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Staphylococcus aureusgamma-hemolysin CB (HlgCB) is a core-genome encoded pore-forming toxin that targets the C5a receptor, similarly as the phage-encoded Panton-Valentine Leucocidin. Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in HlgC and HlgB yields between isolates. Interestingly, when testing the hypothesis that HlgCB might be associated with severeS. aureusCAP, we found that a high level of HlgCB synthesis was associated with mortality in a rabbit model of pneumonia. To decipher the molecular basis for the variation inhlgCB andhlgB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. HlgCB amount and HlgC/HlgB ratio were found to both depend on promotor activity, mRNA stability and translatability, and on the presence of an individualhlgB mRNA processed from thehlgCB transcript. Strikingly, toe-printing andin vitrotranslation assays revealed that a single SNP in the 5’-UTR ofhlgCB mRNA strongly impairedhlgC translation in the USA300 strain, leading to a strong decrease in HlgC but not in HlgB; the level of HlgB is likely to have been maintained by the presence of the processedhlgB mRNA. This work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect, where subtle genomic variations have a major impact on phenotype and virulence.Author SummaryThe Gram-positive bacteriumStaphylococcus aureuscan provoke a wide range of infections due to its ability to produce a large diversity of virulence factors, including immune evasion molecules, adhesins, and toxins. Some of these toxin-encoding genes are localized in mobile genetic elements, and are thus not present in all strains, whilst others are encoded in the core-genome and present in all strains. Gamma-hemolysin CB is a core-genome encoded toxin but its amount varies between community-acquired pneumonia isolates. The regulation mechanisms underlying this variation however, are not well characterized. Here, we show that gamma-hemolysin expression levels vary largely among clinical strains and that, when highly produced, it induces high mortality in a rabbit model of pneumonia. The molecular basis for the variation in gamma-hemolysin expression depends on multiple mechanisms including promoter strength, transcript stability and processing, and translatability (i.e. the amount of protein that is synthetized by the ribosome for a given transcript). Incredibly, all these factors rely on a subtle genetic modification. This work emphasizes the importance of the disparity in virulence factor expression among clinical isolates and points the extreme complexity of the molecular mechanisms underlying their regulation, rendering the prediction of virulence for a clinical isolate difficult.

International audience; ObjectivesRheumatoid arthritis (RA) is a debilitating disease, but patient management and treatment have been revolutionized since the advent of bDMARDs. However, about one third of RA patients do not respond to specific bDMARD treatment without clear identified reasons. Different bDMARDs must be tried until the right drug is found. Here, we sought to identify a predictive protein signature to stratify patient responsiveness to rituximab (RTX) among patients with an insufficient response to a first anti-TNFα treatment.MethodsSerum samples were collected at baseline before RTX initiation. A proteomics study comparing responders and nonresponders was conducted to identify and select potential predictive biomarkers whose concentration was measured by quantitative assays. Logistic regression was performed to determine the best biomarker combination to predict good or nonresponse to RTX (EULAR criteria after 6 months’ treatment).ResultsEleven biomarkers potentially discriminating between responders and nonresponders were selected following discovery proteomics. Quantitative immunoassays and univariate statistical analysis showed that fetuin-A and thyroxine binding globulin (TBG) presented a good capacity to discriminate between patient groups. A logistic regression analysis revealed that the combination of fetuin-A plus TBG could accurately predict a patient’s responsiveness to RTX with an AUC of 0.86, sensitivity of 80%, and a specificity of 79%.ConclusionIn RA patients for whom a first anti-TNFα treatment has failed, the serum abundance of fetuin-A and TBG before initiating RTX treatment is an indicator for their response status at 6 months. ClinicalTrials.gov identifier: NCT01000441.

The roof plate-specific spondin-leucine-rich repeat-containing G-protein-coupled receptor 4/5 (Lgr4/5)-zinc and ring finger 3/ring finger protein 43 module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether hepatic epithelial cell-specific loss of the Wnt/β-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice on feeding with normal diet or high-fat diet (HFD). Livers of 6-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis compared with control mice. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with control mice. An ex vivo primary hepatocyte culture assay and a comprehensive BA characterization in liver, plasma, bile, and feces demonstrated that normal diet-fed Lgr4/5dLKO mice revealed impaired BA secretion, predisposing to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD.

International audience; The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.

Background & AimsThe Rspo-Lgr4/5-Znrf3/Rnf43 module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether liver-specific loss of the Wnt/β-catenin modulators Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4/5 (Lgr4/5) promotes nonalcoholic fatty liver disease (NAFLD).MethodsMice with liver-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were fed with normal diet (ND) or high fat diet (HFD). Livers of these mice were analyzed for lipid and fibrotic content by tissue staining and immunohistochemistry (IHC), and lipoproteins, inflammation and liver enzyme markers were measured in blood. Mechanistic insights into hepatic lipid accumulation were obtained by using ex vivo primary hepatocyte cultures derived from the Lgr4/5dLKO mice. Lipid analysis of mouse livers was performed by mass spectrometry (MS)-based untargeted lipidomic analysis.ResultsWe demonstrated that liver-specific ablation of Lgr4/5-mediated Wnt signaling resulted in hepatic steatosis, impaired bile acid (BA) secretion and predisposition to liver fibrosis. Under HFD conditions, we observed progressive intrahepatic fat accumulation, developing into macro-vesicular steatosis. Serum lipoprotein levels in HFD-fed Lgr4/5dLKO mice were decreased, rather than increased, suggesting that accumulation of fat in the liver was due to impaired lipid secretion by hepatocytes. Our lipidome analysis revealed a severe alteration of several lipid species in livers of Lgr4/5dLKO mice, including triacylglycerol estolides (TG-EST), a storage form of bioactive free fatty acid (FA) esters of hydroxy FAs (FAHFAs).ConclusionsLoss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to deregulation of lipoprotein pathways, loss of BA secretion, intrinsic alterations of lipid homeostasis and the onset of NAFLD.Lay summaryThe Wnt/β-catenin pathway plays an important role during development and tissue homeostasis. Loss of Wnt/β-catenin activity in mouse liver leads to loss of liver zonation, but the impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We show that livers of mice developed steatosis upon deletion of the positive pathway regulators Lgr4/5. Livers of knock-out (KO) mice exhibited altered lipid composition due to impaired lipid secretion. Furthermore, livers of these mice developed a nonalcoholic steatohepatitis (NASH)-like phenotype and fibrotic features derived from activated hepatic stellate cells. Our data demonstrate a protective role of Wnt/β-catenin pathway activity towards the development of NAFLD.HighlightsAbrogation of hepatic Wnt/β-catenin activity and liver zonation upon Lgr4/5 deletion in mice led to hepatic steatosis.Liver fat accumulation was caused by impaired lipid secretion from hepatocytes.Steatotic livers contained increased levels of diverse lipid species, including polyunsaturated fatty acids and triglycerol-estolides.These data confirmed that a decrease in Wnt/β-catenin signaling led to the development of nonalcoholic fatty liver disease (NAFLD) in mice.

Summary Many factors can influence results in clinical research, in particular bias in the distribution of samples prior to biochemical preparation. Well Plate Maker is a user-friendly application to design single- or multiple-well plate assays. It allows multiple group experiments to be randomized and therefore helps to reduce possible batch effects. Although primarily fathered to optimize the design of clinical sample analysis by high throughput mass spectrometry (e.g. proteomics or metabolomics), it includes multiple options to limit edge-of-plate effects, to incorporate control samples or to limit cross-contamination. It thus fits the constraints of many experimental fields. Availability and implementation Well Plate Maker is implemented in R and available at Bioconductor repository (https://bioconductor.org/packages/wpm) under the open source Artistic 2.0 license. In addition to classical scripting, it can be used through a graphical user interface, developed with Shiny technology.

Immunoassays have been used for decades in clinical laboratories to quantify proteins in serum and plasma samples. However, their limitations make them inappropriate in some cases. Recently, mass spectrometry (MS) based proteomics analysis has emerged as a promising alternative method when seeking to assess panels of protein biomarkers with a view to providing protein profiles to monitor health status. Up to now, however, translation of MS-based proteomics to the clinic has been hampered by its complexity and the substantial time and human resources necessary for sample preparation. Plasma matrix is particularly tricky to process as it contains more than 3000 proteins with concentrations spanning an extreme dynamic range (1010). To address this preanalytical challenge, we designed a microfluidic device (PepS) automating and accelerating blood sample preparation for bottom-up MS-based proteomics analysis. The microfluidic cartridge is operated through a dedicated compact instrument providing fully automated fluid processing and thermal control. In less than 2 h, the PepS device allows bedside plasma separation from whole blood, volume metering, depletion of albumin, protein digestion with trypsin, and stabilization of tryptic peptides on solid-phase extraction sorbent. For this first presentation, the performance of the PepS device was assessed using discovery proteomics and targeted proteomics, detecting a panel of three protein biomarkers routinely assayed in clinical laboratories (alanine aminotransferase 1, C-reactive protein, and myoglobin). This innovative microfluidic device and its associated instrumentation should help to streamline and simplify clinical proteomics studies.

Wilson disease (WD) is an autosomal recessive disorder of copper (Cu) metabolism. The gene responsible for WD, ATP7B, is involved in the cellular transport of Cu, and mutations in the ATP7B gene induce accumulation of Cu in the liver and ultimately in the brain. In a pilot study, the natural variations of copper stable isotope ratios (65Cu/63Cu) in the serum of WD patients have been shown to differ from that of healthy controls. In the present study, we challenged these first results by measuring the 65Cu/63Cu ratios in the blood of treated (n = 25), naïve patients (n = 11) and age matched healthy controls (n = 75). The results show that naïve patients and healthy controls exhibit undistinguishable 65Cu/63Cu ratios, implying that the Cu isotopic ratio cannot serve as a reliable diagnostic biomarker. The type of treatment (d-penicillamine vs. triethylenetetramine) does not affect the 65Cu/63Cu ratios in WD patients, which remain constant regardless of the type and duration of the treatment. In addition, the 65Cu/63Cu ratios do not vary in naïve patients after the onset of the treatment. However, the 65Cu/63Cu ratios decrease with the degree of liver fibrosis and the gradient of the phenotypic presentation, i.e. presymptomatic, hepatic and neurologic. To get insights into the mechanisms at work, we study the effects of the progress of the WD on the organism by measuring the Cu concentrations and the 65Cu/63Cu ratios in the liver, feces and plasma of 12 and 45 week old Atp7b−/− mice. The evolution of the 65Cu/63Cu ratios is marked by a decrease in all tissues. The results show that 63Cu accumulates in the liver preferentially to 65Cu due to the preferential cellular entry of 63Cu and the impairment of the 63Cu exit by ceruloplasmin. The hepatic accumulation of monovalent 63Cu+ is likely to fuel the production of free radicals, which is potentially an explanation of the pathogenicity of WD. Altogether, the results suggest that the blood 65Cu/63Cu ratio recapitulates WD progression and is a potential prognostic biomarker of WD.

Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson’s disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson’s disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.

© 2020 The Author(s)., [Introduction]: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. [Areas covered]: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. [Expert commentary]: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine., This paper was funded by the European cooperation in science and technology - COST action No. CA16113 - CliniMARK: ‘good biomarker practice’ to increase the number of clinically validated biomarkers.

Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b

Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b−/− mouse, a genetic and phenotypic model of WD, was developed to provide new insights into the pathogenic mechanisms of WD. Many plasma proteins are secreted by the liver, and impairment of liver function can trigger changes to the plasma proteome. High standard proteomics workflows can identify such changes. Here, we explored the plasma proteome of the Atp7b−/− mouse using a mass spectrometry (MS)-based proteomics workflow combining unbiased discovery analysis followed by targeted quantification. Among the 367 unique plasma proteins identified, 7 proteins were confirmed as differentially abundant between Atp7b−/− mice and wild-type littermates, and were directly linked to WD pathophysiology (regeneration of liver parenchyma, plasma iron depletion, etc.). We then adapted our targeted proteomics assay to quantify human orthologues of these proteins in plasma from copper-chelator-treated WD patients. The plasma proteome changes observed in the Atp7b−/− mouse were not confirmed in these samples, except for alpha-1 antichymotrypsin, levels of which were decreased in WD patients compared to healthy individuals. Plasma ceruloplasmin was investigated in both the Atp7b−/− mouse model and human patients; it was significantly decreased in the human form of WD only. In conclusion, MS-based proteomics is a method of choice to identify proteome changes in murine models of disrupted metal homeostasis, and allows their validation in human cohorts.

Rheumatoid arthritis (RA) is a debilitating disease, but patient management and treatment have been revolutionized since the advent of bDMARDs. However, about one third of RA patients do not respond to specific bDMARD treatment without clear identified reasons. Different bDMARDs must be tried until the right drug is found. Here, we sought to identify a predictive protein signature to stratify patient responsiveness to rituximab (RTX) among patients with an insufficient response to a first anti-TNFα treatment.Serum samples were collected at baseline before RTX initiation. A proteomics study comparing responders and nonresponders was conducted to identify and select potential predictive biomarkers whose concentration was measured by quantitative assays. Logistic regression was performed to determine the best biomarker combination to predict good or nonresponse to RTX (EULAR criteria after 6 months' treatment).Eleven biomarkers potentially discriminating between responders and nonresponders were selected following discovery proteomics. Quantitative immunoassays and univariate statistical analysis showed that fetuin-A and thyroxine binding globulin (TBG) presented a good capacity to discriminate between patient groups. A logistic regression analysis revealed that the combination of fetuin-A plus TBG could accurately predict a patient's responsiveness to RTX with an AUC of 0.86, sensitivity of 80%, and a specificity of 79%.In RA patients for whom a first anti-TNFα treatment has failed, the serum abundance of fetuin-A and TBG before initiating RTX treatment is an indicator for their response status at 6 months. ClinicalTrials.gov identifier: NCT01000441. Key Points • Proteomic analysis revealed 11 putative predictive biomarkers to discriminate rituximab responder vs. nonresponder RA patients. • Fetuin-A and TBG are significantly differentially expressed at baseline in rituximab responder vs. nonresponder RA patients. • Algorithm combining fetuin-A and TBG accurately predicts response to rituximab in RA patients with insufficient response to TNFi.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

In discovery proteomics experiments, tandem mass spectrometry and data-dependent acquisition (DDA) are classically used to identify and quantify peptides and proteins through database searching. This strategy suffers from known limitations such as under-sampling and lack of reproducibility of precursor ion selection in complex proteomics samples, leading to somewhat inconsistent analytical results across large datasets. Data-independent acquisition (DIA) based on fragmentation of all the precursors detected in predetermined isolation windows can potentially overcome this limitation. DIA promises reproducible peptide and protein quantification with deeper proteome coverage and fewer missing values than DDA strategies. This approach is particularly attractive in the field of clinical biomarker discovery, where large numbers of samples must be analyzed. Here, we describe a DIA workflow for non-depleted serum analysis including a straightforward approach through which to construct a dedicated spectral library, and indications on how to optimize chromatographic and mass spectrometry analytical methods to produce high-quality DIA data and results.

Secretome proteomics for the discovery of cancer biomarkers holds great potential to improve early cancer diagnosis. In this context, a knowledge‐based approach relying on mechanistic criteria related to the type of cancer should help to identify candidates from available “omics” information. Numerous bioinformatics tools, databases and “omics” datasets are available, but are often widely disseminated. In addition biomedical researchers with little programming experience or no in‐house bioinformatics support can find these tools difficult to access and use. With the aim of accelerating the discovery process for novel biomarkers, we have developed a set of tools we made available via a Galaxy‐based instance thereby providing a centralized access to a unified framework to assist end‐users biologists. These tools we implemented proceed by a step‐by‐step strategy to mine transcriptomics and proteomics databases for information relating to tissue‐specificity, allow the selection of proteins that are part of the secretome, and combine this information with proteomics datasets to rank the most promising candidate biomarkers for early cancer diagnosis. Using pancreatic cancer as a case study, this strategy produced a list of 24 candidate biomarkers suitable for experimental assessment by MS‐based proteomics. Among these proteins, three (SYCN, REG1B and PRSS2) were previously reported as circulating candidate biomarkers of pancreatic cancer. Further refinement of this list allowed us to prioritize 14 candidate biomarkers along with their associated proteotypic peptides for further investigation, using targeted MS‐based proteomics. The bioinformatics tools and the workflow implementing this strategy for the selection of candidate biomarkers are freely accessible at http://www.proteore.org for researchers who wish to reuse them in their own quests for biomarker discovery.

In discovery proteomics experiments, tandem mass spectrometry and data-dependent acquisition (DDA) are classically used to identify and quantify peptides and proteins through database searching. This strategy suffers from known limitations such as under-sampling and lack of reproducibility of precursor ion selection in complex proteomics samples, leading to somewhat inconsistent analytical results across large datasets. Data-independent acquisition (DIA) based on fragmentation of all the precursors detected in predetermined isolation windows can potentially overcome this limitation. DIA promises reproducible peptide and protein quantification with deeper proteome coverage and fewer missing values than DDA strategies. This approach is particularly attractive in the field of clinical biomarker discovery, where large numbers of samples must be analyzed. Here, we describe a DIA workflow for non-depleted serum analysis including a straightforward approach through which to construct a dedicated spectral library, and indications on how to optimize chromatographic and mass spectrometry analytical methods to produce high-quality DIA data and results.

Hepatocellular carcinoma (HCC) is recognized as the fifth most common neoplasm and currently represents the second leading form of cancer-related death worldwide. Despite great progress has been done in the understanding of its pathogenesis, HCC represents a heavy societal and economic burden as most patients are still diagnosed at advanced stages and the 5-year survival rate remain below 20%. Early detection and revolutionary therapies that rely on the discovery of new molecular biomarkers and therapeutic targets are therefore urgently needed to develop precision medicine strategies for a more efficient management of patients. Areas covered: This review intends to comprehensively analyse the proteomics-based research conducted in the last few years to address some of the principal still open riddles in HCC biology, based on the identification of molecular drivers of tumor progression and metastasis. Expert commentary: The technical advances in mass spectrometry experienced in the last decade have significantly improved the analytical capacity of proteome wide studies. Large-scale protein and protein variant (post-translational modifications) identification and quantification have allowed detailed dissections of molecular mechanisms underlying HCC progression and are already paving the way for the identification of clinically relevant proteins and the development of their use on patient care.

A major class of clinical biomarkers is constituted of intracellular proteins which are leaking into the blood following ischemia, exposure to toxic xenobiotics or mechanical aggression. Their ectopic presence in plasma/serum is an indicator of tissue damage and raises a warning signal. These proteins, referred to as cytolysis biomarkers, are generally of cytoplasmic origin and as such, are devoid of glycosylation. In contrast, most plasma/serum proteins originate from the hepatic secretory pathway and are heavily glycosylated (at the exception of albumin). Recent advances in targeted proteomics have supported the parallelized evaluation of new blood biomarkers. However, these analytical methods must be combined with prefractionation strategies that reduce the complexity of plasma/serum matrix. In this article, we present the glycodepletion method, which reverses the hydrazide-based glycocapture concept to remove plasma/serum glycoproteins from plasma/serum matrix and facilitates the detection of cytolysis biomarkers. Glycodepletion was integrated to a targeted proteomics pipeline to evaluate 4 liver cytolysis biomarker candidates in the context of acetaminophen-induced acute hepatitis.

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS.

There is a need for multiplex, specific and quantitative methods to speed-up the development of acute kidney injury biomarkers and allow a more specific diagnosis. Targeted proteomic analysis combined with stable isotope dilution has recently emerged as a powerful option for the parallelized evaluation of candidate biomarkers. This article presents the development of a targeted proteomic assay to quantify 4 acute kidney injury biomarker candidates in urine samples. The proteins included in the assessed panel consisted of myo-inositol oxygenase (MIOX), phosphoenolpyruvate carboxykinase 1 (PCK1), neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP). The proteomic assay combined an antibody-free sample preparation and a liquid chromatography-selected reaction monitoring (LC-SRM) analysis pipeline. For accurate quantification of the selected candidates, we used PSAQ (Protein Standard Absolute Quantification) standards which are isotopically labeled versions of the target proteins. When added directly to the biological samples, these standards improve detection specificity and quantification accuracy. The multiplexed assay developed for the 4 biomarker candidates showed excellent analytical performance, in line with the recommendations of health authorities. Tests on urine from two small patient cohorts and a group of healthy donors confirmed the relevance of NGAL and L-FABP as biomarkers for AKI diagnosis. The assay is readily adaptable to other biomarker candidates and should be very useful for the simultaneous and accurate quantification of multiple biomarkers.

Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.

The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical biomarker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies.

This paper presents an adaptive method which enables estimation of protein quantity present in complex mixtures (urine, plasma) analyzed by an LC-MS (Liquid Chromatography and Mass Spectrometry) system. The proposed approach is based on the inversion of a parametric state space model describing the chromatographic column behaviour. Efficiency of the algorithm is tested on synthetic and real dataset

Staphylococcal enterotoxins are major causing agents of food-borne diseases. Their detection in food remnants for risk assessment or food poisoning outbreaks investigation suffers from a lack in comprehensive immunological tools. In this study, we demonstrate that the combination of immunocapture and Protein Standard Absolute Quantification (PSAQ) strategy, which uses isotope-labeled enterotoxins as internal standards for MS-based analysis, is powerful to specifically identify and quantify these contaminating agents in food matrices. This approach is believed to significantly improve the elucidation of staphylococcal food poisoning outbreaks.

Au Mexique, l’ouverture commerciale et la liberalisation des marches agricoles posent la question du devenir de l’agriculture paysanne et de son insertion competitive sur les marches. Cette question est traitee ici a partir d’une etude de cas dans des ejidos du sud-Veracruz, ou l’intensification recente des systemes de production cerealiers s’est accompagnee d’un processus d’integration marchande des exploitations pilote par le secteur prive. Il s’agit de rendre compte des mecanismes ayant permis le passage de systemes largement tournes vers l’autoconsommation a des systemes intensifs a vocation commerciale, en insistant sur les roles majeurs joues par l’Etat dans l’incitation au rapprochement des petits producteurs avec le secteur prive. L’analyse s’appuie sur des enquetes realisees dans les ejidos des terres basses du fleuve Tesechoacan, a proximite de la ville d’Isla. La dynamique d’evolution des systemes de production est analysee de facon croisee avec les changements intervenus dans le domaine de l’intervention publique d’une part, et les transformations institutionnelles qui l’ont accompagnee d’autre part (organisation paysanne, integration marchande).

Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.

The development of rapid methods for unambiguous identification and precise quantification of protein toxins in various matrices is essential for public health surveillance. Nowadays, analytical strategies classically rely on sensitive immunological assays, but mass spectrometry constitutes an attractive complementary approach thanks to direct measurement and protein characterization ability. We developed here an innovative multiplex immuno-LC-MS/MS method for the simultaneous and specific quantification of the three potential biological warfare agents, ricin, staphylococcal enterotoxin B, and epsilon toxin, in complex human biofluids and food matrices. At least 7 peptides were targeted for each toxin (43 peptides in total) with a quadrupole-Orbitrap high-resolution instrument for exquisite detection specificity. Quantification was performed using stable isotope-labeled toxin standards spiked early in the sample. Lower limits of quantification were determined at or close to 1 ng·mL(-1). The whole process was successfully applied to the quantitative analysis of toxins in complex samples such as milk, human urine, and plasma. Finally, we report new data on toxin stability with no evidence of toxin degradation in milk in a 48 h time frame, allowing relevant quantitative toxin analysis for samples collected in this time range.

Background: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. Materials & methods: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. Results: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.

The preparation of 6-chloro-2-iodo-9-tetrahydropyranylpurine ( 2 ), was achieved in three high yield steps from 6-chloropurine. This derivative was then selectively substituted at C-6 by reaction with a benzylthiol to give 3 , a versatile intermediate for the synthesis of 2,6,9-trisubstituted purines. Reaction of 3 in palladium-catalyzed cross-coupling reactions, (including Sonogashira coupling at room temperature), as well as nucleophilic substitutions with amines occurred selectively at C-2. The 6-thiobenzyl substituent was activated through oxidation to the corresponding sulfone and replaced by various benzyl or phenyl amines. This strategy was subsequently adapted to solid support, wherein 23 is connected to Merrifield resin via a 6-thiovaleric ester linker. The presence of the linker, in combination with the use of palladacycle type catalysts improved the yield of palladium(0)-catalyzed Suzuki and Sonogashira cross-coupling reactions. This strategy opens a new route to combinatorial chemistry library synthesis of trisubstituted purines on the solid support.

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.

To validate a proposed solid support synthesis strategy for the construction of 2,6,9-trisubstituted purine based CDK inhibitors, the N-9 THP protected 6-benzylthio-2-iodopurine 11 was reacted with piperidine-2-methanol to give 12 . Alternatively, intermediate 11 was converted to the C-2 acetylenyl substituted purine 16 in five steps, involving N-9 alkylation (Mitsunobu reaction), a Pd(0)–CuI-catalyzed acetylene coupling, selective activation of the 6-sulfur substituent and its displacement by ArCH2NH2.

(−)-Mesembrine 11 was synthesized in 7 steps, in 19.4% overall yield and with ee>95%. The key step of the sequence is a stereoselective alkylation reaction of dianion derived from C2 symmetric imidazolines allowing efficient formation of quaternary benzylic center. Chiral auxiliaries derived from (S,S)-1,2-diamino-1,2-diphenylethane 1a and (S,S)-1,2-diaminocyclohexane 1b were compared. This synthesis provided unambiguous correlation of the newly formed stereocenter.

International audience; The egcSEs comprise five genetically linked staphylococcal enterotoxins, SEG, SEI, SElM, SElN, and SElO and two pseudotoxins which constitute an operon present in up to 80% of Staphylococcus aureus isolates. A preparation containing these proteins was recently used to treat advanced lung cancer with pleural effusion. We investigated the hypothesis that egcSEs induce nitrous oxide (NO) and associated cytokine production and that these agents may be involved in tumoricidal effects against a broad panel of clinically relevant human tumor cells. Preliminary studies showed that egcSEs and SEA activated T cells (range: 11-25%) in a concentration dependent manner. Peripheral blood mononuclear cells (PBMCs) stimulated with equimolar quantities of egcSEs expressed NO synthase and generated robust levels of nitrite (range: 200-250 μM), a breakdown product of NO; this reaction was inhibited by NG-monomethyl-L-arginine (L-NMMA) (0.3 mM), an NO synthase antagonist. Cell free supernatants (CSFs) of all egcSE-stimulated PBMCs were also equally effective in inducing concentration dependent tumor cell apoptosis in a broad panel of human tumor cells. The latter effect was due in part to the generation of NO and TNF-α since it was significantly abolished by L-NMMA, anti-TNF-α antibodies, respectively, and a combination thereof. A hierarchy of tumor cell sensitivity to these CFSs was as follows: lung carcinoma > osteogenic sarcoma > melanoma > breast carcinoma >neuroblastoma. Notably, SEG induced robust activation of NO/TNFα-dependent tumor cell apoptosis comparable to the other egcSEs and SEA despite TNF-α and IFN-γ levels that were 2 and 8 fold lower, respectively, than the other egcSEs and SEA. Thus, egcSEs produced by S. aureus induce NO synthase and the increased NO formation together with TNF-α appear to contribute to egcSE-mediated apoptosis against a broad panel of human tumor cells.

Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable-isotope-labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ™) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here.

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.

Depuis le milieu des annees 1990, le Mexique est engage dans un processus d’insertion competitive qui met en question le devenir des petites exploitations familiales composant la trame de son secteur agricole. Sur la base d’une enquete realisee aupres de 320 menages ruraux, cet article explore les formes de reconfiguration des organisations economiques familiales a partir d’une analyse de leurs revenus. Il en examine la structure et la distribution en reference a l’hypothese d’evolution trimodale des formes d’insertion dans les marches proposee par le Rapport sur le developpement dans le monde 2008 de la Banque mondiale : la specialisation agricole via l’insertion dans les chaines de valeur agro-industrielles ; la diversification des activites rurales agricoles et non agricoles ; et la sortie de l’agriculture par la migration. L’article s’attache au role des programmes publics d’appui aux menages ruraux dans la reorganisation des structures d’activite, ainsi que dans les dynamiques de differenciation socio-spatiale associees au processus d’insertion competitive.

Depuis le debut des annees 1990, l’agriculture mexicaine connait de profondes restructurations qui s’inscrivent dans un ensemble de reformes visant a accelerer l’insertion competitive des exploitations sur les marches agricoles, notamment dans le cadre de l’Accord de libre-echange nord-americain liant le Mexique, les Etats-Unis et le Canada depuis le 1er janvier 1994. Dans le domaine foncier, la revision, en 1991, de l’article 27 de la Constitution mexicaine a signifie la fin des redistributions foncieres – a l’origine de la formation des ejidos –, et la reintegration aux marches fonciers des terres distribuees dans le cadre de la reforme agraire (autorisation de leur cession definitive et/ou temporaire et de leur mise en gage). En instaurant par ailleurs un cadre institutionnel propice a l’emergence de marches fonciers dans les ejidos via un important programme de titrage individuel des droits fonciers, la reforme devait contribuer a la mise en place de nouvelles dynamiques productives, en forcant la reallocation des ressources productives, notamment la terre, vers les structures les plus competitives. Les concentrations foncieres aux mains des plus « productifs » sont donc desormais possibles et meme souhaitees, et cela, en totale contradiction avec le Code agraire qui interdit toujours la grande propriete au Mexique. Les questions foncieres liees aux transformations productives et au changement institutionnel sont traitees ici a partir d’une etude de cas conduite dans differents ejidos des terres basses du Sud-Veracruz (bassins du Tesechoacan et du Moyen-Coatzacoalcos), ou l’on observe depuis le milieu des annees 1990 un developpement important de la production mecanisee de mais et de sorgho destinee aux marches. Il s’agit notamment, apres la revision constitutionnelle de 1991, de dresser un etat des lieux des dynamiques foncieres liees a la diffusion de la cerealiculture commerciale, lesquelles se manifestent par un accroissement marque de la demande sur les marches fonciers internes et externes aux ejidos. Il s’agit aussi de montrer comment l’acces des petits producteurs aux differentes ressources productives (terre, intrants et capital technique) s’organise aujourd’hui a differentes echelles, au sein des ejidos d’une part, mais egalement autour de l’intervention de nouveaux acteurs issus du secteur prive (agro-industriels, entrepreneurs et operateurs agricoles, fournisseurs d’intrants et de services, etc.). Que ces derniers prennent part ou non a un recent phenomene d’acquisitions foncieres – qui n’a toutefois ni l’ampleur ni les caracteristiques des grandes appropriations foncieres ayant actuellement lieu dans certains pays du Sud –, ils jouent un role de plus en plus central dans les strategies d’expansion des petites exploitations familiales et dans les processus locaux d’integration commerciale. Dans le cas du bassin du Tesechoacan, le modele d’integration impulse par les acteurs prives s’apparente a une forme d’agriculture sous contrat (sur le modele developpe par l’agrobusiness), tandis que dans celui du Moyen-Coatzacoalcos, le schema fonctionne avant tout sur la base de relations personnalisees de type clienteliste. Dans les deux cas toutefois, les logiques d’intervention de ces nouveaux acteurs prives s’appuient sur un controle accru des exploitations familiales, tant en amont (acces aux ressources productives) qu’en aval (mise en marche) de la production. Par ailleurs, la presence du secteur prive beneficie en premier lieu a une categorie emergente d’ejidataires « capitalises », dont le rapprochement avec ces nouveaux acteurs se construit autour de la prise de terres en location. L’une des consequences est une differenciation socio-economique acceleree au sein des ejidos. Au final, l’analyse des processus productifs en cours dans les terres basses du Sud-Veracruz permet de mettre en evidence une tendance a l’accumulation et a la concentration de terres, aux mains d’une categorie minoritaire d’ejidataires bien dotes en capital technique d’une part, et d’entrepreneurs prives controlant les acces aux ressources productives et aux marches d’autre part.

Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.

In the field of analytical chemistry, stable isotope dilution assays are extensively used in combination with liquid chromatography-mass spectrometry (LC-MS) to provide confident quantification results. Over the last decade, the principle of isotope dilution has been adopted by the proteomic community in order to accurately quantify proteins in biological samples. In these experiments, a protein's concentration is deduced from the ratio between the MS signal of a tryptic peptide and that of a stable isotope-labeled analog, which serves as an internal standard. The first isotope dilution standards introduced in proteomics were chemically synthesized peptides incorporating a stable isotope-tagged amino acid. These isotopically labeled peptide standards, which are currently widely used, are generally added to samples after protein isolation and digestion. Thus, if protein enrichment is necessary, they do not allow correction for protein losses that may occur during sample pre-fractionation, nor do they allow the tryptic digestion yield to be taken into account. To reduce these limitations we have developed the PSAQ (Protein Standard Absolute Quantification) strategy using full-length stable isotope-labeled proteins as quantification standards. These standards and the target proteins share identical biochemical properties. This allows standards to be spiked into samples at an early stage of the analytical process. Thanks to this possibility, the PSAQ method provides highly accurate quantification results, including for samples requiring extensive biochemical pre-fractionation. In this chapter, we describe the production of full-length stable isotope-labeled proteins (PSAQ standards) using cell-free expression devices. The purification and quality control of protein standards, crucial for good-quality and accurate measurements, are also detailed. Finally, application of the PSAQ method to a typical protein quantification assay is presented.

In the field of analytical chemistry, stable isotope dilution assays are extensively used in combination with liquid chromatography-mass spectrometry (LC-MS) to provide confident quantification results. Over the last decade, the principle of isotope dilution has been adopted by the proteomic community in order to accurately quantify proteins in biological samples. In these experiments, a protein's concentration is deduced from the ratio between the MS signal of a tryptic peptide and that of a stable isotope-labeled analog, which serves as an internal standard. The first isotope dilution standards introduced in proteomics were chemically synthesized peptides incorporating a stable isotope-tagged amino acid. These isotopically labeled peptide standards, which are currently widely used, are generally added to samples after protein isolation and digestion. Thus, if protein enrichment is necessary, they do not allow correction for protein losses that may occur during sample pre-fractionation, nor do they allow the tryptic digestion yield to be taken into account. To reduce these limitations we have developed the PSAQ (Protein Standard Absolute Quantification) strategy using full-length stable isotope-labeled proteins as quantification standards. These standards and the target proteins share identical biochemical properties. This allows standards to be spiked into samples at an early stage of the analytical process. Thanks to this possibility, the PSAQ method provides highly accurate quantification results, including for samples requiring extensive biochemical pre-fractionation. In this chapter, we describe the production of full-length stable isotope-labeled proteins (PSAQ standards) using cell-free expression devices. The purification and quality control of protein standards, crucial for good-quality and accurate measurements, are also detailed. Finally, application of the PSAQ method to a typical protein quantification assay is presented.

Purine bound resins 1a – c were obtained by the reaction of 6-thiopurines 2 or 6-chloropurines 3 – 5 with Merrifield-Cl or -SH resins (DMF, 70°C, base). S -Oxidation of resins 1c and reaction of the desired sulfone with p -methoxybenzylamine to give 6 , proved effective for release of the purine from the resin and simultaneous C-6 substitution. Reaction of resin 1c with pyrrolidine and pyrrolidine-2-methanol prior to S -oxidation led to the C-2 amine substituted resin bound purines 8 and 9 . Activation of sulfur in these intermediates, followed by reaction with Ar–CH 2 –NH 2 gave the 2,6,9-trisubstituted purines 10 – 14 in 42–60% yields.

Experiments to effect Sonogashira (Ph 3 P) 2 Cl 2 Pd–CuI-based coupling of 3-methylpentyn-3-ol at C-2 of a 2-iodo-6-thiopurine derivative, bound via the sulfur atom to a Merrifield resin failed. In contrast, the analogous reaction with Pd(dppe)Cl 2 and trans-di(μ-acetato)bis[ o -(di- o -tolylphosphino)benzyl]dipalladium catalysts were successful (20–25%), indicating that (Ph 3 P) 2 Pd(0) loses its ligands irreversibly on contact with the resin bound purine. The coupling yield was improved considerably (58%) using a Merrifield resin in which a valeric acid linker is interposed between the purine and the resin.

To validate a proposed solid support synthesis strategy for the construction of 2,6,9-trisubstituted purine based CDK inhibitors, the N-9 THP protected 6-benzylthio-2-iodopurine 11 was reacted with piperidine-2-methanol to give 12 . Alternatively, intermediate 11 was converted to the C-2 acetylenyl substituted purine 16 in five steps, involving N-9 alkylation (Mitsunobu reaction), a Pd(0)–CuI-catalyzed acetylene coupling, selective activation of the 6-sulfur substituent and its displacement by ArCH2NH2.