Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC-MS.

Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.

Utilizing Nonequilibrium Isotope Enrichments to Dramatically Increase Turnover Measurement Ranges in Single Biopsy Samples from Humans.

The synthesis of new proteins and the degradation of old proteins in vivo can be quantified in serial samples using metabolic isotope labeling to measure turnover. Because serial biopsies in humans are impractical, we set out to develop a method to calculate the turnover rates of proteins from single human biopsies. This method involved a new metabolic labeling approach and adjustments to the calculations used in previous work to calculate protein turnover. We demonstrate that using a nonequilibrium isotope enrichment strategy avoids the time dependent bias caused by variable lag in label delivery to different tissues observed in traditional metabolic labeling methods. Turnover rates are consistent for the same subject in biopsies from different labeling periods, and turnover rates calculated in this study are consistent with previously reported values. We also demonstrate that by measuring protein turnover we can determine where proteins are synthesized. In human subjects a significant difference in turnover rates differentiated proteins synthesized in the salivary glands versus those imported from the serum. We also provide a data analysis tool, DeuteRater-H, to calculate protein turnover using this nonequilibrium metabolic 2H2O method.

Improved Sensitivity for Protein Turnover Quantification by Monitoring Immonium Ion Isotopologue Abundance.

We describe an analytical strategy allowing for the direct quantification of stable isotope label incorporation in newly synthesized proteins following administration of the stable isotope tracer deuterium oxide. We present a demonstration of coupling high-resolution mass spectrometry, metabolic stable isotope labeling, and MS/MS-based isotopologue quantification for the measurement of protein turnover. Stable isotope labeling with deuterium oxide, followed by immonium ion isotopologue quantification, is a more sensitive strategy for determining protein fractional synthesis rates compared to peptide centric mass isotopomer distribution analysis approaches when labeling time and/or stable isotope tracer exposure is limited and, as such, offers a great advantage for human studies.

Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood.

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR. CONCLUSION: Using a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88).

Characterization of individual mouse cerebrospinal fluid proteomes.

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the CNS. Characterization of murine CSF proteomes can provide a valuable resource for studying CNS injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through nonterminal CSF extractions in C57Bl/6 mice and high-resolution 2D-LC MS/MS analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. We identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis. The data are available in the ProteomeXchange with identifier PXD000248 (http://proteomecentral.proteomexchange.org/dataset/PXD000248).

Proteomic profiles of five strains of oxygenic photosynthetic cyanobacteria of the genus Cyanothece.

Members of the cyanobacterial genus Cyanothece exhibit considerable variation in physiological and biochemical characteristics. The comparative assessment of the genomes and the proteomes has the potential to provide insights on differences among Cyanothece strains. By applying Sequedex, an annotation-independent method for ascribing gene functions, we confirmed significant species-specific differences of functional genes in different Cyanothece strains, particularly in Cyanothece PCC7425. Using a shotgun proteomics approach based on prefractionation and tandem mass spectrometry, we detected ∼28-48% of the theoretical Cyanothece proteome, depending on the strain. The expression of a total of 642 orthologous proteins was observed in all five Cyanothece strains. These shared orthologous proteins showed considerable correlations in their abundances across different Cyanothece strains. Functional classification indicated that the majority of proteins involved in central metabolic functions such as amino acid, carbohydrate, protein, and RNA metabolism, photosynthesis, respiration, and stress responses were observed to a greater extent in the core proteome, whereas proteins involved in membrane transport, iron acquisition, regulatory functions, flagellar motility, and chemotaxis were observed to a greater extent in the unique proteome. Considerable differences were evident across different Cyanothece strains. Notably, the analysis of Cyanothece PCC7425, which showed the highest number of unique proteins (682), provided direct evidence of evolutionary differences in this strain. We conclude that Cyanothece PCC7425 diverged significantly from the other Cyanothece strains or evolved from a different lineage.

Morphine produces immunosuppressive effects in nonhuman primates at the proteomic and cellular levels.

Morphine has long been known to have immunosuppressive properties in vivo, but the molecular and immunologic changes induced by it are incompletely understood. To explore how these changes interact with lentiviral infections in vivo, animals from two nonhuman primate species (African green monkeys and pigtailed macaques) were provided morphine and studied using a systems biology approach. Biological specimens were obtained from multiple sources (e.g. lymph node, colon, cerebrospinal fluid, and peripheral blood) before and after the administration of morphine (titrated up to a maximum dose of 5 mg/kg over a period of 20 days). Cellular immune, plasma cytokine, and proteome changes were measured and morphine-induced changes in these parameters were assessed on an interorgan, interindividual, and interspecies basis. In both species, morphine was associated with decreased levels of Ki-67(+) T-cell activation but with only minimal changes in overall T-cell counts, neutrophil counts, and NK cell counts. Although changes in T-cell maturation were observed, these varied across the various tissue/fluid compartments studied. Proteomic analysis revealed a morphine-induced suppressive effect in lymph nodes, with decreased abundance of protein mediators involved in the functional categories of energy metabolism, signaling, and maintenance of cell structure. These findings have direct relevance for understanding the impact of heroin addiction and the opioids used to treat addiction as well as on the potential interplay between opioid abuse and the immunological response to an infective agent.

Proteome analyses of strains ATCC 51142 and PCC 7822 of the diazotrophic cyanobacterium Cyanothece sp. under culture conditions resulting in enhanced H2 production.

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N(2)-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N(2)-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H(2)-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H(2) production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H(2) production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H(2) production.

Mass spectrometry-based proteomics: existing capabilities and future directions.

Mass spectrometry (MS)-based proteomics is emerging as a broadly effective means for identification, characterization, and quantification of proteins that are integral components of the processes essential for life. Characterization of proteins at the proteome and sub-proteome (e.g., the phosphoproteome, proteoglycome, or degradome/peptidome) levels provides a foundation for understanding fundamental aspects of biology. Emerging technologies such as ion mobility separations coupled with MS and microchip-based-proteome measurements combined with MS instrumentation and chromatographic separation techniques, such as nanoscale reversed phase liquid chromatography and capillary electrophoresis, show great promise for both broad undirected and targeted highly sensitive measurements. MS-based proteomics increasingly contribute to our understanding of the dynamics, interactions, and roles that proteins and peptides play, advancing our understanding of biology on a systems wide level for a wide range of applications including investigations of microbial communities, bioremediation, and human health.

Metabonomic profiling of TASTPM transgenic Alzheimer's disease mouse model.

Identification of molecular mechanisms underlying early stage Alzheimer's disease (AD) is important for the development of new therapies against and diagnosis of AD. In this study, nontargeted metabonomics of TASTPM transgenic AD mice was performed. The metabolic profiles of both brain and plasma of TASTPM mice were characterized using gas chromatography-mass spectrometry and compared to those of wild-type C57BL/6J mice. TASTPM mice were metabolically distinct compared to wild-type mice (Q2Y=0.587 and 0.766 for PLS-DA models derived from brain and plasma, respectively). A number of metabolites were found to be perturbed in TASTPM mice in both brain (D-fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D-galactose, linoleic acid, arachidonic acid, palmitic acid and D-gluconic acid). In addition, enzyme immunoassay confirmed that selected endogenous steroids were significantly perturbed in brain (androstenedione and 17-OH-progesterone) and plasma (cortisol and testosterone) of TASTPM mice. Ingenuity pathway analysis revealed that perturbations related to amino acid metabolism (brain), steroid biosynthesis (brain), linoleic acid metabolism (plasma) and energy metabolism (plasma) accounted for the differentiation of TASTPM and wild-type mice. Our results provided insights on the pathogenesis of APP-induced AD and reinforced the role of TASTPM in drug and biomarker development.

Cerebrospinal fluid proteome of patients with acute Lyme disease.

During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS), leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry-based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.

Conserved waters mediate structural and functional activation of family A (rhodopsin-like) G protein-coupled receptors.

G protein-coupled receptors with seven transmembrane alpha-helices (GPCRs) comprise the largest receptor superfamily and are involved in detecting a wide variety of extracellular stimuli. The availability of high-resolution crystal structures of five prototypical GPCRs, bovine and squid rhodopsin, engineered A(2A)-adenosine, beta(1)- and beta(2)-adrenergic receptors, permits comparative analysis of features common to these and likely all GPCRs. We provide an analysis of the distribution of water molecules in the transmembrane region of these GPCR structures and find conserved contacts with microdomains demonstrated to be involved in receptor activation. Colocalization of water molecules associating with highly conserved and functionally important residues in several of these GPCR crystal structures supports the notion that these waters are likely to be as important to proper receptor function as the conserved residues. Moreover, in the absence of large conformational changes in rhodopsin after photoactivation, we propose that ordered waters contribute to the functional plasticity needed to transmit activation signals from the retinal-binding pocket to the cytoplasmic face of rhodopsin and that fundamental features of the mechanism of activation, involving these conserved waters, are shared by many if not all family A receptors.

Structural waters define a functional channel mediating activation of the GPCR, rhodopsin.

Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H(2)O(18) solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.

Comparative analysis of GPCR crystal structures.

The phototransduction cascade is perhaps the best understood model system for G protein-coupled receptor (GPCR) signaling. Phototransduction links the absorption of a single photon of light to a decrease in cytosolic cGMP. Depletion of the cGMP pool induces closure of cGMP-gated cation channels resulting in the hyperpolarization of photoreceptor cells and consequently a neuronal response. Many biochemical and both low- and high-resolution structural approaches have been utilized to increase our understanding of rhodopsin, the key molecule of this signaling cascade. Rhodopsin, a member of the GPCR or seven-transmembrane spanning receptor superfamily, is composed of a chromophore, 11-cis-retinal that is covalently bound by a protonated Schiff base linkage to the apo-protein opsin at Lys(296) (in bovine opsin). Upon absorption of a photon, isomerization of the chromophore to an all-trans-retinylidene conformation induces changes in the rhodopsin structure, ultimately converting it from an inactive to an activated state. This state allows it to activate the heterotrimeric G protein, transducin, by triggering nucleotide exchange. To fully understand the structural and functional aspects of rhodopsin it is necessary to critically examine crystal structures of its different photointermediates. In this review we summarize recent progress on the structure and activation of rhodopsin in the context of other GPCR structures.

Different properties of the native and reconstituted heterotrimeric G protein transducin.

Visual signal transduction serves as one of the best understood G protein-coupled receptor signaling systems. Signaling is initiated when a photon strikes rhodopsin (Rho) causing a conformational change leading to productive interaction of this G protein-coupled receptor with the heterotrimeric G protein, transducin (Gt). Here we describe a new method for Gt purification from native bovine rod photoreceptor membranes without subunit dissociation caused by exposure to photoactivated rhodopsin (Rho*). Native electrophoresis followed by immunoblotting revealed that Gt purified by this method formed more stable heterotrimers and interacted more efficiently with membranes containing Rho* or its target, phosphodiesterase 6, than did Gt purified by a traditional method involving subunit dissociation and reconstitution in solution without membranes. Because these differences could result from selective extraction, we characterized the type and amount of posttranslational modifications on both purified native and reconstituted Gt preparations. Similar N-terminal acylation of the Gtalpha subunit was observed for both proteins as was farnesylation and methylation of the terminal Gtgamma subunit Cys residue. However, hydrogen/deuterium exchange experiments revealed less incorporation of deuterium into the Gtalpha and Gtbeta subunits of native Gt as compared to reconstituted Gt. These findings may indicate differences in conformation and heterotrimer complex formation between the two preparations or altered stability of the reconstituted Gt that assembles differently than the native protein. Therefore, Gt extracted and purified without subunit dissociation appears to be more appropriate for future studies.

Metarhodopsin-II stabilization by crosslinked Gtalpha C-terminal peptides and implications for the mechanism of GPCR-G protein coupling.

Metarhodopsin-II is the light-excited form of rhodopsin that triggers G protein function. Metarhodopsin-II is stabilized when the N-terminus of the carboxyl (340-350) tail peptide of the alpha-subunit of transducin (Gtalpha) is crosslinked to rhodopsin cysteine 140 or the 340-350 peptide C-terminus of Gtalpha is crosslinked to rhodopsin cysteine 316. When the N-terminus of the peptide is coupled to C316 the MI<-->MII equilibrium is not affected. The evidence suggests that the N-terminus of the 340-350 region of Gtalpha is located near C140 when transducin stabilizes metarhodopsin-II and alternative explanations are suggested for the effectiveness of the 340-350 Gtalpha tail peptide when bound to C316.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease.

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2-0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.

Approach to cerebrospinal fluid (CSF) biomarker discovery and evaluation in HIV infection.

Central nervous system (CNS) infection is a nearly universal facet of systemic HIV infection that varies in character and neurological consequences. While clinical staging and neuropsychological test performance have been helpful in evaluating patients, cerebrospinal fluid (CSF) biomarkers present a valuable and objective approach to more accurate diagnosis, assessment of treatment effects and understanding of evolving pathobiology. We review some lessons from our recent experience with CSF biomarker studies. We have used two approaches to biomarker analysis: targeted, hypothesis-driven and non-targeted exploratory discovery methods. We illustrate the first with data from a cross-sectional study of defined subject groups across the spectrum of systemic and CNS disease progression and the second with a longitudinal study of the CSF proteome in subjects initiating antiretroviral treatment. Both approaches can be useful and, indeed, complementary. The first is helpful in assessing known or hypothesized biomarkers while the second can identify novel biomarkers and point to broad interactions in pathogenesis. Common to both is the need for well-defined samples and subjects that span a spectrum of biological activity and biomarker concentrations. Previously-defined guide biomarkers of CNS infection, inflammation and neural injury are useful in categorizing samples for analysis and providing critical biological context for biomarker discovery studies. CSF biomarkers represent an underutilized but valuable approach to understanding the interactions of HIV and the CNS and to more objective diagnosis and assessment of disease activity. Both hypothesis-based and discovery methods can be useful in advancing the definition and use of these biomarkers.