Corona Isolation Method Matters: Capillary Electrophoresis Mass Spectrometry Based Comparison of Protein Corona Compositions Following On-Particle versus In-Solution or In-Gel Digestion

Abstract

Increased understanding of the role of the nanomaterial protein corona in driving nanomaterial uptake into, and impacts on, cells and organisms, and the consequent need for characterization of the corona, has led to a flourishing of methods for isolation and analysis of the constituent proteins over the past decade. However, despite over 700 corona studies to date, very little is understood in terms of which methods provide the most precise and comprehensive characterization of the corona. With the increasing importance of the modeling of corona formation and its correlation with biological impacts, it is timely to properly characterize and validate the isolation approaches used to determine the protein corona. The current work introduces Capillary Electrophoresis with Electro Spray Ionization Mass Spectrometry (CESI-MS) as a novel method for protein corona characterizations and develops an on-particle tryptic digestion method, comparing peptide solubilization solutions and characterizing the recovery of proteins from the nanomaterial surface. The CESI-MS was compared to the gold standard nano-LC-MS for corona analysis and maintained a high degree of reproducibility, while increasing throughput by >3-fold. The on-particle digestion is compared to an in-solution digestion and an in-gel digestion of the protein corona. Interestingly, a range of different protein classes were found to be recovered to greater or lesser extents among the different methods. Apolipoproteins were detected at lower concentrations when a surfactant was used to solubilize peptides, whereas immunoglobulins in general have a high affinity for nanomaterials, and thus show lower recovery using on-particle digestion. The optimized on-particle digestion was validated using 6 nanomaterials and proved capable of recovering in excess of 97% of the protein corona. These are important factors to consider when designing corona studies and modeling corona formation and impacts, highlighting the significance of a comprehensive validation of nanomaterial corona analysis methods.

Keywords: CE-MS; capillary electrophoresis; mass-spectrometry; nanoparticles; protein corona; proteomics; reproducibility.

Figure 1

Analysis of SiO₂ NM protein corona enzymatically digested on-particle using trypsin in the presence of 100 mM ammonium bicarbonate (pH 8.0). (A) Base peak electropherograms of experimental replicates, red boxes indicate visual discrepancies between replicates. Venn diagrams of peptides (B) identified via MSMS and (C) detected as precursor mass in the three experimental replicates. (D) Reproducibility of peptide quantifications shown as a histogram of relative standard deviations (RSDs) calculated for each individual peptide.

Figure 2

Technical reproducibility of CESI-MS analyses when analyzing a digested SiO₂ NM protein corona. (A) Base peak electropherograms of technical replicates, Venn diagrams of peptides (B) identified via MSMS and (C) detected as precursor mass in the three technical replicates. (D) Reproducibility of peptide quantifications shown as histogram of relative standard deviations.

Figure 3

Carryover analysis of CESI-MS when analyzing a digested SiO₂ NM protein corona followed by a water blank. (A) Base peak electropherograms of SiO₂ NM protein corona above base peak electropherogram of subsequent blank injection. (B) Peptides identified in each sample injection and subsequent blank.

Figure 4

Impact of solubilizing agents during on-particle digestion of the SiO₂ NM corona on the total numbers of (A) proteins and (B) peptides identified in three experimental replicates, and (C) on the reproducibility in peptide quantification within triplicate experiments.

Figure 5

Pair-wise comparisons of protein abundances in SiO₂ NM corona depending on the solubilizing agent used, (A) Rapigest SF™ treated versus untreated samples, (B) Rapigest SF™ versus urea treated samples, (C) Rapigest SF™ versus Rapigest SF™ plus urea treated samples. Proteins labeled in orange show a statistically significant difference in abundance between the two experimental triplicates with a ratio of at least 2.

Figure 6

Analysis of the left-over proteins after on-particle digestion of corona on SiO₂ NMs. (A) SDS-PAGE of analytes bound onto the SiO₂ NMs after on-particle digestion. (B) Box plot illustrating protein abundances determined for each individual protein in the original on-particle digestion and in the in-gel control. (C) Calculated protein abundance ratios (on-particle/in-gel control) revealed significantly higher ratios for immunoglobulins remaining attached to the NMs compared to other proteins.

Figure 7

Analysis of SiO₂ NMs protein corona using an in-gel digestion and an in-solution digestion-based approach. Base peak electropherograms of protein corona digested (A) in-gel and (D) in-solution. The impact of the different sample preparation (corona isolation) procedures on the total numbers of (B) proteins and (C) peptides identified. Venn diagrams of non-immunoglobulins (E) and immunoglobulins (F) identified by the on-particle and in-gel approach. All experiments were performed in triplicate.

Figure 8

Protein composition in ppm from SiO₂ NM coronas as determined either by (A) on-particle digestion combined with CESI-MS analysis, or (B) in-gel digestion combined with CESI-MS analysis. (C) Relationship between protein compositions calculated for the most abundant proteins (>10,000 ppm) in both approaches.