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ABSTRACT: 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: Proteomics from protein structures to clinical applications (CNPN 2012).
Journal of proteomics 11/2012; · 5.07 Impact Factor
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ABSTRACT: There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. While to date protein quantification in biological samples is routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of the orbitrap mass analyzer equipped with a quadrupole mass filter as front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring, SIM) and in MS/MS mode (parallel reaction monitoring, PRM). While the ability of the quadrupole to select a restricted m/z range allows overcoming the dynamic range limitations associated with trapping devices, the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performances in terms of selectivity, dynamic range, and sensitivity. These high performances are further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60 minute experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and to cope with the pressing demand of systems biology or biomarker evaluation studies.
Molecular & Cellular Proteomics 09/2012; · 7.40 Impact Factor
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ABSTRACT: 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.
Proteomics 04/2012; 12(8):1122-33. · 4.43 Impact Factor
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ABSTRACT: 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.
Nature Protocol 01/2012; 7(5):859-71. · 8.36 Impact Factor
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Dimitri Heintz, Sebastien Gallien,
Vincent Compagnon,
Anne Berna,
Masashi Suzuki,
Shigeo Yoshida,
Toshiya Muranaka,
Alain Van Dorsselaer,
Christine Schaeffer,
Thomas J Bach,
Hubert Schaller
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ABSTRACT: Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type Arabidopsis thaliana and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC-MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the hmgr1-1 mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the Arabidopsis membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis.
Journal of Proteome Research 12/2011; 11(2):1228-39. · 5.11 Impact Factor
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Babafemi Taiwo,
Lu Zheng, Sebastien Gallien,
Roy M Matining,
Daniel R Kuritzkes,
Cara C Wilson,
Baiba I Berzins,
Edward P Acosta,
Barbara Bastow,
Peter S Kim,
Joseph J Eron
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ABSTRACT: To explore darunavir/ritonavir (DRV/r) plus raltegravir (RAL) combination therapy in antiretroviral-naive patients.
Phase IIb, single-arm, open-label, multicenter study.
One hundred and twelve antiretroviral-naive, HIV-1-infected patients received DRV/r 800/100 mg once daily and RAL 400 mg twice daily. Primary endpoint was virologic failure by week 24. Virologic failure was defined as confirmed viral load of 1000 copies/ml or more at week 12, or an increase of more than 0.5 log(10) copies/ml in viral load from week 4 to 12, or a confirmed viral load of more than 50 copies/ml at or after week 24. Protease and integrase genes were sequenced in patients experiencing virologic failure.
Virologic failure rate was 16% [95% confidence interval (CI) 10-24] by week 24 and 26% (95% CI 19-36) by week 48 in an intent-to-treat analysis. Viral load at virologic failure was 51-200 copies/ml in 17/28 failures. Adjusting for age and sex, virologic failure was associated with baseline viral load of more than 100,000 copies/ml [hazard ratio 3.76, 95% CI (1.52-9.31), P = 0.004] and lower CD4 cell count [0.77 per 100 cells/μl increase (95% CI 0.61-0.98), P = 0.037]. When trough RAL concentrations were included as a time-varying covariate in the analysis, virologic failure remained associated with baseline viral load more than 100,000 copies/ml [hazard ratio = 4.67 (95% CI 1.93-11.25), P < 0.001], whereas RAL level below detection limit in plasma at one or more previous visits was associated with increased hazard [hazard ratio = 3.42 (95% CI 1.41-8.26), P = 0.006]. All five participants with integrase mutations during virologic failure had baseline viral load more than 100,000 copies/ml.
DRV/r plus RAL was effective and well tolerated in most patients, but virologic failure and integrase resistance were common, particularly in patients with baseline viral load more than 100,000 copies/ml.
AIDS (London, England) 08/2011; 25(17):2113-22. · 4.91 Impact Factor
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ABSTRACT: Population sequencing was performed for persons identified with persistent low-level viremia in 2 clinical trials. Persistent low-level viremia (defined as plasma HIV-1 RNA level >50 and <1000 copies/mL in at least 2 determinations over a 24-week period, after at least 24 weeks of antiretroviral therapy) was observed in 65 (5.6%) of 1158 patients at risk. New resistance mutations were detected during persistent low-level viremia in 37% of the 54 evaluable cases. The most common mutations were M184I/V (14 cases), K103N (9), and M230L (3). Detection of new mutations was associated with higher HIV-1 RNA levels during persistent low-level viremia.
The Journal of Infectious Diseases 08/2011; 204(4):515-20. · 6.41 Impact Factor
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ABSTRACT: Selected reaction monitoring (SRM) performed on triple quadrupole mass spectrometers has been the reference quantitative technique to analyze small molecules for several decades. It is now emerging in proteomics as the ideal tool to complement shotgun qualitative studies; targeted SRM quantitative analysis offers high selectivity, sensitivity and a wide dynamic range. However, SRM applied to proteomics presents singularities that distinguish it from small molecules analysis. This review is an overview of SRM technology and describes the specificities and the technical aspects of proteomics experiments. Ongoing developments aiming at increasing multiplexing capabilities of SRM are discussed; they dramatically improve its throughput and extend its field of application to directed or supervised discovery experiments.
Biological Mass Spectrometry 03/2011; 46(3):298-312. · 3.41 Impact Factor
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ABSTRACT: HIV-infected patients with opportunistic infections receive therapeutic regimens that often contain numerous drugs and are then exposed to potential drug-to-drug interactions. We report here an unusual case of relapse of toxoplasmic encephalitis in an HIV-infected patient, possibly due to a drug-to-drug interaction between dapsone and minocycline.
Scandinavian Journal of Infectious Diseases 08/2009; 41(9):700-2. · 1.72 Impact Factor
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ABSTRACT: 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.
Molecular & Cellular Proteomics 07/2009; 8(9):2159-69. · 7.40 Impact Factor
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ABSTRACT: 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.
Genome Research 11/2008; 19(1):128-35. · 13.61 Impact Factor
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ABSTRACT: In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with undetected frameshifts and in-frame stop codons. These interrupted coding sequences (ICDSs) may really be present in the organism or may result from misannotation based on sequencing errors. The reality or otherwise of these sequences has major implications for all subsequent functional characterization steps, including module prediction, comparative genomics and high-throughput proteomic projects.
We show here, using Mycobacterium smegmatis as a model species, that a significant proportion of these ICDSs result from sequencing errors. We used a resequencing procedure and mass spectrometry analysis to determine the nature of a number of ICDSs in this organism. We found that 28 of the 73 ICDSs investigated correspond to sequencing errors.
The correction of these errors results in modification of the predicted amino acid sequences of the corresponding proteins and changes in annotation. We suggest that each bacterial ICDS should be investigated individually, to determine its true status and to ensure that the genome sequence is appropriate for comparative genomics analyses.
Genome biology 02/2007; 8(2):R20. · 6.63 Impact Factor
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ABSTRACT: We report an unusual case of Plasmodium falciparum malaria in a European returning from tropical regions associating an anterior ischemic optic neuropathy and an asymptomatic centropontine myelinolysis. The transient antiphospholipid antibodies detected in the patient may have played a role in the ischemic process at the origin of this unusual clinical association.
The Journal of infection 02/2007; 54(1):e1-3. · 4.13 Impact Factor
