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

• IMAC on Fe(III) stearate collapsed Langmuir-Blodgett films.
• A new multi-step elution scheme provides improved recovery and specificity of IMAC.
• Two-fold higher phosphoproteome coverage.
• Improved sensitivity and linearity for quantitative applications.

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.