skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Measurements of heterogeneity in proteomics analysis of the nanoparticle protein corona across core facilities
Abstract Robust characterization of the protein corona—the layer of proteins that spontaneously forms on the surface of nanoparticles immersed in biological fluids—is vital for prediction of the safety, biodistribution, and diagnostic/therapeutic efficacy of nanomedicines. Protein corona identity and abundance characterization is entirely dependent on liquid chromatography coupled to mass spectroscopy (LC-MS/MS), though the variability of this technique for the purpose of protein corona characterization remains poorly understood. Here we investigate the variability of LC-MS/MS workflows in analysis of identical aliquots of protein coronas by sending them to different proteomics core-facilities and analyzing the retrieved datasets. While the shared data between the cores correlate well, there is considerable heterogeneity in the data retrieved from different cores. Specifically, out of 4022 identified unique proteins, only 73 (1.8%) are shared across the core facilities providing semiquantitative analysis. These findings suggest that protein corona datasets cannot be easily compared across independent studies and more broadly compromise the interpretation of protein corona research, with implications in biomarker discovery as well as the safety and efficacy of our nanoscale biotechnologies.  more » « less
Award ID(s):
2046159
PAR ID:
10416496
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
Nature Communications
Volume:
13
Issue:
1
ISSN:
2041-1723
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals. 
    more » « less
  2. Elofsson, Arne (Ed.)
    Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a powerful protein characterization technique that provides insights into protein dynamics and flexibility at the peptide level. However, analyzing HDX-MS data presents a significant challenge due to the wealth of information it generates. Each experiment produces data for hundreds of peptides, often measured in triplicate across multiple time points. Comparisons between different protein states create distinct datasets containing thousands of peptides that require matching, rigorous statistical evaluation, and visualization. Our open-source R package, HDXBoxeR, is a comprehensive tool designed to facilitate statistical analysis and comparison of multiple sets among samples and time points for different protein states, along with data visualization. 
    more » « less
  3. Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions. 
    more » « less
  4. Polyfluoroalkyl substances (PFASs) and para-phenylenediamines (PPDs) are emerging classes of anthropogenic contaminants that are environmentally persistent (most often found in ground and surface water sources), bioaccumulative, and harmful to human health. These chemicals are currently regulated in the US by the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA), and the Occupational Safety and Health Administration (OSHA). Analysis of these contaminants is currently spearheaded by mass spectrometry (MS) coupled to liquid chromatography (LC) because of their high sensitivity and separation capabilities. Although effective, a major flaw in LC-MS analysis is its large consumption of solvents and the amount of time required for each experiment. Direct analysis in real time mass spectrometry (DART-MS) is a new technique that offers high sensitivity and permits rapid analysis with little to no sample preparation. Herein, we present the qualitative and quantitative analysis of PFASs and PPDs by high-resolution DART-MS, interfaced with ion mobility (IM) and tandem mass spectrometry (MS/MS) characterization, demonstrating the utility of this multidimensional approach for the fast separation and detection of environmental contaminants. 
    more » « less
  5. Abstract We make fundamental observations of the particle size variability of magnetic properties from 71 core tops that span the southern Greenland and Norwegian Seas. These data provide the first detailed regional characterization of how bulk magnetic properties vary with sediment texture, sediment source, and sediment transport. Magnetic susceptibility (MS) and hysteresis parameters were measured on the bulk sediment and the five constituent sediment particle size fractions (clay, fine silt, medium silt, coarse silt, and sand). The median MS value of the medium silt size fraction is ~3–5 times higher than that of the sand and clay size fractions and results in a strong sensitivity of bulk MS to sediment texture. Hysteresis properties of the clay size fraction are relatively homogeneous and contrast that silt and sand size fractions which show regional differences across the study area. These coarser fractions are more transport limited and using medium silt hysteresis measurements and low temperature MS behavior we establish three endmembers that effectively explain the variability observed across the region. We model the response of bulk magnetic properties to changes in sediment texture and suggest that variations in sediment source are required to explain the bulk magnetic property variability observed in cores across the southern Greenland and Norwegian Seas. These findings imply that sediment source has a greater influence on driving bulk magnetic property variability across this region than has previously been assumed. 
    more » « less