TY - JOUR

T1 - Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics

AU - Gladilovich, Vladimir

AU - Greifenhagen, Uta

AU - Sukhodolov, Nikolai

AU - Selyutin, Artem

AU - Singer, David

AU - Thieme, Domenika

AU - Majovsky, Petra

AU - Shirkin, Alexey

AU - Hoehenwarter, Wolfgang

AU - Bonitenko, Evgeny

AU - Podolskaya, Ekaterina

AU - Frolov, Andrej

N1 - Funding Information: We thank Prof. Dr. Ralf Hoffmann for financial support and helpful discussions, as well as Nicole Hünecke (IPB Halle), Vemula Venukumar and Dr. Ravi Chand Bollineni (Universität Leipzig) for preparation of cell lysates. Financial support from the “Deutsche Forschungsgemeinschaft” to AF (FR 3117/2-1), Ernst Schering Foundation to UG, the European Fund for Regional Structure Development (EFRE, European Union and Free State Saxony) for RH, Russian Federal medico-biological Agency (Contract 64/2-12/К) and Russian Academy of Sciences to VG, NS, AS and EP and the “Bundesministerium für Bildung and Forschung” (BMBF) to DS and RH is gratefully acknowledged. Publisher Copyright: © 2016 Elsevier B.V. Copyright: Copyright 2016 Elsevier B.V., All rights reserved.

PY - 2016/4/22

Y1 - 2016/4/22

N2 - 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.

AB - 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.

KW - Immobilized metal affinity chromatography (IMAC)

KW - Iron oxide

KW - Langmuir-Blodgett films

KW - Perfluorooctanoic acid

KW - Phosphopeptides

KW - Phosphoproteomics

UR - http://www.scopus.com/inward/record.url?scp=84979462460&partnerID=8YFLogxK

U2 - 10.1016/j.chroma.2016.03.044

DO - 10.1016/j.chroma.2016.03.044

M3 - Article

C2 - 27016113

VL - 1443

SP - 181

EP - 190

JO - Journal of Chromatography

JF - Journal of Chromatography

SN - 0021-9673

ER -