Top-down characterization of endogenous protein complexes with native proteomics.

Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.

Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes.

Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma.

Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry.

Fragmentation of intact proteins in the gas phase is influenced by amino acid composition, the mass and charge of precursor ions, higher order structure, and the dissociation technique used. The likelihood of fragmentation occurring between a pair of residues is referred to as the fragmentation propensity and is calculated by dividing the total number of assigned fragmentation events by the total number of possible fragmentation events for each residue pair. Here, we describe general fragmentation propensities when performing top-down mass spectrometry (TDMS) using denaturing or native electrospray ionization. A total of 5311 matched fragmentation sites were collected for 131 proteoforms that were analyzed over 165 experiments using native top-down mass spectrometry (nTDMS). These data were used to determine the fragmentation propensities for 399 residue pairs. In comparison to denatured top-down mass spectrometry (dTDMS), the fragmentation pathways occurring either N-terminal to proline or C-terminal to aspartic acid were even more enhanced in nTDMS compared with other residues. More generally, 257/399 (64%) of the fragmentation propensities were significantly altered (P ≤ 0.05) when using nTDMS compared with dTDMS, and of these, 123 were altered by 2-fold or greater. The most notable enhancements of fragmentation propensities for TDMS in native versus denatured mode occurred (1) C-terminal to aspartic acid, (2) between phenylalanine and tryptophan (F|W), and (3) between tryptophan and alanine (W|A). The fragmentation propensities presented here will be of high value in the development of tailored scoring systems used in nTDMS of both intact proteins and protein complexes. Graphical Abstract ᅟ.

Metabologenomics: Correlation of Microbial Gene Clusters with Metabolites Drives Discovery of a Nonribosomal Peptide with an Unusual Amino Acid Monomer.

For more than half a century the pharmaceutical industry has sifted through natural products produced by microbes, uncovering new scaffolds and fashioning them into a broad range of vital drugs. We sought a strategy to reinvigorate the discovery of natural products with distinctive structures using bacterial genome sequencing combined with metabolomics. By correlating genetic content from 178 actinomycete genomes with mass spectrometry-enabled analyses of their exported metabolomes, we paired new secondary metabolites with their biosynthetic gene clusters. We report the use of this new approach to isolate and characterize tambromycin, a new chlorinated natural product, composed of several nonstandard amino acid monomeric units, including a unique pyrrolidine-containing amino acid we name tambroline. Tambromycin shows antiproliferative activity against cancerous human B- and T-cell lines. The discovery of tambromycin via large-scale correlation of gene clusters with metabolites (a.k.a. metabologenomics) illuminates a path for structure-based discovery of natural products at a sharply increased rate.

Investigation of the HIV-1 matrix interactome during virus replication.

PURPOSE: Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. EXPERIMENTAL DESIGN: A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2 -transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. RESULTS: A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions.

Granulocyte-macrophage colony stimulating factor exerts protective and immunomodulatory effects in cortical trauma.

Neurodegeneration after traumatic brain injury is facilitated by innate and adaptive immunity and can be harnessed to affect brain repair. In mice subjected to controlled cortical impact (CCI), we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers in the cervical lymph nodes coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI.

Quantitative proteomics by SWATH-MS reveals altered expression of nucleic acid binding and regulatory proteins in HIV-1-infected macrophages.

Human immunodeficiency virus type 1 (HIV-1) infection remains a worldwide epidemic, and innovative therapies to combat the virus are needed. Developing a host-oriented antiviral strategy capable of targeting the biomolecules that are directly or indirectly required for viral replication may provide advantages over traditional virus-centric approaches. We used quantitative proteomics by SWATH-MS in conjunction with bioinformatic analyses to identify host proteins, with an emphasis on nucleic acid binding and regulatory proteins, which could serve as candidates in the development of host-oriented antiretroviral strategies. Using SWATH-MS, we identified and quantified the expression of 3608 proteins in uninfected and HIV-1-infected monocyte-derived macrophages. Of these 3608 proteins, 420 were significantly altered upon HIV-1 infection. Bioinformatic analyses revealed functional enrichment for RNA binding and processing as well as transcription regulation. Our findings highlight a novel subset of proteins and processes that are involved in the host response to HIV-1 infection. In addition, we provide an original and transparent methodology for the analysis of label-free quantitative proteomics data generated by SWATH-MS that can be readily adapted to other biological systems.