute of Analytical Instrumentation, Russian Academy of Science, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Podolskaya
authors.affiliation: Universität Leipzig, Faculty of Chemistry and Mineralogy, Germany; Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Germany. Electronic address: afrolov@ipb-halle.de.
authors.forename: Andrej
authors.initials: A
authors.lastname: Frolov
mesh_terms: D002846:Chromatography, Affinity; D005290:Ferric Compounds / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007501:Iron / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013228:Stearates / Q000737:chemistry*
chemical_list: D005290:Ferric Compounds; D010750:Phosphoproteins; D013228:Stearates; D007501:Iron
keywords: Immobilized metal affinity chromatography (IMAC); Iron oxide; Langmuir-Blodgett films; Perfluorooctanoic acid; Phosphopeptides; Phosphoproteomics