Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para-aminobenzoate in Chlamydia trachomatis.

Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of para-aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen Chlamydia trachomatis. The pathway used by Chlamydiae for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from l-tyrosine. As a member of the emerging superfamily of heme oxygenase-like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is ∼14 Šfrom the dimetal site. We propose that this self-sacrificial reaction occurs through O2 activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the "substrate" Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in C. trachomatis, which will inform the design of novel therapeutics.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.

Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via "flow-through affinity chromatography" using peptide-based adsorbents.

The growth of advanced analytics in manufacturing monoclonal antibodies (mAbs) has highlighted the challenges associated with the clearance of host cell proteins (HCPs). Of special concern is the removal of "persistent" HCPs, including immunogenic and mAb-degrading proteins, that co-elute from the Protein A resin and can escape the polishing steps. Responding to this challenge, we introduced an ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow-through affinity chromatography. This study describes their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity of ~30 mg of HCPs per mL of resin as well as dynamic capacities up to 16 and 22 mg/ml at 1- and 2-min residence times, respectively. When evaluated against cell culture harvests with different mAb and HCP titers and properties, LigaGuard™ afforded high HCP clearance, with logarithmic removal values (LRVs) up to 1.5, and mAb yield above 90%. Proteomic analysis of the effluents confirmed the removal of high-risk HCPs, including cathepsins, histones, glutathione-S transferase, and lipoprotein lipases. Finally, combining LigaGuard™ for HCP removal with affinity adsorbents for product capture afforded a global mAb yield of 85%, and HCP and DNA LRVs > 4.

A bottom-up proteomics workflow for a system containing multiple organisms.

RATIONALE: Discovery proteomics has been popularized to be essential in the investigator's biological toolbox. Many biological problems involve the interplay of multiple organisms. Herein, a bottom-up proteomics workflow was developed to study a system containing multiple organisms to promote a thorough understanding of how each interacts with the others. METHODS: A label-free quantification proteomics workflow was developed with nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). This protocol describes a bottom-up proteomics workflow used to study differential protein expression in the context of fleas (Ctenocephalides felis felis) experimentally infected by the bacterium Bartonella henselae, the etiological agent of Cat Scratch Disease (CSD). RESULTS: Step-by-step instructions are provided for protein extraction, protein cleanup, total protein measurement, nanoLC-MS/MS data acquisition, and data analysis using Proteome Discoverer software. Comprehensive and exhaustive details are included to promote the adoption of this proteomics workflow in other laboratories. CONCLUSION: A proteomics protocol is detailed for a system containing multiple proteomes from different taxonomic lineages using CSD (cats bitten by fleas infected with Bartonella henselae) as a model. The operating protocol can be readily applied to other label-free proteomics work involving multiple proteomes from taxonomically distinct organisms.

Discovery proteomics of human placental tissue.

We describe a label-free proteomics protocol for the interrogation of the placental proteome. Step-by-step directions, including tissue cleanup and preparation, proteolytic digestion, nanoLC-MS/MS data collection and data analysis, are provided. The workflow has been applied toward exploring differential protein expression patterns in placentas from women who have been exposed to drugs during pregnancy relative to those who have not. We collected 20 tissue specimens, each representing a combination of spatially diverse sections across the placenta. These specimens were analyzed in the work described here, to survey information across the entire organ. This protocol can be scaled up or down as needed.

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis.

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow-through mode using ion exchange or mixed-mode chromatography. Recent studies have highlighted the presence of "problematic HCP" species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb-producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide-based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many "problematic HCPs" by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next-generation adsorbents for safer and cost-effective manufacturing of biologics.

Dynamics of the striped bass (Morone saxatilis) ovary proteome reveal a complex network of the translasome.

We evaluated changes in the striped bass (Morone saxatilis) ovary proteome during the annual reproductive cycle using label-free quantitative mass spectrometry and a novel machine learning analysis based on K-means clustering and support vector machines. Modulated modularity clustering was used to group co-variable proteins into expression modules and Gene Ontology (GO) biological process and KEGG pathway enrichment analyses were conducted for proteins within those modules. We discovered that components of the ribosome along with translation initiation and elongation factors generally decrease as the annual ovarian cycle progresses toward ovulation, concomitant with a slight increase in components of the 26S-proteasome. Co-variation within more than one expression module of components from these two multi-protein complexes suggests that they are not only co-regulated, but that co-regulation occurs through more than one sub-network. These components also co-vary with subunits of the TCP-1 chaperonin system and enzymes of intermediary metabolic pathways, suggesting that protein folding and cellular bioenergetic state play important roles in protein synthesis and degradation. We provide further evidence to suggest that protein synthesis and degradation are intimately linked, and our results support function of a proteasome-ribosome supercomplex known as the translasome.

Discovery Proteomics and Absolute Protein Quantification Can Be Performed Simultaneously on an Orbitrap-Based Mass Spectrometer.

Mass spectrometry (MS) has steadily moved into the forefront of quantification-centered protein research. Protein cleavage isotope dilution MS is a proven way for quantifying proteins by using an isotope-labeled analogue of a peptide fragment of the parent protein as an internal standard. Parallel reaction monitoring (PRM) has become the go-to approach for such quantification on an Orbitrap-based instrument as it is assumed that the instrument sensitivity is enhanced. We performed a comparative study on data-dependent acquisition (DDA) and PRM-based workflows to quantify egg yolk protein precursors or vitellogenins (VTGs) Aa, Ab, and C in striped bass (Morone saxatilis). VTG proportions serve as a developmental measure of egg quality, possibly changing with the environment, and have been studied as an indicator of the health of North Carolina stocks. Based on single-factor analysis of variance comparisons of mean VTG amounts across fish from the same sample groupings, our results indicate that there is no statistical difference between MS1-based and MS2-based VTG quantification. We further conclude that DDA is able to deliver both discovery data and absolute quantification data in the same experiment.

Myeloid cell MHC I expression drives CD8+ T cell activation in nonalcoholic steatohepatitis.

Background & aims: Activated CD8+ T cells are elevated in Nonalcoholic steatohepatitis (NASH) and are important for driving fibrosis and inflammation. Despite this, mechanisms of CD8+ T cell activation in NASH are largely limited. Specific CD8+ T cell subsets may become activated through metabolic signals or cytokines. However, studies in NASH have not evaluated the impact of antigen presentation or the involvement of specific antigens. Therefore, we determined if activated CD8+ T cells are dependent on MHC class I expression in NASH to regulate fibrosis and inflammation. Methods: We used H2Kb and H2Db deficient (MHC I KO), Kb transgenic mice, and myeloid cell Kb deficient mice (LysM Kb KO) to investigate how MHC class I impacts CD8+ T cell function and NASH. Flow cytometry, gene expression, and histology were used to examine hepatic inflammation and fibrosis. The hepatic class I immunopeptidome was evaluated by mass spectrometry. Results: In NASH, MHC class I isoform H2Kb was upregulated in myeloid cells. MHC I KO demonstrated protective effects against NASH-induced inflammation and fibrosis. Kb mice exhibited increased fibrosis in the absence of H2Db while LysM Kb KO mice showed protection against fibrosis but not inflammation. H2Kb restricted peptides identified a unique NASH peptide Ncf2 capable of CD8+ T cell activation in vitro. The Ncf2 peptide was not detected during fibrosis resolution. Conclusion: These results suggest that activated hepatic CD8+ T cells are dependent on myeloid cell MHC class I expression in diet induced NASH to promote inflammation and fibrosis. Additionally, our studies suggest a role of NADPH oxidase in the production of Ncf2 peptide generation.

Using Proteomic Approaches to Unravel the Response of Ctenocephalides felis felis to Blood Feeding and Infection With Bartonella henselae.

Among the Ctenocephalides felis felis-borne pathogens, Bartonella henselae, the main aetiological agent of cat scratch disease (CSD), is of increasing comparative biomedical importance. Despite the importance of B. henselae as an emergent pathogen, prevention of the diseases caused by this agent in cats, dogs and humans mostly relies on the use of ectoparasiticides. A vaccine targeting both flea fitness and pathogen competence is an attractive choice requiring the identification of flea proteins/metabolites with a dual effect. Even though recent developments in vector and pathogen -omics have advanced the understanding of the genetic factors and molecular pathways involved at the tick-pathogen interface, leading to discovery of candidate protective antigens, only a few studies have focused on the interaction between fleas and flea-borne pathogens. Taking into account the period of time needed for B. henselae replication in flea digestive tract, the present study investigated flea-differentially abundant proteins (FDAP) in unfed fleas, fleas fed on uninfected cats, and fleas fed on B. henselae-infected cats at 24 hours and 9 days after the beginning of blood feeding. Proteomics approaches were designed and implemented to interrogate differentially expressed proteins, so as to gain a better understanding of proteomic changes associated with the initial B. henselae transmission period (24 hour timepoint) and a subsequent time point 9 days after blood ingestion and flea infection. As a result, serine proteases, ribosomal proteins, proteasome subunit α-type, juvenile hormone epoxide hydrolase 1, vitellogenin C, allantoinase, phosphoenolpyruvate carboxykinase, succinic semialdehyde dehydrogenase, glycinamide ribotide transformylase, secreted salivary acid phosphatase had high abundance in response of C. felis blood feeding and/or infection by B. henselae. In contrast, high abundance of serpin-1, arginine kinase, ribosomal proteins, peritrophin-like protein, and FS-H/FSI antigen family member 3 was strongly associated with unfed cat fleas. Findings from this study provide insights into proteomic response of cat fleas to B. henselae infected and uninfected blood meal, as well as C. felis response to invading B. henselae over an infection time course, thus helping understand the complex interactions between cat fleas and B. henselae at protein levels.

Development of Peptide Ligands for Targeted Capture of Host Cell Proteins from Cell Culture Production Harvests.

Capture of host cell proteins (HCPs) from cell culture production harvests is critical to ensure the maximum levels specified by international regulatory bodies of product purity for therapeutic monoclonal antibodies (mAbs). Peptide ligands that selectively target the whole spectrum of the HCPs, while letting the mAb product flow through unbound, are an ideal complement to the affinity-based capture step via Protein A chromatography. In this work, we describe the development of HCP-binding peptide ligands, especially focusing on the steps of (1) peptide selection via library screening and (2) quantification of HCP removal via proteomics by mass spectrometry.

The soluble proteome of the Drosophila antenna.

The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems. Identification of proteins contained in the third antennal segment, the main olfactory organ, has previously relied primarily on immunohistochemistry, and although such studies and in situ hybridization studies are informative, they focus generally on one or few gene products at a time, and quantification is difficult. In addition, purification of native proteins from the antenna is challenging because it is small and encased in a hard cuticle. Here, we describe a simple method for the large-scale detection of soluble proteins from the Drosophila antenna by chromatographic separation of tryptic peptides followed by tandem mass spectrometry with femtomole detection sensitivities. Examination of the identities of these proteins indicates that they originate both from the extracellular perilymph and from the cytoplasm of disrupted cells. We identified enzymes involved with intermediary metabolism, proteins associated with regulation of gene expression, nucleic acid metabolism and protein metabolism, proteins associated with microtubular transport, 8 odorant-binding proteins, protective enzymes associated with antibacterial defense and defense against oxidative damage, cuticular proteins, and proteins of unknown function, which represented about one-third of all soluble proteins. The procedure described here opens the way for precise quantification of any target protein in the Drosophila antenna and should be readily applicable to antennae from other insects.

Development of a nanoLC LTQ orbitrap mass spectrometric method for profiling glycans derived from plasma from healthy, benign tumor control, and epithelial ovarian cancer patients.

We report the development of split-less nano-flow liquid chromatography mass spectrometric analysis of glycans chemically cleaved from glycoproteins in plasma. Porous graphitized carbon operating under reverse-phase conditions and an amide-based stationary phase operating under hydrophilic interaction conditions are quantitatively compared for glycan separation. Both stationary phases demonstrated similar column efficiencies and excellent retention time reproducibility without an internal standard to correct for retention time shift. The 95% confidence intervals of the mean retention times were +/-4 s across 5 days of analysis for both stationary phases; however, the amide stationary phase was observed to be more robust. The high mass measurement accuracy of less than 2 ppm and fragmentation spectra provided highly confident identifications along with structural information. In addition, data are compared among samples derived from 10 healthy controls, 10 controls with a differential diagnosis of benign gynecologic tumors, and 10 diseased epithelial ovarian cancer patients (EOC). Two fucosylated glycans were found to be up-regulated in healthy controls and provided an accurate diagnostic value with an area under the receiver operator characteristic curve of 0.87. However, these same glycans provided a significantly less diagnostic value when used to differentiate EOC from benign tumor control samples with an area under the curve of 0.73.

Construction of coordination-driven self-assembled [5 + 5] pentagons using metal-carbonyl dipyridine ligands.

The coordination-driven self-assembly of two metal-carbonyl-cluster-coordinated dipyridyl donors, (4-C(5)H(4)N)(2)C[triple bond]CCo(2)(CO)(6) (1) and (4-C(5)H(4)N)(2)C[triple bond]CMo(2)Cp(2)(CO)(4) (2), with a linear diplatinum(II) acceptor ligand was investigated. The structures of the resulting self-assembled polygons were found to be controlled by the steric bulk of the metal-carbonyl cluster adduct. The use of a sterically less imposing ligand 1 resulted in a pentagon-hexagon mixture, which was characterized by electrospray ionization time-of-flight mass spectroscopy. The exclusive formation of a [5 + 5] pentagon was achieved by the self-assembly of the bulkier molybdenum donor ligand 2 with a linear organoplatinum(II) acceptor ligand. Molecular force field modeling was used to study the structural details of the pentagonal and hexagonal architectures. The first Fe(3)-Co(6)-Pt(6) trimetal [3 + 3] hexagon was also synthesized via the combination of 1 with a 120 degrees ferrocenyldiplatinum(II) acceptor.

Calibration laws based on multiple linear regression applied to matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

Operation of any mass spectrometer requires implementation of mass calibration laws to translate experimentally measured physical quantities into a m/z range. While internal calibration in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) offers several attractive features, including exposure of calibrant and analyte ions to identical experimental conditions (e.g. space charge), external calibration affords simpler pulse sequences and higher throughput. The automatic gain control method used in hybrid linear trap quadrupole (LTQ) FT-ICR-MS to consistently obtain the same ion population is not readily amenable to matrix-assisted laser desorption/ionization (MALDI) FT-ICR-MS, due to the heterogeneous nature and poor spot-to-spot reproducibility of MALDI. This can be compensated for by taking external calibration laws into account that consider magnetic and electric fields, as well as relative and total ion abundances. Herein, an evaluation of external mass calibration laws applied to MALDI-FT-ICR-MS is performed to achieve higher mass measurement accuracy (MMA).

Investigations with O-linked protein glycosylations by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

Posttranslational modifications such as glycosylation can play a fundamental role in signaling pathways that transform an ordinary cell into a malignant one. The development of a protocol to detect these changes in the preliminary stages of disease can lead to a sensitive and specific diagnostic for the early detection of malignancies such as ovarian cancer in which differential glycan patterns are linked to etiology and progression. Small variations in instrument parameters and sample preparation techniques are known to have significant influence on the outcome of an experiment. For an experiment to be effective and reproducible, these parameters must be optimized for the analyte(s) under study. We present a detailed examination of sample preparation and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) analysis of O-linked glycans globally cleaved from mucin glycoproteins. Experiments with stable isotope-labeled biomolecules allowed for the establishment of appropriate acquisition times and excitation voltages for MALDI-FT-ICR-MS of oligosaccharides. Quadrupole ion guide optimization studies with mucin glycans identified conditions for the comprehensive analysis of the entire mass range of O-linked carbohydrates in this glycoprotein. Separately optimized experimental parameters were integrated in a method that allowed for the effective study of O-linked glycans.

Studying O-linked protein glycosylations in human plasma.

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.

Carbohydrate analysis by desorption electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

We report the use of desorption electrospray ionization hybrid Fourier transform ion cyclotron resonance mass spectrometry (DESI-FT-ICR-MS) for the analysis of carbohydrates. Spectra of neat carbohydrates are presented along with their mass measurement accuracies and limits of detection. Furthermore, a comparison is made between the analyses of O-linked glycans from mucin by DESI-FT-ICR-MS and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. Finally, glycans from mucin are identified by using the high mass measurement accuracy and tandem MS capabilities afforded by the hybrid FT-ICR-MS platform.

Effect of matrix crystal structure on ion abundance of carbohydrates by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

Sample preparation techniques for carbohydrate analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are explored, with particular emphasis on analyte/matrix co-crystallization procedures. While carbohydrates are known to prefer 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix of choice, these analytes are quite specific about matrix crystal structure, which in turn is dependent on the rate of drying of analyte/matrix spots on the MALDI target. With N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (sialic acid or NeuAc) as test monosaccharides, significant increases in ion abundances are demonstrated with 2,5-DHB/NeuAc spots (>10-fold improvement) and 2,5-DHB/GlcNAc spots ( approximately 5-fold improvement) with active drying. The fine structure of crystals generated in active and passive drying was investigated using powder diffraction. Passively dried samples were shown to consist of an ordered polymorph, crystallizing in the space group P2(1)/a, while the actively dried samples produced a disordered phase crystallizing in the space group Pa. These data provide the wherewithal to engineer a matrix best suited for carbohydrate analyses.

Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum.

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.