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Intramuscular long-acting antiretroviral drugs can improve adherence to lifelong antiretroviral treatment. Nevertheless, adipose tissue thickness and distribution play a critical role with injectable drugs. We describe a virological failure with cabotegravir and rilpivirine in a Black African woman with human immunodeficiency virus type 1 with gynoid fat distribution (ie, adipose tissue prevailing in the pelvis and hip area) and body mass index

Custom pathway and substrate-kinase libraries for tumor-specific pathway enrichment analyses.

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry–based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled internal standard peptides to guide data acquisition of low-abundance tyrosine phosphopeptides. SureQuant pTyr allowed for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, improving the robustness and usability of targeted mass spectrometry assays. We established the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient-specific oncogenic-driving mechanisms. While in some cases pTyr profiles aligned with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlighted instances of new insights gained using pTyr characterization and emphasized the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enables pTyr-based measurements for applications in precision medicine.Significance:SureQuant pTyr is a mass spectrometry–based targeted method that enables sensitive and selective targeted quantitation of several hundred low-abundance tyrosine phosphorylated peptides commonly dysregulated in cancer, including oncogenic signaling networks.

Supplementary figures, table legends, and methods.

IMPORTANCE: Patients with solid cancer are more susceptible to develop SARS-CoV-2 infection and severe complications; the immunogenicity in patients treated with anticancer agents remains unknown. OBJECTIVE: To assess the immune humoral response to 2 or 3 doses of the BNT162b2 (BioNTech; Pfizer) vaccine in patients treated with anticancer agents. DESIGN, SETTING, AND PARTICIPANTS: A prospective observational cohort study was conducted between February 1 and May 31, 2021. Adults treated with anticancer agents who received 2 or 3 doses of vaccine were included; of these, individuals with a weak humoral response 1 month after the second dose received a third injection. INTERVENTIONS: Quantitative serologic testing of antibodies specific for SARS-CoV-2 was conducted before vaccination and during follow-up. MAIN OUTCOMES AND MEASURES: Humoral response was evaluated with a threshold of anti–SARS-CoV-2 spike protein antibody levels at 1000 arbitrary units (AU)/mL to neutralize less-sensitive COVID-19 variants. RESULTS: Among 163 patients (median [range] age, 66 [27-89] years, 86 men [53%]) with solid tumors who received 2 or 3 doses of vaccine, 122 individuals (75%) were treated with chemotherapy, 15 with immunotherapy (9%), and 26 with targeted therapies (16%). The proportions of patients with an anti-S immunoglobulin G titer greater than 1000 AU/mL were 15% (22 of 145) at the time of the second vaccination and 65% (92 of 142) 28 days after the second vaccination. Humoral response decreased 3 months after the second dose. Treatment type was associated with humoral response; in particular, time between vaccine and chemotherapy did not interfere with the humoral response. Among 36 patients receiving a third dose of vaccine, a serologic response greater than 1000 AU/mL occurred in 27 individuals (75%). CONCLUSIONS AND RELEVANCE: The results of this cohort study appear to support the use of a third vaccine dose among patients with active cancer treatment for solid tumors.

Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.

Objectives We assessed virological outcomes of rilpivirine use in France from 2012 to 2017, in three groups of people living with HIV (PLHIV): (i) antiretroviral (ARV)-naive PLHIV; (ii) ARV-experienced PLHIV switching to rilpivirine while failing therapy; and (iii) ARV-experienced PLHIV switching to rilpivirine while virologically controlled. Methods Virological success (VS) was defined as a plasma HIV-1 viral load (VL) 50 copies/mL or one VL >50 copies/mL followed by a treatment switch prior to the next VL measurement. The cumulative incidence of VS was assessed considering rilpivirine discontinuation, loss to follow-up and death as competing risks, while estimates of cumulative incidence of VF accounted for loss to follow-up and death. Results Among the 2166 ARV-naive PLHIV initiating rilpivirine, the 4 year cumulative incidence of VS was 91.0% and was associated with baseline VL. Among the 2125 ARV-experienced PLHIV switching to rilpivirine while failing therapy, the 4 year cumulative incidence of VS was 82.5% and was associated with lower VL, higher CD4 and less than three prior ARVs. Among the 11 828 ARV-experienced PLHIV switching to rilpivirine while virologically controlled, the 4 year cumulative incidence of VF was 9.6%. The risk of VF was lower among MSM, for PLHIV with CD4 ≥ 500 cell/mm3, without a prior AIDS event, or with a longer VL suppression at baseline. Conclusions Rilpivirine-containing regimens yielded high rates of viral suppression in most participants, while it was ineffective when used outside the marketing authorization in naive participants.

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, enhances target detection success rates, and improves the robustness and usability of targeted acquisition. We establish the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient specific oncogenic driving mechanisms. While in some cases pTyr profiles align with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlight instances of new insights gained using pTyr characterization and emphasize the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enable pTyr-based measurements for applications in precision medicine.SummaryA targeted, mass spectrometry-based method, termed “SureQuant pTyr,” enables highly sensitive and reproducible profiling of tyrosine phosphorylation levels in human colorectal tumors and reveals dysregulated signaling networks for enhanced tumor characterization and biomarker identification.

International audience; Objectives:To assess whether low-dose ritonavir-boosted darunavir (darunavir/r) in combination with two NRTIs could maintain virological suppression in patients on a standard regimen of darunavir/r + two NRTIs.Design:A multicentre, Phase II, non-comparative, single-arm, open-label study.Setting:Tertiary care hospitals in France.Subjects:One hundred HIV-1-infected adults with no darunavir or NRTI resistance-associated mutations (RAMs) and a plasma HIV RNA level ≤50 copies/mL for ≥12 months on once-daily darunavir/r (800/100 mg) + two NRTIs for ≥6 months were switched to darunavir/r 400/100 mg with the same NRTIs.Primary outcome measure:Proportion of patients with treatment success: plasma HIV RNA level ≤50 copies/mL up to 48 weeks without any change in the study regimen, in a modified ITT (mITT) analysis.Results:At baseline, most patients were male (78%), with a median age of 43 years, median duration of HIV RNA ≤50 copies/mL of 35 months and median CD4 T cell count of 633 cells/mm3. Seventy-six percent received tenofovir/emtricitabine and 24% abacavir/lamivudine. Five patients were excluded from the mITT analysis. The rate of treatment success through to week 48 was 91.6% (87/95; 95% CI 84.1%-96.3%). No RAM was detected in three amplifiable genotypes. A total of 212 adverse events (AEs) occurred in 64 patients (64%); 9 AEs were serious, none leading to treatment discontinuation.Conclusions:In HIV-infected patients well suppressed with darunavir/r (800/100 mg) and two NRTIs, a reduction of the darunavir dose to 400 mg/day maintained virological efficacy and was safe over 48 weeks.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrated robust, sensitive, and reproducible data generation across eleven sites in nine countries on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.

Targeted analyses performed on high-resolution accurate mass spectrometers hyphenated to liquid chromatographs have opened new opportunities in quantitative proteomics. A new extension of the parallel reaction monitoring (PRM) technique uses the internal standards added to samples to dynamically control the data acquisition and maximize the analysis of the endogenous peptides. This account describes the hyphenation of ultra-HPLC with a high acquisition rate quadrupole-orbitrap instrument to properly sample the sharp elution profiles of UHPLC separations, possible with the IS-PRM technique.

Targeted mass spectrometry-based approaches are nowadays widely used for quantitative proteomics studies and more recently have been implemented on high resolution/accurate mass (HRAM) instruments resulting in a considerable performance improvement. More specifically, the parallel reaction monitoring technique (PRM) performed on quadrupole-Orbitrap mass spectrometers, leveraging the high resolution and trapping capabilities of the instrument, offers a clear advantage over the conventional selected reaction monitoring (SRM) measurements executed on triple quadrupole instruments. Analyses performed in HRAM mode allow for an improved discrimination between signals derived from analytes and those resulting from matrix interferences translating in the reliable quantification of low abundance components. The purpose of the study defines various implementation schemes of PRM, namely: (i) exploratory experiments assessing the detectability of very large sets of peptides (100-1000), (ii) wide-screen analyses using (crude) internal standards to obtain statistically meaningful (relative) quantitative analyses, and (iii) precise/accurate quantification of a limited number of analytes using calibrated internal standards. Each of the three implementation schemes requires specific acquisition methods with defined parameters to appropriately control the acquisition during the actual peptide elution. This tutorial describes the different PRM approaches and discusses their benefits and limitations in terms of quantification performance and confidence in analyte identification.

International audience; A key concern for reduced-dose antiretroviral regimens in maintenance strategies in HIV-infected patients is virological efficacy, as assessed by the suppression of HIV-RNA in blood and reservoirs, such as the genital tract. The risk of minority variants emerging in reservoirs with different antiretroviral drug penetrations and immune pressures is also a matter of concern [1–4]. Thus, antiviral activity and drug penetration into deep compartments must be considered in reduced-dose strategies.

Blood cells and plasma are important media for the four serotypes of dengue virus (DENV1-4) spreading into an infected person. Thus, interactions with human plasma proteins are expected to be decisive in the course of the viral infection. Affinity purification followed by MS analysis (AP/MS) was used to isolate and identify plasma-derived proteins capable to interact with a recombinant protein comprising the domain III of the envelope protein of DENV2 (DIIIE2). The elution of the AP potently inhibits DENV2 infection. Twenty-nine proteins were identified using a label-free approach as specifically captured by DIIIE2. Of these, a direct interaction with C reactive protein, thrombin and Inter-alpha-inhibitor complexes was confirmed by ELISA. Results provide further evidence of a significant representation of proteins from complement and coagulation cascades on DENV2 interactome in human plasma and stand out the domain III of the viral envelope protein as participant on these interactions. A functional clustering analysis highlights the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. Biological significance Early cycles of dengue virus replication take place in human blood cells. Thus, the characterization of the interactome of dengue virus proteins in human plasma can lead to the identification of pivotal interactions for the infection that can eventually constitute the target for the development of methods to control dengue virus-caused disease. In this work we identified 29 proteins from human plasma that potentially interact with the envelope protein of dengue 2 virus either directly or through co-complex formation. C reactive protein, thrombin and Inter-alpha-inhibitor complexes were validated as interactors of the domain III of the envelope protein of dengue 2. Results highlight the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. This finding together with the participation of domain III of the envelope protein on the interactions with human plasma proteins should contribute to a better understanding of dengue virus interactome in human plasma. Such knowledge can contribute to the development of more effective treatments to infected persons.

The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.

The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measurement and fast acquisition, have enabled new approaches for targeted quantitative proteomics. More specifically, analyses performed on quadrupole-orbitrap mass spectrometers operated in parallel reaction monitoring (PRM) mode leverage the intrinsic high resolving power and trapping capabilities. The PRM technique offers unmatched degrees of selectivity and analytical sensitivity, typically required to analyze peptides in complex samples, such as those encountered in biomedical research or clinical studies. The features of PRM have provoked a paradigm change in targeted experiments, by decoupling acquisition and data processing. It has resulted in a new analytical workflow comprising distinct methods for each step, thus enabling much larger flexibility. The PRM technique was further enhanced by a new data acquisition scheme, allowing dynamic parameter settings. The potential of the technique may radically impact future quantitative proteomics studies.

First-line raltegravir-containing combination antiretroviral therapy in 30 patients living with HIV-2 was well tolerated and yielded a median CD4 gain at 1 year of 87 cells/µL, and HIV-2 RNA, Background New options for first-line treatment of human immunodeficiency virus type 2 (HIV-2) infection are needed. We evaluated an integrase inhibitor (raltegravir)–containing regimen. Methods Antiretroviral therapy (ART)–naive adults with symptomatic infection by HIV-2 only, CD4 count 50 cells/μL/year over the past 3 years, or a confirmed plasma HIV-2 RNA (pVL) load ≥100 copies/mL were eligible for this noncomparative trial. The composite primary endpoint was survival at 48 weeks without any of the following: CD4 gain from baseline 6 copies by PCR in 32%. At week 48, the composite endpoint of success was reached in 40% [95% confidence interval, 22.7%–59.4%]. Failure was mainly (50%) due to CD4 gain 6 copies by PCR in 12% of participants. Median CD4 gain was 87 cells/µL (IQR, 38–213 cells/µL; n = 28). No serious adverse reactions were reported. Conclusions Raltegravir-containing ART is a safe option for first-line treatment of HIV-2 infection, yielding a comparable success rate to protease inhibitors. Clinical Trials Registration NCT 01605890.

The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the “on-matrix” digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. Significance False negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix (“on-matrix” digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by the domain III of the viral envelope glycoprotein. Fifty nine proteins were identified as putative interaction partners of Dengue virus (IPs) either due to direct binding or by co-isolation with interacting proteins. Collectively the IPs identified from the pull-down with the recombinant domain III proteins representing the four viral serotypes, 29% were identified only after “on-matrix” digestion which demonstrate the usefulness of this method of recovering bait-bound proteins. Results highlight a particular importance of “on-matrix” digestion procedure for comparative studies where a stronger interaction with one of the interest baits could prevent a bound protein to elute under standard conditions thus leading to misinterpretation as absent in the interactome of this particular bait. The analysis of the Interaction Network indicates that Dengue virus interactome mediated by the domain III of the envelope protein is rather conserved in the viral complex suggesting a key role of these interactions for viral infection thus making candidates to explore for potential biomarkers of clinical outcome in DENV-caused disease. Interestingly, some particular IPs exhibit significant differences in the strength of the interaction with the viral serotypes representing interactions that involve more variable regions in the surface of the domain III. Since such variable regions are the consequence of the interaction with antibodies generated by human immune response; this result relates the interaction with proteins from human plasma with the interplay of the virus and the human immune system.

Mass-spectrometry-based proteomic approaches are increasingly applied to biological and clinical studies. Initially used by specialized laboratories, the technology has matured and gained acceptance by the community, using various analytical processes and platforms. To facilitate data comparison and integration across laboratories, there is a need to harmonize analytical processes to ensure the generation of reliable proteomic data sets. This is especially critical in the context of large initiatives, such as the Human Proteome Project promoted by the Human Proteome Organization (HUPO). Quality control is a first step toward the harmonization of proteomics data sets. We have developed a procedure to routinely assess the uniformity of proteomics analyses. It relies on a simple protocol based on three proteins and two sets of isotopically labeled peptides, one being added prior to tryptic digestion and the second one prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The proposed method evaluates in a single step both the sample preparation, by measuring the relative amounts of endogenous peptides and their isotopically labeled counterparts, and the LC-MS platform performance, by monitoring the main LC-MS attributes for reference peptides. The procedure is simple and easy to implement into routine workflows typically employed by the proteomics community.

The evaluation of biomarker candidates, involving quantitative measurement of a large number of proteins in bodily fluids, remains the main obstruction in the development of a biomarker validation pipeline. Although immunoassays are commonly used, high-throughput and multiplex-capable methods are required for expediting the evaluation process. MS-based approaches employing targeted proteomic strategies provide not only a sensitive, but in addition a precise quantification tool, which is versatile, systematic, and scalable. Its capability of multiplexing hundreds of targets facilitate a cost-effective and rapid evaluation and is especially useful during the early stage of the process where a large list of candidate biomarkers must be triaged before entering validation studies. The robustness requirement for the methods also mandates a high degree of selectivity to analyze complex clinical samples. Improvement in the selectivity of LC-MS methods has been achieved by adopting high-resolution and high-accuracy mass analyzers to perform quantitative analyses with a novel method called parallel reaction monitoring. This article discusses the design and performance of biomarker evaluation studies using targeted proteomics strategies and the implementation of recent technology developments.

The recent development of hybrid mass spectrometers with high resolution and accurate mass capabilities has opened new avenues in quantitative proteomics. A systematic study was performed to assess the quantification performances of a novel quadrupole-Orbitrap instrument operated in MS/MS mode (parallel reaction monitoring). It included the analyses of 35 isotopically labeled peptides spiked in urine samples to establish their dilution curves. The results were evaluated by replicating the analyses on a triple quadrupole instrument operated in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode to assess and compare the gain in selectivity resulting from high resolution fragment ion analysis. The high resolving power dramatically increased the selectivity of measurements by separating ions of interest from interferences, which occurred in several cases, and thus improved the quantification performance. In addition, an experiment to assess the "co-habitation" of fragment ions in specific regions of the LC-MS/MS spectral space of a complex proteome digest was carried out. The study included the evaluation of the fragmentation patterns acquired under various experimental conditions (i.e., quadrupole isolation windows and Orbitrap resolving powers) for more than 200 peptides, which provided an experimental baseline to guide the development of methods for parallel reaction monitoring acquisition. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Background Despite its overall efficacy, combined antiretroviral therapy (cART) has failed to control human immunodeficiency virus (HIV) infection of the central nervous system (CNS). New acute and chronic neurological complications continue to be reported. Methods We conducted a retrospective study of 14 HIV-infected patients with documented encephalitis, which was initially attributed to an undetermined origin. Brain magnetic resonance imaging (MRI) uniformly revealed unusual, multiple linear gadolinium-enhanced perivascular lesions. Results All patients had manifested acute or subacute neurological symptoms; the brain MRIs indicating diffuse brain damage. The mean duration of HIV infection was approximately 10 years, and 8 patients were immunovirologically stable. Cerebrospinal fluid abnormalities with mildly elevated protein and pleocytosis with >90% lymphocytes, predominantly CD8, were found in all but 1 patient. The mean cerebral spinal fluid HIV load was 5949 copies/mL. Six patients reported a minor infection a few days prior to neurological symptoms, 2 patients presented criteria for the immune reconstitution inflammatory syndrome of the CNS, 2 were in virological escape, and 1 developed encephalitis after interruption of cART. Brain biopsies revealed inflammatory encephalitis associated with astrocytic and microglial activation as well as massive perivascular infiltration by polyclonal CD8(+) lymphocytes. All patients had been treated with glucocorticosteroids. The long-term therapeutic response varied from excellent, with no sequalae (n = 5), to moderate, with cognitive disorders (n = 4). The mean survival time was 8 years; however, 5 patients died within 13 months of initiation of treatment. Conclusions CD8 encephalitis in HIV-infected patients receiving cART is a clinical entity that should be added to the list of HIV complications.

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.

Comprehensive proteomic characterizations were performed aiming to create proteome reference maps for somatic and zygotic embryos of Cyclamen persicum. Separation by two dimensional isoelectric focusing - sodium dodecyl sulfate polyacrylamide gel electrophoresis led to a resolution of more than 800 protein spots for each tissue. Approximately 70% of the spots likewise appeared in both zygotic and in somatic embryo's protein fractions. However, differential gel electrophoresis analyses revealed pronounced differences in abundances for the majority of proteins present in both tissues. MS analyses for 300 reproducible spots in total (263 of the zygotic embryos' protein fraction and 37 spots appearing specifically in the somatic embryos' proteome) led to identification of 261 proteins, 35 of which were specific or highly abundant in gels of the somatic embryo's tissue. Most identified proteins were found to be involved in glycolysis or gluconeogenesis and stress response pathways.

Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of considerable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl coenzyme A, is attacked by dearomatizing benzoyl-CoA reductases (BCRs). An ATP-dependent BCR has been characterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately anaerobic model organism Geobacter metallireducens identified genes putatively coding for a completely different dearomatizing BCR. The corresponding BamBCDEFGHI complex is predicted to comprise soluble molybdenum or tungsten, selenocysteine, and FeS cluster-containing components. To elucidate key processes involved in the degradation of aromatic compounds in obligately anaerobic bacteria, differential membrane protein abundance levels from G. metallireducens grown on benzoate and acetate were determined by the MS-based spectral counting approach. A total of 931 proteins were identified by combining one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with liquid chromatography-tandem mass spectrometry. Several membrane-associated proteins involved in the degradation of aromatic compounds were newly identified including proteins with similarities to modules of NiFe/heme b-containing and energy-converting hydrogenases, cytochrome bd oxidases, dissimilatory nitrate reductases, and a tungstate ATP-binding cassette transporter system. The transcriptional regulation of differentially expressed genes was analyzed by quantitative reverse transcription-PCR; in addition benzoate-induced in vitro activities of hydrogenase and nitrate reductase were determined. The results obtained provide novel insights into the poorly understood degradation of aromatic compounds in obligately anaerobic bacteria.

The progress in sequencing technologies irrigates biology with an ever-increasing number of genome sequences. In most cases, the gene repertoire is predicted in silico and conceptually translated into proteins. As recently highlighted, the predicted genes exhibit frequent errors, particularly in start codons, with a serious impact on subsequent biological studies. A new “ortho-proteogenomic” approach is presented here for the annotation refinement of multiple genomes at once. It combines comparative genomics with an original proteomic protocol that allows the characterization of both N-terminal and internal peptides in a single experiment. This strategy was applied to the Mycobacterium genus with Mycobacterium smegmatis as the reference, and identified 946 distinct proteins, including 443 characterized N termini. These experimental data allowed the correction of 19% of the characterized start codons, the identification of 29 proteins missed during the annotation process, and the curation, thanks to comparative genomics, of 4328 sequences of 16 other Mycobacterium proteomes.

The four serotypes of dengue virus (DENV1-4) are the causal agents of the emerging disease Dengue Fever and its severe forms. DENV is inoculated into human blood through a mosquito bite. Thus, plasma is an important media for DENV dissemination in infected persons and several important interactions should take place for the virus with human plasma proteins that strongly influence or may determine the course of the infection. This dataset contains 239 proteins identified in the elution fractions of human plasma subjected to DE-52 anion exchange chromatography. Data on DENV2 infection of Huh 7.5 cells in presence of the human plasma fraction is also presented. Keywords: Human plasma, Anion exchange chromatography, Dengue virus

The wide diversity of proteins expressed in a cell or a tissue as a result of gene variants, RNA editing or PTMs results in several hundred thousand distinct functional proteins called proteoforms. The large-scale analysis of proteomes has been driven by bottom-up MS approaches. This allowed to identify and quantify large numbers of gene products and perform PTM profiling which yielded a significant number of biological discoveries. Trypsin is the gold standard enzyme for the production of peptides in bottom-up approaches. Several investigators argued recently that the near exclusive use of trypsin provided only a partial view of the proteome and hampered the discovery of new isoforms. The use of multiple proteases in a complementary fashion can increase sequence coverage providing more extensive PTM and sequence variant profiling. Here the various approaches to characterize proteoforms are discussed, including the use of alternative enzymes to trypsin in shotgun approaches to expand the observable sequence space by LC-MS/MS. The technical considerations associated with the use of alternative enzymes are discussed.

International audience; Arsenic-resistant prokaryote diversity is far from being exhaustively explored. In this study, the arsenic-adapted prokaryotic community present in a moderately arsenic-contaminated site near Sainte-Marie-aux-Mines (France) was characterized, using metaproteomic and 16S rRNA-encoding gene amplification. High prokaryotic diversity was observed, with a majority of Proteobacteria, Acidobacteria and Bacter-oidetes, and a large archaeal community comprising Euryarchaeaota and Thaumarchaeota. Metaproteomic analysis revealed that Proteobacteria, Planctomycetes and Cyanobacteria are among the active bacteria in this ecosystem. Taken together, these results highlight the unsuspected high diversity of the arsenic-adapted prokaryotic community, with some phyla never having been described in highly arsenic-exposed sites.

Targeted high-resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared with the reference method of selected reaction monitoring performed on triple quadrupole instruments. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data acquisition scheme enabled the analysis of moderate-to-large peptide numbers while retaining a high performance level. This new method, called internal standard triggered-parallel reaction monitoring (IS-PRM), relies on added internal standards and the on-the-fly adjustment of acquisition parameters to drive in real-time measurement of endogenous peptides. The acquisition time management was designed to maximize the effective time devoted to measure the analytes in a time-scheduled targeted experiment. The data acquisition scheme alternates between two PRM modes: a fast low-resolution "watch mode" and a "quantitative mode" using optimized parameters ensuring data quality. The IS-PRM method exhibited a highly effective use of the instrument time. Applied to the analysis of large peptide sets (up to 600) in complex samples, the method showed an unprecedented combination of scale and analytical performance, with limits of quantification in the low amol range. The successful analysis of various types of biological samples augurs a broad applicability of the method, which is likely to benefit a wide range of proteomics experiments.

Quantitative proteomics has benefited from the recent development of mass spectrometers capable of high-resolution and accurate-mass (HR/AM) measurements. While targeted experiments are routinely performed on triple quadrupole instruments in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode, the quadrupole-orbitrap mass spectrometers allow quantification in MS/MS mode, also known as parallel reaction monitoring (PRM). This technique is characterized by higher selectivity and better confidence in the assignment of the precursor and fragment ions, and thus translates into an improved analytical performance. More fundamentally, PRM introduces a change of the overall paradigm of targeted experiments, by the decoupling of the acquisition and data processing. They rely on two distinct steps, with a simplified acquisition method in conjunction with a flexible, iterative, post-acquisition data processing. This account describes in detail the different steps of a PRM experiment, which include the design of the acquisition method, the confirmation of the identity of the analytes founded upon a full MS/MS fragmentation pattern, and the quantification based on the extraction of specific fragment ions (selected post-acquisition) using tight mass tolerance. The different types of PRM experiments, defined as large-scale screening or precise targeted quantification using calibrated internal standards, together with the considerations on the selection of experimental parameters are discussed.

High resolution/accurate mass hybrid mass spectrometers have considerably advanced shotgun proteomics and the recent introduction of fast sequencing capabilities has expanded its use for targeted approaches. More specifically, the quadrupole-orbitrap instrument has a unique configuration and its new features enable a wide range of experiments. An overview of the analytical capabilities of this instrument is presented, with a focus on its application to quantitative analyses. The high resolution, the trapping capability and the versatility of the instrument have allowed quantitative proteomic workflows to be redefined and new data acquisition schemes to be developed. The initial proteomic applications have shown an improvement of the analytical performance. However, as quantification relies on ion trapping, instead of ion beam, further refinement of the technique can be expected.

MS-based approaches using targeted methods have been widely adopted by the proteomics community to study clinical questions such as the evaluation of biomarkers. At present, the most widely used targeted MS method is the SRM technique typically performed on a triple quadrupole instrument. However, the high analytical demands for performing clinical studies in combination with the extreme complexity of the samples involved are a serious challenge. The segmentation of the biomarker evaluation workflow has only partially alleviated these issues by differently balancing the analytical requirements and throughput at different stages of the process. The recent introduction of targeted high-resolution and accurate-mass analyses on fast sequencing mass spectrometers operated in parallel reaction monitoring (PRM) mode offers new avenues to conduct clinical studies and thus overcome some of the limitations of the triple quadrupole instrument. This article discusses the attributes and specificities of the PRM technique, in terms of experimental design, execution, and data analysis, and the implications for biomarker evaluation. The benefits of PRM on data quality and the impact on the consistency of results are highlighted and the definitive progress on the overall output of clinical studies, including high throughput, is discussed.

Biological systems are not only controlled by the abundance of individual proteins, but also by the formation of complexes and the dynamics of protein-protein interactions. The identification of the components of protein complexes can be obtained by shotgun proteomics using affinity purification coupled to mass spectrometry. Such studies include the analyses of several samples and experimental controls in order to discriminate true specific interactions from unspecific interactions and contaminants. However, shotgun proteomics have limited quantification capabilities for low abundant proteins on large sample sets due to the undersampling and the stochastic precursor ion selection. In this context, targeted proteomics constitutes a powerful analytical tool to systematically detect and quantify peptides in multiple samples, for instance those obtained from affinity purification experiments. Hypothesis-driven strategies have mainly relied on the selected reaction monitoring (SRM) technique performed on triple quadrupole instruments, which enables highly selective and sensitive measurements of peptides, acting as surrogates of the pre-selected proteins, over a wide range of concentrations. More recently, novel quantitative methods based on high resolution instruments, such as the parallel reaction monitoring (PRM) technique implemented on the quadrupole-orbitrap instrument, have arisen and provided alternatives to perform quantitative analyses with enhanced selectivity.The application of targeted proteomics to protein-protein interaction experiments from plasma and other physiological fluid samples and the inclusion of parallel reaction monitoring (PRM), combined with other recent technology developments opens a vast area for clinical application of proteomics. It is anticipated that it will reveal valuable information about specific, individual, responses against drugs, exogenous proteins or pathogens.

Targeted methods have gained acceptance among proteomics community to perform quantitative experiments. However, the current reference to conduct such experiments relies on selected reaction monitoring (SRM) analyses performed on triple quadrupole mass spectrometers, although it suffers from some limitations. First, the low resolution quadrupole mass analyzers do not present enough selectivity to discriminate the analytes from interferences commonly encountered in biological samples. Second, the number of peptides monitored in one single experiment often remains limited. The introduction of high resolution/accurate mass instruments with fast sequencing capabilities has enabled the development of novel quantitative methods. More specifically, the new quadrupole-orbitrap mass spectrometer operated in parallel reaction monitoring (PRM) mode showed detection and quantification performances similar or better than those obtained in SRM, due to the increased selectivity of the high-resolution orbitrap mass analyzer. The versatility of the instrument, with its ability to multiplex the selection of precursor ions and to operate with varying quadrupole isolation windows, has enabled the design of large-scale experiments, which require the optimization of several acquisition parameters to maintain high performance. It includes the adjustments of the fill time of the trapping device and the tight scheduling of elution times of the peptides, ideally adjusted on-the-fly. Biological significance The present study constitutes a valuable baseline for the proteomics community to better control the trade-off between sensitivity and number of analyzed peptides in large-scale parallel reaction monitoring (PRM) experiments performed on a quadrupole-orbitrap instrument. A standard acquisition method requires careful setting of the parameters, namely the fill time in accordance with the control of the resolving power and the degree of multiplexing on one hand, and the quadrupole selection window on the other hand. This study helps in establishing acquisition parameters for large-scale PRM experiments, while maintaining sufficient sensitivity. In addition, the real-time correction of the scheduled peptide monitoring windows, to compensate for possible elution time drift, was explored. This approach supports the use of narrowed monitoring windows in PRM analyses, which greatly scale up the number of peptides targeted in a single LC–MS experiment. The broad application of the presented approaches by the community is likely to allow the establishment of an unprecedented scale for targeted proteomics, thus matching the pressing demand of systems biology or biomarker evaluation studies. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

nodeficiency virus was negative, total lymphocyte count and levels of gammaglobulins were normal, and the patient did not report having had any noteworthy infections in the past several years. Despite the initiation of intravenous acyclovir (10 mg/ kg 3 times daily) on the day of admission, his clinical state worsened with persistent high fever and severe dyspnea (respiratory rate: 40 bpm and oxygen saturation 94%, despite a flow rate of 6 liter/min). High-resolution computed tomography revealed small nodules scattered randomly throughout the lung parenchyma, patchy ground-glass opacities and basal alveolar consolidations ( fig. 2 ). At day 4, intravenous corticosteroids were started (methylprednisolone 1 mg/kg) and progressively tapered down within a week, leading rapidly to apyrexia and normalization of all respiratory parameters within 4 days. The patient experienced a full recovery. A 35-year-old heavy smoker presented with a 3-day history of fever, pruritic rash, cough and progressive dyspnea. His 3-year-old son had contracted chickenpox 15 days before admission. The father had no history of varicella or chickenpox vaccination. Upon admission, his temperature was 38.7 ° C, oxygen saturation 96% and respiratory rate 32 bpm. Physical examination revealed an exanthematous rash with lesions in different stages of development (vesicles, pustules and crusty lesions, see fig. 1 ) and bilateral basal subcrepitant rales. Blood tests were normal except for a plasma C-reactive protein level of 22 mg/l. Chest radiography performed at admission showed bilateral interstitial nodular infiltrates. Varicella zoster virus DNA was detected by polymerase chain reaction in the serum. The patient had no evidence of an underlying immunocompromised state: serological test for human immuPublished online: May 9, 2013

Advances in proteomics have always been tightly linked to the development of mass spectrometry instrumentation. While for over one decade liquid chromatography/mass spectrometry based proteomics has relied on shotgun approaches, new hypothesis-driven methods have emerged. The latter allows more systematic analyses of peptides (used as surrogate for the proteins) with a high degree of sensitivity. Thus, they facilitate large-scale quantitative clinical studies, aiming at evaluating and validating biomarker panels in bodily fluids, such as blood or urine.

Large-scale proteomics applications using SRM analysis on triple quadrupole mass spectrometers present new challenges to LC-MS/MS experimental design. Despite the automation of building large-scale LC-SRM methods, the increased numbers of targeted peptides can compromise the balance between sensitivity and selectivity. To facilitate large target numbers, time-scheduled SRM transition acquisition is performed. Previously published results have demonstrated incorporation of a well-characterized set of synthetic peptides enabled chromatographic characterization of the elution profile for most endogenous peptides. We have extended this application of peptide trainer kits to not only build SRM methods but to facilitate real-time elution profile characterization that enables automated adjustment of the scheduled detection windows. Incorporation of dynamic retention time adjustments better facilitate targeted assays lasting several days without the need for constant supervision. This paper provides an overview of how the dynamic retention correction approach identifies and corrects for commonly observed LC variations. This adjustment dramatically improves robustness in targeted discovery experiments as well as routine quantification experiments.