Human plasma proteome association and cytotoxicity of nano-graphene oxide grafted with stealth polyethylene glycol and poly(2-ethyl-2-oxazoline)

Polyethylene glycol (PEG) is a gold standard against protein fouling. However, recent studies have revealed surprising adverse effects of PEG, namely its immunogenicity and shortened bio-circulation upon repeated dosing. This highlights a crucial need to further examine ‘stealth’ polymers for controlling the protein ‘corona’, a new challenge in nanomedicine and bionanotechnology. Poly(2-ethyl-2-oxazoline) (PEtOx) is another primary form of stealth polymer that, despite its excellent hydrophilicity and biocompatibility, has found considerably less applications compared with PEG. Herein, we performed label-free proteomics to compare the associations of linear PEG- and PEtOx-grafted nano-graphene oxide (nGO) sheets with human plasma proteins, complemented by cytotoxicity and haemolysis assays to compare the cellular interactions of these polymers. Our data revealed that nGO-PEG enriched apolipoproteins, while nGO-PEtOx displayed a preferred binding with pro-angiogenic and structural proteins, despite high similarities in their respective top-10 enriched proteins. In addition, nGO-PEG and nGO-PEtOx exhibited similar levels of enrichment of complement proteins. Both PEG and PEtOx markedly reduced nGO toxicity to HEK 293 cells while mitigating nGO haemolysis. This study provides the first detailed profile of the human plasma protein corona associated with PEtOx-grafted nanomaterials and, in light of the distinctions of PEtOx in chemical adaptability, in vivo clearance and immunogenicity, validates the use of PEtOx as a viable stealth alternative to PEG for nanomedicines and bionanotechnologies

Balancing sufficiency and impact in reporting standards for mass spectrometry imaging experiments

Reproducibility, or a lack thereof, is an increasingly important topic across many research fields. A key aspect of reproducibility is accurate reporting of both experiments and the resulting data. Herein, we propose a reporting guideline for mass spectrometry imaging (MSI). Previous standards have laid out guidelines sufficient to guarantee a certain quality of reporting; however, they set a high bar and as a consequence can be exhaustive and broad, thus limiting uptake.To help address this lack of uptake, we propose a reporting supplement-Minimum Information About a Mass Spectrometry Imaging Experiment (MIAMSIE)-and its abbreviated reporting standard version, MSIcheck. MIAMSIE is intended to improve author-driven reporting. It is intentionally not exhaustive, but is rather designed for extensibility and could therefore eventually become analogous to existing standards that aim to guarantee reporting quality. Conversely, its abbreviated form MSIcheck is intended as a diagnostic tool focused on key aspects in MSI reporting.We discuss how existing standards influenced MIAMSIE/MSIcheck and how these new approaches could positively impact reporting quality, followed by test implementation of both standards to demonstrate their use. For MIAMSIE, we report on author reviews of four articles and a dataset. For MSIcheck, we show a snapshot review of a one-month subset of the MSI literature that indicated issues with data provision and the reporting of both data analysis steps and calibration settings for MS systems. Although our contribution is MSI specific, we believe the underlying approach could be considered as a general strategy for improving scientific reporting

MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney

Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.13 page(s

Nanoparticle–proteome in vitro and in vivo

The protein corona is a concept central to a range of disciplines exploiting the bio–nano interface. As the literature continues to expand in this field, it is essential to condense and contextualize the in vitro and in vivo proteome databases accumulated over the past decade: a goal which this review intends to achieve for the benefit of nanomedicine and nanobiotechnology. The parameters used for our review are the physicochemical characteristics of the nanoparticles, their surface ligands, the biological matrix from which a corona was formed, methods employed, plus the top-ten enriched corona proteins. In addition, the protein coronal networks and their implications in vivo are highlighted for selected studies

Plasma proteome association and catalytic activity of stealth polymer-grafted iron oxide nanoparticles

Polyethylene glycol (PEG) is widely used as an antifouling and stealth polymer in surface engineering and nanomedicine. However, recent research has revealed adverse effects of bioaccumulation and immunogenicity following the administration of PEG, prompting this proteomic examination of the plasma protein coronae association with superparamagnetic iron oxide nanoparticles (IONPs) grafted with brushed PEG (bPEG) and an alternative, brushed phosphorylcholine (bPC). Using label-free quantitation by liquid chromatography tandem-mass spectrometry, this study determines protein abundances for the in vitro hard coronae of bare, bPC-, and bPEG-grafted IONPs in human plasma. This study also shows unique protein compositions in the plasma coronae of each IONP, including enrichment of coagulation factors and immunogenic complement proteins with bPEG, and enhanced binding of apolipoproteins with bPC. Functional analysis reveals that plasma protein coronae elevate the horseradish peroxidase-like activities of the bPC- and bPEG-IONPs by approximately twofold, an effect likely mediated by the diverse composition and physicochemical properties of the polymers as well as their associated plasma proteins. Taken together, these observations support the rational design of stealth polymers based on a quantitative understanding of the interplay between IONPs and the plasma proteome, and should prove beneficial for the development of materials for nanomedicine, biosensing, and catalysis

Raw N-glycan mass spectrometry imaging data on formalin-fixed mouse kidney

Provided is the annotated raw data for N-glycan mass spectrometry imaging (MSI) annotations in thin cross-sections of formalin-fixed and paraffin-embedded murine kidney. Relevant meta-data have been provided in this brief and the raw MSI data can be accessed using ProteomeXchange with the PRoteomics IDEntifications (PRIDE) identifier PXD009808. This brief is the first in a set of submissions from our group which will make raw data publicly accessible for existing and future MSI studies

Secondary Structural Changes in Proteins as a Result of Electroadsorption at Aqueous-Organogel Interfaces

The electroadsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated anti-parallel β-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI ToF MS measurement, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamic simulations, which modelled the structure of lysozyme at an aqueous-organic interface. Based on these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins.<br /

Tryptic Peptide Reference Data Sets for MALDI Imaging Mass Spectrometry on Formalin-fixed Ovarian Cancer Tissues

MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed <i>m</i>/<i>z</i> species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC–MS/MS. The sample preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This resulted in 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per sample. From this, a total of 840 proteins and, on average, 297 proteins per sample could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several <i>m</i>/<i>z</i> species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer

Tryptic Peptide Reference Data Sets for MALDI Imaging Mass Spectrometry on Formalin-fixed Ovarian Cancer Tissues

MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed <i>m</i>/<i>z</i> species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC–MS/MS. The sample preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This resulted in 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per sample. From this, a total of 840 proteins and, on average, 297 proteins per sample could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several <i>m</i>/<i>z</i> species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer