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.
Highlights
A decade ago, the term nanomaterial (NM) protein corona was coined to describe the layer of absorbed proteins acquired by NMs in contact with biological or environmental fluids [1,2]
The released proteins would be separated using SDS-PAGE electrophoresis followed by excision of several individual protein bands of interest and their digestion and analysis using nano-liquid chromatography-mass spectrometry [4,7,8]
The aim of this study is to introduce CESI-MS as a potent new tool for protein corona characterization, as such it is compared to the current gold standard analytical platform, nano-LC-MS
Summary
A decade ago, the term nanomaterial (NM) protein corona was coined to describe the layer of absorbed proteins acquired by NMs in contact with biological or environmental fluids [1,2]. To extend the quality and depth of proteins characterized in the corona, methods for global analysis of the corona have been implemented These include precipitating proteins from the NM surface using commercial reagent kits [9] or using SDS-PAGE starting buffer incubations prior to the removal of SDS using commercial surfactant/detergent filters to prevent LC-MS fouling [10]. An alternative method that mitigates these specific risks is to perform a tryptic digest on the intact NP-corona complex, a so-called on-particle digestion This method has begun to acquire traction within the corona community [11,12], and by using fewer steps between formation of the NM-corona complex and the digested peptide sample being ready to analyze, there is less risk of introducing errors (e.g., loss of proteins), while improving throughput. Without a full understanding of the relative performance of these methods it is difficult to further optimize them to improve protein coverage and provide a complete and reproducible description of NM coronas for correlation with signaling impacts
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