Coupling functionalized cobalt ferrite nanoparticle enrichment with online LC/MS/MS for top-down phosphoproteomics

Abstract

Phosphorylation plays pivotal roles in cellular processes and dysregulated phosphorylation is considered as an underlying mechanism in many human diseases. Top-down mass spectrometry (MS) analyzes intact proteins and provides a comprehensive analysis of protein phosphorylation. However, top-down MS-based phosphoproteomics is challenging due to the difficulty in enriching low abundance intact phosphoproteins as well as separating and detecting the enriched phosphoproteins from complex mixtures. Herein, we have designed and synthesized the next generation functionalized superparamagnetic cobalt ferrite (CoFe₂O₄) nanoparticles (NPs), and have further developed a top-down phosphoproteomics strategy coupling phosphoprotein enrichment enabled by the functionalized CoFe₂O₄ NPs with online liquid chromatography (LC)/MS/MS for comprehensive characterization of phosphoproteins. We have demonstrated the highly specific enrichment of a minimal amount of spike-in β-casein from a complex tissue lysate as well as effective separation and quantification of its phosphorylated genetic variants. More importantly, this integrated top-down phosphoproteomics strategy allows for enrichment, identification, quantification, and comprehensive characterization of low abundance endogenous phosphoproteins from complex tissue extracts on a chromatographic time scale.

Fig. 1. Schematic illustration of the top-down phosphoproteomics strategy integrating intact phosphoprotein enrichment using functionalized magnetic nanoparticles (NPs) with online LC/MS/MS. (a) Proteins were extracted from tissue (or cell), and subjected to enrichment by functionalized CoFe₂O₄ NPs. (b) Phosphoproteins bound on magnetic NPs were then pulled down by a magnet for subsequent elution. (c) The loading mixture (LM), flow through (FT), and elution (E) were further separated and analyzed by top-down LC/MS. Enriched phosphoproteins are then comprehensively characterized by online LC/MS/MS. (d) Details of the CoFe₂O₄ NPs functionalized with GAPT-Zn ligands that specifically bind to phosphate groups.

Fig. 2. (a) Schematic illustration of the synthesis of functionalized CoFe₂O₄ NPs. The GAPT-Zn chelating groups possess strong binding ability and preference for phosphate dianions at neutral pH. (b) TEM image of as-prepared CoFe₂O₄–OA/OE NPs. Inset shows the size distribution (9.96 ± 1.03 nm) of the NPs. (c) PXRD pattern of as-synthesized CoFe₂O₄–OA/OE NPs. Blue dash lines indicate a typical diffraction pattern of a ferrite structure. (d) FTIR and (e) TGA analysis of as-synthesized CoFe₂O₄–OA/OE (black), functionalized CoFe₂O₄–NH₃/PEG (red), and functionalized CoFe₂O₄–GAPT (blue) NPs.

Fig. 3. SDS-PAGE analysis with (a) Sypro Ruby and (b) Pro-Q Diamond based detection of spike-in β-casein and other endogenous phosphoproteins specifically enriched from highly complex swine heart tissue extracts (loading mixture). Each loading mixture (LM) contains different concentration of 0.03 μg μL^–1 (I), 0.05 μg μL^–1 (II), 0.07 μg μL^–1 (III), and 0.1 μg μL^–1 (IV) of spiked-in β-casein. Equal amount (10 μg) of the loading mixture, flow-through (FT), and elution after enrichment (E) was loaded on the gel. M: marker; LM: loading mixture; FT: flow through; E: elution.

Fig. 4. Top-down LC/MS analysis of multiple β-casein variants after phosphoprotein enrichment from a swine heart tissue extract with 0.05 μg μL^–1 of spike-in β-casein. (a) Base peak chromatograms of LM (loading mixture; dark blue), FT (flow through; light blue), and E (elution; red). (i), (ii), and (iii), represent extracted ion chromatograms of β-casein variants. (b) Average mass spectra and deconvoluted spectra of LM (dark blue), FT (light blue), and E (red) samples from 41.3 to 41.7 min (highlighted in grey). The intensity in the region from 23 940 Da to 24 040 Da was magnified 3 times. NL: normalized level. (c) Cartoon illustration of the three β-casein variants B (i), A1 (ii), and A2 (iii), each carrying five phosphorylations.

Fig. 5. LC/MS detection of well characterized cardiac phosphoproteins enabled by highly specific NP-based enrichment from a complex tissue extract. Representative endogenous phosphoproteins, (a) cardiac troponin I (cTnI) and (b) tropomyosin (Tm) were shown by comparing normalized deconvoluted average MS spectra of loading mixture (LM), flow through (FT), and elution from 37.5–37.8 min and 41.3–41.4 min, respectively. Mass difference of 28 Da corresponding to polymorphism V116A was observed for cTnI.

Fig. 6. LC/MS/MS analysis of a representative previously uncharacterized phosphoprotein, hepatoma-derived growth factor, enabled by effective CoFe₂O₄ NP-based enrichment from a complex swine heart tissue extract. (a) MS spectra of loading mixture (LM, dark blue), flow through (FT, light blue), and elution (E, red) from 32.3 min to 32.7 min; (b) the corresponding deconvoluted spectra. (c) Fragment ion map of online MS/MS analysis with CID and ETD from triply phosphorylated precursor ion (29+). Grey “M” indicates methionine excision and red “S” indicates phosphorylation sites. Red numbers above the underlined red sequence reveal modifications with their additional mass. (d) Representative fragment ions from both online CID and ETD LC/MS/MS of the targeted protein.