A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism.

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

Structural and Functional Analysis of a ß2-Adrenergic Receptor Complex with GRK5.

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

A lethal mitonuclear incompatibility in complex I of natural hybrids.

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells.

Recombinant adeno-associated virus (rAAV) vectors could be manufactured by plasmid transfection into human embryonic kidney 293 (HEK293) cells or baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. However, systematic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. rAAV from Sf9 cells (Sf9-rAAV) at 2-50 L and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized. HEK-rAAV had ∼40-fold lower yields but ∼10-fold more host cell DNA measured by droplet digital PCR and next-generation sequencing, respectively. The electron microscope observed a lower full/empty capsid ratio in HEK-rAAV (70.8%) than Sf9-rAAV (93.2%), while dynamic light scattering and high-performance liquid chromatography analysis showed that HEK-rAAV had more aggregation. Liquid chromatography tandem mass spectrometry identified different post-translational modification profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher tissue culture infectious dose/viral genome than HEK-rAAV, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, as measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.

A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development.

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

Enhancing Data Reliability in TOMAHAQ for Large-Scale Protein Quantification.

The analytical scale of most mass-spectrometry-based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post-acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Elucidating the "Gravome": Quantitative Proteomic Profiling of the Response to Chronic Hypergravity in Drosophila.

Altered gravity conditions, such as experienced by organisms during spaceflight, are known to cause transcriptomic and proteomic changes. We describe the proteomic changes in whole adult Drosophila melanogaster (fruit fly) but focus specifically on the localized changes in the adult head in response to chronic hypergravity (3 g) treatment. Canton S adult female flies (2 to 3 days old) were exposed to chronic hypergravity for 9 days and compared with 1 g controls. After hypergravity treatment, either whole flies (body + head) or fly-head-only samples were isolated and evaluated for quantitative comparison of the two gravity conditions using an isobaric tagging liquid chromatography-tandem mass spectrometry approach. A total of 1948 proteins from whole flies and 1480 proteins from fly heads were differentially present in hypergravity-treated flies. Gene Ontology analysis of head-specific proteomics revealed host immune response, and humoral stress proteins were significantly upregulated. Proteins related to calcium regulation, ion transport, and ATPase were decreased. Increased expression of cuticular proteins may suggest an alteration in chitin metabolism and in chitin-based cuticle development. We therefore present a comprehensive quantitative survey of proteomic changes in response to chronic hypergravity in Drosophila, which will help elucidate the underlying molecular mechanism(s) associated with altered gravity environments.

Comparison of Pandemrix and Arepanrix, two pH1N1 AS03-adjuvanted vaccines differentially associated with narcolepsy development.

Narcolepsy onset in children has been associated with the 2009 influenza A H1N1 pandemic and vaccination with Pandemrix. However it was not clearly observed with other adjuvanted pH1N1 vaccines such as Arepanrix or Focetria. Our aim was to characterize the differences between Pandemrix and Arepanrix that might explain the risk for narcolepsy after Pandemrix vaccination using 2D-DIGE and mass spectrometry (MS). We found that Pandemrix (2009 batch) and Arepanrix (2010 batch) showed 5 main viral proteins: hemagglutinin HA1 and HA2 subunits, neuraminidase NA, nucleoprotein NP, and matrix protein MA1 and non-viral proteins from the Gallus gallus growth matrix used in the manufacturing of the vaccines. Latticed patterns of HA1, HA2 and NA indicated charge and molecular weight heterogeneity, a phenomenon likely caused by glycosylation and sulfation. Overall, Pandemrix contained more NP and NA, while Arepanrix displayed a larger diversity of viral and chicken proteins, with the exception of five chicken proteins (PDCD6IP, TSPAN8, H-FABP, HSP and TUB proteins) that were relatively more abundant in Pandemrix. Glycosylation patterns were similar in both vaccines. A higher degree of deamidation and dioxidation was found in Pandemrix, probably reflecting differential degradation across batches. Interestingly, HA1 146N (residue 129N in the mature protein) displayed a 10-fold higher deamidation in Arepanrix versus Pandemrix. In recent vaccine strains and Focetria, 146N is mutated to D which is associated with increased production yields suggesting that 146N deamidation may have also occurred during the manufacturing of Arepanrix. The presence of 146N in large relative amounts in Pandemrix and the wild type virus and in lower relative quantities in Arepanrix or other H1N1 vaccines may have affected predisposition to narcolepsy.

Limited Proteolysis-Mass Spectrometry Reveals Aging-Associated Changes in Cerebrospinal Fluid Protein Abundances and Structures.

Cerebrospinal fluid (CSF) proteins and their structures have been implicated repeatedly in aging and neurodegenerative diseases. Limited proteolysis-mass spectrometry (LiP-MS) is a method that enables proteome-wide screening for changes in both protein abundance and structure. To screen for novel aging-associated changes in the CSF proteome, we performed LiP-MS on CSF from young and old mice with a modified analysis pipeline. We found 38 protein groups change in abundance with aging, most dominantly immunoglobulins of the IgM subclass. We discovered six high-confidence candidates that appeared to change in structure with aging, of which Kng1, Itih2, Lp-PLA2, and 14-3-3 proteins have binding partners or proteoforms known previously to change in the brain with Alzheimer's disease. Intriguingly, using orthogonal validation by Western blot we found the LiP-MS hit Cd5l forms a covalent complex with IgM in mouse and human CSF whose abundance increases with aging. SOMAmer probe signals for all six LiP-MS hits in human CSF, especially 14-3-3 proteins, significantly associate with several clinical features relevant to cognitive function and neurodegeneration. Together, our findings show that LiP-MS can uncover age-related structural changes in CSF with relevance to neurodegeneration.

Ions in size-selected aqueous nanodrops: sequential water molecule binding energies and effects of water on ion fluorescence.

The effects of water on ion fluorescence were investigated, and average sequential water molecule binding energies to hydrated ions, M(z)(H(2)O)(n), at large cluster size were measured using ion nanocalorimetry. Upon 248-nm excitation, nanodrops with ~25 or more water molecules that contain either rhodamine 590(+), rhodamine 640(+), or Ce(3+) emit a photon with average energies of approximately 548, 590, and 348 nm, respectively. These values are very close to the emission maxima of the corresponding ions in solution, indicating that the photophysical properties of these ions in the nanodrops approach those of the fully hydrated ions at relatively small cluster size. As occurs in solution, these ions in nanodrops with 8 or more water molecules fluoresce with a quantum yield of ~1. Ce(3+) containing nanodrops that also contain OH(-) fluoresce, whereas those with NO(3)(-) do not. This indirect fluorescence detection method has the advantages of high sensitivity, and both the size of the nanodrops as well as their constituents can be carefully controlled. For ions that do not fluoresce in solution, such as protonated tryptophan, full internal conversion of the absorbed 248-nm photon occurs, and the average sequential water molecule binding energies to the hydrated ions can be accurately obtained at large cluster sizes. The average sequential water molecule binding energies for TrpH(+)(H(2)O)(n) and a doubly protonated tripeptide, [KYK + 2H](2+)(H(2)O)(n), approach asymptotic values of ~9.3 (n ≥ 11) and ~10.0 kcal/mol (n ≥ 25), respectively, consistent with a liquidlike structure of water in these nanodrops.

Average sequential water molecule binding enthalpies of M(H2O)(19-124)2+ (M = Co, Fe, Mn, and Cu) measured with ultraviolet photodissociation at 193 and 248 nm.

The average sequential water molecule binding enthalpies to large water clusters (between 19 and 124 water molecules) containing divalent ions were obtained by measuring the average number of water molecules lost upon absorption of an UV photon (193 or 248 nm) and using a statistical model to account for the energy released into translations, rotations, and vibrations of the products. These values agree well with the trend established by more conventional methods for obtaining sequential binding enthalpies to much smaller hydrated divalent ions. The average binding enthalpies decrease to a value of ~10.4 kcal/mol for n > ~40 and are insensitive to the ion identity at large cluster size. This value is close to that of the bulk heat of vaporization of water (10.6 kcal/mol) and indicates that the structure of water in these clusters may more closely resemble that of bulk liquid water than ice, owing either to a freezing point depression or rapid evaporative cooling and kinetic trapping of the initial liquid droplet. A discrete implementation of the Thomson equation using parameters for liquid water at 0 °C generally fits the trend in these data but provides values that are ~0.5 kcal/mol too low.

Nanocalorimetry in mass spectrometry: a route to understanding ion and electron solvation.

A gaseous nanocalorimetry approach is used to investigate effects of hydration and ion identity on the energy resulting from ion-electron recombination. Capture of a thermally generated electron by a hydrated multivalent ion results in either loss of a H atom accompanied by water loss or exclusively loss of water. The energy resulting from electron capture by the precursor is obtained from the extent of water loss. Results for large-size-selected clusters of Co(NH(3))(6)(H(2)O)(n3)(+) and Cu(H(2)O)(n2)(+) indicate that the ion in the cluster is reduced on electron capture. The trend in the data for Co(NH(3))(6)(H(2)O)(n3)(+) over the largest sizes (n >/= 50) can be fit to that predicted by the Born solvation model. This agreement indicates that the decrease in water loss for these larger clusters is predominantly due to ion solvation that can be accounted for by using a model with bulk properties. In contrast, results for Ca(H(2)O)(n2)(+) indicate that an ion-electron pair is formed when clusters with more than approximately 20 water molecules are reduced. For clusters with n = approximately 20-47, these results suggest that the electron is located near the surface, but a structural transition to a more highly solvated electron is indicated for n = 47-62 by the constant recombination energy. These results suggest that an estimate of the adiabatic electron affinity of water could be obtained from measurements of even larger clusters in which an electron is fully solvated.

Age-related neuroinflammation and pathology in the locus coeruleus and hippocampus: beta-adrenergic antagonists exacerbate impairment of learning and memory in aged mice.

The locus coeruleus (LC) provides the primary noradrenergic input to the forebrain and hippocampus, and may be vulnerable to degeneration and contribute to age-related cognitive decline and neuroinflammation. Additionally, inhibition of noradrenergic transmission by brain-permeable beta-blockers could exacerbate cognitive impairment. This study examined effects of age and acute beta-blocker administration on LC and hippocampus pathology, neuroinflammation and learning and memory behavior in mice. Male mice, 3 and 18 months old, were administered propranolol (beta-blocker) or mabuterol (beta-adrenergic agonist) acutely around behavioral assessment. Terminal inflammatory markers in plasma, hippocampus and LC were assessed alongside histopathology. An increase in hippocampal and LC microgliosis and inflammatory proteins in the hippocampus was detected in aged mice. We report pathological hyperphosphorylation of the postsynaptic NMDA receptor subunit 2B (NR2B) in the hippocampus, suggesting neuronal hyperexcitability. Furthermore, the aged proteome revealed an induction in proteins related to energy metabolism, and mitochondria dysfunction in the LC and hippocampus. In a series of hippocampal dependent behavioral assessment tasks acute beta-adrenergic agonist or beta blocker administration altered learning and memory behavior in both aged and young mice. In Y-maze, propranolol and mabuterol differentially altered time spent in novel versus familiar arms in young and aged mice. Propranolol impaired Novel Object Recognition in both young and aged mice. Mabuterol enhanced trace learning in fear conditioning. Aged mice froze more to context and less to cue. Propranolol impaired contextual recall in aged mice. Concluding, aged mice show LC and hippocampus pathology and heightened effects of beta-adrenergic pharmacology on learning and memory.

Electron hydration and ion-electron pairs in water clusters containing trivalent metal ions.

The hydrated electron is one of the most fundamental nucleophiles in aqueous solution, yet it is a transient species in liquid water, making it challenging to study. The solvation thermodynamics of the electron are important for determining the band structure and properties of water and aqueous solutions. However, a wide range of values for the electron solvation enthalpy (-1.0 to -1.8 eV) has been obtained from previous methods, primarily because of the large uncertainty as to the value for the absolute proton solvation enthalpy. In the gas phase, electron interactions with water can be investigated in stable water clusters that contain an excess electron, or an electron and a solvent-separated monovalent or divalent metal ion. Here, we report the generation of stable water clusters that contain an excess electron and a solvent-separated trivalent metal ion that are formed upon electron capture by hydrated trivalent lanthanide clusters. From the number of water molecules lost upon electron capture, adiabatic recombination energies are obtained for La(H(2)O)(n)(3+) (n = 42-160). The trend in recombination energies as a function of hydration extent is consistent with a structural transition from a surface-located excess electron at smaller sizes (n or= approximately 60). The recombination enthalpies for n > 60 are extrapolated as a function of the geometrical dependence on cluster size to infinite size to obtain the bulk hydration enthalpy of the electron (-1.3 eV). This extrapolation method has the advantages that it does not require estimates of the absolute proton or hydrogen hydration enthalpies.

"Weighing" photon energies with mass spectrometry: effects of water on ion fluorescence.

We report a new, highly sensitive method for indirectly measuring fluorescence from ions with a discrete number of water molecules attached. Absorption of a 248 nm photon by hydrated protonated proflavine, PH(+)(H(2)O)(n) (n = 13-50), results in two resolved product ion distributions that correspond to full internal conversion of the photon energy (loss of approximately 11 water molecules) and to partial internal conversion of the photon energy and emission of a lower energy photon (loss of approximately 6 water molecules). In addition to fluorescence, a long-lived triplet state with a half-life of approximately 0.5 s (for n = 50) is formed. The energy of the emitted photon can be obtained from the number of water molecules lost from the precursor to form each distribution. The photon energies generally red shift from approximately 450 to 580 nm with increasing cluster size (the onset of the PH(+)(aq) fluorescence spectrum is 600 nm and the maximum is 518 nm) consistent with preferential stabilization of the first excited singlet state versus the ground state. The fluorescence quantum yield of PH(+)(H(2)O)(n) for n > or = 30 is 0.36 +/- 0.02, the same as that in bulk solution, and increases dramatically with decreasing cluster sizes, due to less efficient conversion of electronic-to-vibrational energy. The high sensitivity of this method should make it possible to perform Forster resonance energy transfer experiments with gas-phase biomolecules in a microsolvated environment to investigate how a controlled number of water molecules facilitates dynamical motions in proteins or other molecules of interest.

Direct standard-free quantitation of Tamiflu and other pharmaceutical tablets using clustering agents with electrospray ionization mass spectrometry.

Accurate and rapid quantitation is advantageous to identify counterfeit and substandard pharmaceutical drugs. A standard-free electrospray ionization mass spectrometry method is used to directly determine the dosage in the prescription and over-the-counter drugs Tamiflu, Sudafed, and Dramamine. A tablet of each drug was dissolved in aqueous solution, filtered, and introduced into solutions containing a known concentration of l-tryptophan, l-phenylalanine, or prednisone as a clustering agent. The active ingredient(s) incorporates statistically into large clusters of the clustering agent where effects of differential ionization/detection are substantially reduced. From the abundances of large clusters, the dosages of the active ingredients in each of the tablets were determined to typically better than 20% accuracy even when the ionization/detection efficiency of the individual components differed by over 100x. Although this unorthodox method for quantitation is not as accurate as using conventional standards, it has the advantages that it is fast, it can be applied to mixtures where the identities of the analytes are unknown, and it can be used when suitable standards may not be readily available, such as schedule I or II controlled substances or new designer drugs that have not previously been identified.

Proteomic analysis of platelet-rich and platelet-poor plasma.

BACKGROUND: Autologous blood products, such as platelet-rich plasma (PRP) are commercial products broadly used to accelerate healing of tissues after injuries. However, their content is not standardized and significantly varies in composition, which may lead to differences in clinical efficacy. Also, the underlying molecular mechanisms for therapeutic effects are not well understood. PURPOSE: A proteomic study was performed to compare the composition of low leukocyte PRP, platelet poor plasma (PPP), and blood plasma. Pathway analysis of the proteomic data was performed to evaluate differences between plasma formulations at the molecular level. Low abundance regulatory proteins in plasma were identified and quantified as well as cellular pathways regulated by those proteins. METHODS: Quantitative proteomic analysis, using multiplexed isotopically labeled tags (TMT labeling) and label-free tandem mass spectrometry, was performed on plasma, low leukocyte PRP, and PPP. Plasma formulations were derived from two blood donors (one donor per experiment). Pathway analysis of the proteomic data identified the major differences between formulations. RESULTS: Nearly 600 proteins were detected in three types of blood plasma formulations in two experiments. Identified proteins showed more than 50% overlap between plasma formulations. Detected proteins represented more than 100 canonical pathways, as was identified by pathway analysis. The major pathways and regulatory molecules were linked to inflammation. CONCLUSION: Three types of plasma formulations were compared in two proteomic experiments. The most represented pathways, such as Acute Phase Response, Coagulation, or System of the Complement, had many proteins in common in both experiments. In both experiments plasma sample sets had the same direction of biochemical pathway changes: up- or down-regulation. The most represented biochemical pathways are linked to inflammation.

Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors.

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05-0.0001), in various mouse tissues in vivo (p < 0.03-0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

Mass Spectrometric Characterization of Narcolepsy-Associated Pandemic 2009 Influenza Vaccines.

The onset of narcolepsy, an irreversible sleep disorder, has been associated with 2009 influenza pandemic (pH1N1) infections in China, and with ASO3-adjuvanted pH1N1 vaccinations using Pandemrix in Europe. Intriguingly, however, the increased incidence was only observed following vaccination with Pandemrix but not Arepanrix in Canada. In this study, the mutational burden of actual vaccine lots of Pandemrix (n = 6) and Arepanrix (n = 5) sourced from Canada, and Northern Europe were characterized by mass spectrometry. The four most abundant influenza proteins across both vaccines were nucleoprotein NP, hemagglutinin HA, matrix protein M1, with the exception that Pandemrix harbored a significantly increased proportion of neuraminidase NA (7.5%) as compared to Arepanrix (2.6%). Most significantly, 17 motifs in HA, NP, and M1 harbored mutations, which significantly differed in Pandemrix versus Arepanrix. Among these, a 6-fold higher deamidation of HA146 (p.Asn146Asp) in Arepanrix was found relative to Pandemrix, while NP257 (p.Thr257Ala) and NP424 (p.Thr424Ile) were increased in Pandemrix. DQ0602 binding and tetramer analysis with mutated epitopes were conducted in Pandemrix-vaccinated cases versus controls but were unremarkable. Pandemrix harbored lower mutational burden than Arepanrix, indicating higher similarity to wild-type 2009 pH1N1, which could explain differences in narcolepsy susceptibility amongst the vaccines.