Ion Mobility Mass Spectrometry Coupled with Rapid Protein Threading Predictor Structure Prediction and Collision-Induced Dissociation for Probing Chemokine Conformation and Stability

Unique to ion mobility mass spectrometry (IM-MS) is the ability to provide collision cross section (CCS) data and the capacity to delineate any dissociation and/or unfolding of protein complexes. The strong correlation of the experimentally determined CCS with theory is indicative of the retention of native structure in the gas phase, which in turn, qualifies as a means in evaluating the IM-MS data. The assessment of IM-MS data, however, is currently impeded due to the lack of appropriate structural coordinates to use as input in the in silico calculation of theory. To address this issue, this study involves the use of rapid protein threading predictor (RAPTOR) to generate tertiary structures of closely related monomeric chemokines (MCP-1, MCP-3, MCP-4, and eotaxin) and, subsequently, utilize these models to estimate the theoretical values. Experimental CCS of both the model proteins and chemokines correlate well with theory generated by RAPTOR. All conformations for <i>z</i> = 5+ of chemokines fall within theoretical limits. Of the four chemokines, MCP-4 with <i>z</i> = 6+ appears to adopt an extended conformation, while eotaxin gradually unfolds, and the extended structures of MCP-1 and MCP-3 increase in abundance upon activation. Combining RAPTOR with IM-MS and collision-induced dissociation (CID) enables us to interrogate the conformations of homologous proteins with very similar tertiary structures

Novel Mass Spectrometric Method for Phosphorylation Quantification Using Cerium Oxide Nanoparticles and Tandem Mass Tags

The stoichiometry of protein phosphorylation significantly impacts protein function. The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor the regulation levels of various proteins. This study reports an integrated methodology using cerium oxide nanoparticles and isobaric tandem mass tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation. This protocol was designed to directly measure the dephosphorylation levels for a known phosphorylation site, therefore allowing for quantification of phosphosites. Both the accuracy and precision of the method were verified using standard peptides and protein tryptic digests. This novel method was then applied to quantify phosphorylations on eukaryotic initiation factor 3H (eIF3H), a protein integral to overall eukaryotic protein translation initiation. To date, this is the first report of assessment of protein phosphorylation quantification on eIF3

Differentiation of CC vs CXC Chemokine Dimers with GAG Octasaccharide Binding Partners: An Ion Mobility Mass Spectrometry Approach

Chemokines, 8 kDa proteins implicated in leukocyte migration via oligomerization, bind to glycosaminoglycans (GAGs) during the inflammation response as a means to regulate chemokine migration. Structural characterization of chemokines non-covalently bound to GAGs provides physiologically meaningful data in regard to routine inmmunosurveillance and disease response. In order to analyze the structures resulting from the GAG:chemokine interaction, we employed ion mobility mass spectrometry (IMMS) to analyze monocyte chemoattractant protein‑1 (MCP‑1), a CC chemokine, and interleukin‑8 (IL‑8), a CXC chemokine, along with their individual interactions with GAG heparin octasaccharides. We show that MCP‑1 and IL‑8 are physiologically present as a dimer, with MCP‑1 having two variants of its dimeric form and IL‑8 having only one. We also show that the MCP‑1 dimer adopts two conformations, one extended and one compact, when bound to a dodecasulfated heparin octasaccharide. Binding of MCP‑1 to heparin octasaccharide isomers of varying sulfation patterns results in similar arrival time distribution values, which suggests minimal distinguishing features among the resultant complexes. Additionally, tandem mass spectrometry (MS/MS) showed that the binding of MCP‑1 to a heparin octasaccharide has different dissociation patterns when compared with the corresponding IL‑8 bound dimer. Overall, IMMS and MS/MS were used to better define the structural tendencies and differences associated with CC and CXC dimers when associated with GAG octasaccharides

Sulfolipid-1 Biosynthesis Restricts <i>Mycobacterium tuberculosis</i> Growth in Human Macrophages

<i>Mycobacterium tuberculosis</i> (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host–pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a <i>stf0</i>-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide <i>in vitro</i>, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the <i>stf0</i> mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages