The Protein Interactome of a Nanoparticle Population in Whole Cytoplasm under Near‐Native Conditions: A Pilot Study

Abstract

The surface interactions of nanoparticles (NPs) with proteins are intensively studied because they partly determine the biological fate of these reagents. The protein interaction trajectory of NPs delivered from complex biofluids into the cytosol of vertebrate cells however remains poorly characterized. This topology is explored using isolated whole cytoplasm and carboxylated superparamagnetic iron oxide NPs (SPIONs). SPIONs are introduced into the cytoplasm in naked form or with an adsorbed cargo of serum proteins as an example of a complex NP “corona.” By filtering interactome datasets with information about protein localization and absolute cytosolic concentration, the soluble cytoplasmic proteins that contribute to corona formation in the cytoplasm are identified. These include a subset of glycolytic enzymes and proteins in soluble ribosomes. In the cytoplasm, the proteins of the serum corona are quickly removed from SPIONs by a non‐proteolytic mechanism. Although naked SPIONs and SPIONs with a serum corona capture the same 65 cytosolic proteins, their overall interaction trajectory in the cytoplasm is not identical. In particular, they diverge in engagement with macroscale cell parts such as mitochondria. This knowledge of the interaction landscape of NPs in the cytoplasm is expected to support hypothesis development in projects aimed at improving NP reagents for biomedical applications.

Multienzyme activity profiling for evaluation of cell‐to‐cell variability of metabolic state

“…The proteome of six individual cytoplasmic S7500s was determined by LC‐MS as previously described for whole cells. 42 Relative abundance values as % of total ∑#PSMs were ranked using the PERCENTRANK.EXC function in Excel 2016.…”

“…Whole cytoplasms were dispersed in HR buffer (1 cytoplasm/25 μl HR buffer) and centrifuged at 4°C (7,500 × g, 15 min) for collection of the S7500 supernatant. The proteome of six individual cytoplasmic S7500s was determined by LC‐MS as previously described for whole cells 42 . Relative abundance values as % of total ∑#PSMs were ranked using the PERCENTRANK.EXC function in Excel 2016.…”

Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus

“…Proteome analysis. The proteome of six individual stage VI nuclei and six low speed supernatants of cytoplasmic homogenate were determined by liquid chromatography coupled to mass spectrometry (LC-MS) as previously described for whole cells [44]. For the proteins of interest, relative abundance values as % of total ∑#PSMs were ranked using the PERCEN-TRANK.EXC function in Excel 2016.…”