Lipopolysaccharide Regulates the Macrophage RNA-Binding Proteome.

RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins' (RBPs') effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs. Global RNA sequencing and LFQ proteomics were used to characterize the correlation of transcript and protein abundance changes in response to LPS at different time points with changes in protein-RNA binding. Il1α, MARCKS, and ACOD1 were noted as RBP candidates involved in innate immune signaling. The binding sites of the RBP and RNA conjugates at amino acid resolution were investigated by digesting the cross-linked oligonucleotide from peptides remaining after elution using Nuclease P1. The combined data sets provide directions for further studies of innate immune signaling regulation by RBP interactions with different classes of RNA.

Omics and systems view of innate immune pathways.

Multiomics approaches to studying systems biology are very powerful techniques that can elucidate changes in the genomic, transcriptomic, proteomic, and metabolomic levels within a cell type in response to an infection. These approaches are valuable for understanding the mechanisms behind disease pathogenesis and how the immune system responds to being challenged. With the emergence of the COVID-19 pandemic, the importance and utility of these tools have become evident in garnering a better understanding of the systems biology within the innate and adaptive immune response and for developing treatments and preventative measures for new and emerging pathogens that pose a threat to human health. In this review, we focus on state-of-the-art omics technologies within the scope of innate immunity.

Increased sequence hydrophobicity reduces conformational specificity: A mutational case study of the Arc repressor protein.

The amino-acid sequences of soluble, globular proteins must have hydrophobic residues to form a stable core, but excess sequence hydrophobicity can lead to loss of native state conformational specificity and aggregation. Previous studies of polar-to-hydrophobic mutations in the ß-sheet of the Arc repressor dimer showed that a single substitution at position 11 (N11L) leads to population of an alternate dimeric fold in which the ß-sheet is replaced by helix. Two additional hydrophobic mutations at positions 9 and 13 (Q9V and R13V) lead to population of a differently folded octamer along with both dimeric folds. Here we conduct a comprehensive study of the sequence determinants of this progressive loss of fold specificity. We find that the alternate dimer-fold specifically results from the N11L substitution and is not promoted by other hydrophobic substitutions in the ß-sheet. We also find that three highly hydrophobic substitutions at positions 9, 11, and 13 are necessary and sufficient for oligomer formation, but the oligomer size depends on the identity of the hydrophobic residue in question. The hydrophobic substitutions increase thermal stability, illustrating how increased hydrophobicity can increase folding stability even as it degrades conformational specificity. The oligomeric variants are predicted to be aggregation-prone but may be hindered from doing so by proline residues that flank the ß-sheet region. Loss of conformational specificity due to increased hydrophobicity can manifest itself at any level of structure, depending upon the specific mutations and the context in which they occur.

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step toward identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top-down mass spectrometry to identify proteoforms released during 1 min of mechanical stimulation of mouse skin from naïve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top-down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.

The role of mass spectrometry in the characterization of biologic protein products.

INTRODUCTION: Mass spectrometry (MS) is widely used in the characterization of biomolecules including peptide and protein therapeutics. These biotechnology products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported an increased development of biopharmaceutical products. Areas covered: This review describes recent developments in MS-based biotherapeutic analysis including sequence determination, post-translational modifications (PTMs) and higher order structure (HOS) analysis along with improvements in ionization and dissociation methods. An outlook of emerging applications of MS in the lifecycle of product development such as comparability, biosimilarity and quality control practices is also presented. Expert commentary: MS-based methods have established their utility in the analysis of new biotechnology products and their lifecycle appropriate implementation. In the future, MS will likely continue to grow as one of the leading protein identification and characterization techniques in the biopharmaceutical industry landscape.

Obtaining complementary polypeptide sequence information from a single precursor ion packet via sequential ion mobility-resolved electron transfer and vibrational activation.

Tandem mass spectrometry (MS/MS) is now well-known as a powerful tool for characterizing the primary structures of peptides and proteins; however, in many cases the use of but a single dissociation method provides only a partial view of the amino acid sequences and post-translational modification patterns of polypeptides. While the application of multiple fragmentation methods can be more informative, this introduces the burden of acquiring multiple MS/MS spectra per analyte, thus reducing the effective duty cycle of such methods. In this work, initial proof-of-concept is provided for a method designed to overcome these barriers. This method relies on the complementary fragmentation information that can be provided by performing collision-induced dissociation (CID) and electron transfer dissociation (ETD) in concert, while also taking advantage of an ion mobility (IM) dimension to temporally resolve the occurrence of CID and ETD when applied to a single accumulated packet of precursor ions. In this way, the significant proportion of the precursor ion population that remains unreacted in ETD experiments is subjected to CID rather than being fruitlessly discarded. In addition, the two distinct fragmentation spectra can be extracted from their corresponding IM domains to render readily interpretable individual fragmentation spectra. This scheme was demonstrated for several polypeptides ranging from 1.3 to 8.6 kDa in molecular weight. In each case, IM-resolved CID and ETD events resulted in b/y and c/z ions, respectively, which each covered both unique and overlapping sequence information. These findings demonstrate that the combination of CID and ETD can be achieved with greater utilization of the available ion population and little or no loss of duty cycle.

Phosphoproteome Analysis in Immune Cell Signaling.

Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR7 with immune-response inducing ligands in cultured immune cells. This review will focus on the TLR stimulation of mouse macrophages as an example; however, the technique is applicable to any immortalized immune cell and any soluble stimuli. The methodology includes protocols for metabolic labeling of immune cells (stable isotope labeling of amino acids in cell culture, i.e., SILAC); ligand-initiated stimulation of immune receptors followed by cell lysis; in-solution trypsin digestion of proteins and enrichment of the resulting peptide mix for collecting phosphopeptides, which are then analyzed by high-resolution LC-MS/MS (liquid-chromatography tandem mass spectrometry). © 2020 Wiley Periodicals LLC. Basic Protocol 1: SILAC labeling of mouse macrophages Basic Protocol 2: Stimulation, cell lysis and Western Blotting Basic Protocol 3: Trypsin digestion, fractionation and phosphopeptide enrichment Basic Protocol 4: Quantitative mass spectrometry Alternate Protocol: Culturing SILAC-labeled cells from frozen mouse macrophages cells.

Collision-induced release, ion mobility separation, and amino acid sequence analysis of subunits from mass-selected noncovalent protein complexes.

In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and ß-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.