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Publications (14)44.23 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: In this study, we performed an in-depth characterization of the male pediatric infant urinary proteome by parallel proteomic analysis of normal healthy adult (n =6) and infant (n =6) males and comparison to available published data. A total of 1584 protein groups were identified. Of these, 708 proteins were identified in samples from both cohorts. Although present in both cohorts, 136 of these common proteins were significantly enriched in urine from adults and 94 proteins were significantly enriched in urine from infants. Using Gene Ontology, we found that the infant-enriched or specific subproteome (743 proteins) had an overrepresentation of proteins that are involved in translation and transcription, cellular growth and metabolic processes. In contrast, the adult enriched or specific subproteome (364 proteins) showed an overexpression of proteins involved in immune response and cell adhesion. This study demonstrates that the non-diseased male urinary proteome is quantitatively affected by age, has age-specific subproteomes, and identifies a common subproteome with no age-dependent abundance variations. These findings highlight the importance of age-matching in urinary proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
    No preview · Article · May 2013 · Biochimica et Biophysica Acta
  • No preview · Article · Apr 2012 · The Journal of Urology
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    [Show abstract] [Hide abstract] ABSTRACT: The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined. We studied an isogenic daptomycin-susceptible (DAP(S)) and daptomycin-resistant (DAP(R)) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques. Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAP(R) isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAP(S)/DAP(R) clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAP(R) phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid. Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.
    Full-text · Article · May 2011 · Journal of Antimicrobial Chemotherapy
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    [Show abstract] [Hide abstract] ABSTRACT: This chapter provides an overview of different approaches that can be used for sample preparation of body fluids for proteomics. Sample collection, protein extraction, protease inhibitor supplementation, sample storage, and abundant protein depletion are presented here in the context of various human body fluids. We emphasize that the particular set of techniques chosen for such investigations tightly correlates with the fluid to be analyzed, as no consensus methods are appropriate for all body fluids. However, we stress the need for standardized methods for the individual body fluids which is paramount in obtaining reproducible and robust data when analyzing human body fluids. In addition, we provide examples of optimized sample handling techniques using a systemic (urine) and a proximal body fluid (pancreatic fluid).
    Full-text · Chapter · Jan 2011
  • [Show abstract] [Hide abstract] ABSTRACT: Shotgun proteomics is a rapid and near universal strategy to identify proteins in complex protein mixtures. After protein digestion, the resulting peptide mixture is submitted to two orthogonal techniques: peptides are first separated according to their isoelectric point (pI) by isoelectric focusing (IEF) on immobilized pH gradient (IPG); after peptide extraction, they are then separated in the second dimension according to their hydrophobic properties by reverse phase liquid chromatography (RPLC). Finally, they are detected by tandem mass spectrometry (MS/MS) and proteins are matched by means of bioinformatics software.
    No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract] ABSTRACT: Shotgun proteomics originated as a strategy to identify proteins in complex protein mixtures, but it is also possible to obtain information on relative quantitation with some adjustments to the procedure. After protein digestion, the resulting peptide mixture is labelled with isobaric tags. Then, labelled peptides are submitted to two orthogonal techniques: first, peptides are separated according to their isoelectric point (pI) by Off-Gel electrophoresis (OGE), a relatively new isoelectric focusing (IEF) technique; after peptide purification, they are then separated in a second dimension according to their hydrophobic properties by reversed-phase liquid chromatography (RPLC). Finally, following detection by mass spectrometry (MS) and sequencing by tandem mass spectrometry (MS/MS), proteins are matched by means of bioinformatics software, and protein ratios are calculated by comparing isobaric tagged reporter fragments to highlight the different expression of one protein in one sample relative to other samples.
    No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
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    Ali R Vaezzadeh · Andrew C Briscoe · Hanno Steen · Richard S Lee
    [Show abstract] [Hide abstract] ABSTRACT: Workflows in urinary proteomics studies are often complex and require many steps to enrich, purify, deplete, and separate the complex mixture. Many of these methods are laborious, are time-consuming, and have the potential for error. Although individual steps of these methods have been previously studied, their downstream compatibilities with fractionation technologies such as off-gel electrophoresis have not been investigated. We developed a one-step sample preparation workflow that simultaneously (i) concentrates proteins, (ii) purifies by removing salts and other low molecular weight compounds, and (iii) depletes (albumin) from urine samples. This simple and robust workflow can be multiplexed and is compatible with a diverse range of downstream multidimensional separation technologies. Additionally, because of its high reproducibility and flexibility in processing samples with different volumes and concentrations, it has the potential to be used for standardization of urinary proteomics studies, as well as for studying other body fluids of similar complexity.
    Preview · Article · Oct 2010 · Journal of Proteome Research
  • [Show abstract] [Hide abstract] ABSTRACT: In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.
    No preview · Article · Nov 2009 · Journal of microbiological methods
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    [Show abstract] [Hide abstract] ABSTRACT: Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.
    Full-text · Article · Sep 2009 · Mass Spectrometry Reviews
  • Ali R Vaezzadeh · Hanno Steen · Michael R Freeman · Richard S Lee
    [Show abstract] [Hide abstract] ABSTRACT: Proteomics is a rapidly growing new discipline that has the potential to increase and improve the understanding of protein function and interaction in the context of systems biology. As a translational science it has the potential to identify many new therapeutic targets as well as diagnostic and prognostic biomarkers of disease. Proteomics approaches consist of a combination of powerful technologies such as protein/peptide separation, identification and bioinformatic detection, and quantitation based on powerful computational data processing tools. We present an overview of current proteomics technologies, a review of proteomics applications in urology and a perspective on the future of proteomics in clinical medicine. A literature search was performed on the basic concepts of proteomics and technologies commonly used in this field. Advantages, challenges and limitations of current proteomics approaches are discussed, and proteomics applications in the field of urology are presented. The proteomics approaches to answer clinical questions have only recently been introduced. Many different technologies have been used in this field, which is moving from simple description to quantitative clinical applications. Proteomics offers new approaches to the study of genitourinary tract diseases, and the potential to identify clinically relevant biomarkers and new therapeutic targets.
    No preview · Article · Aug 2009 · The Journal of urology
  • [Show abstract] [Hide abstract] ABSTRACT: The use of isoelectric focusing as first dimension of separation is a new trend in shotgun proteomics. In all applications using this approach, peptides are separated into equitable fractions, whereas theoretical distribution of peptides according to p I is heterogeneous. We present the development of a new tool and strategy that generates a fractionation scheme resulting in almost even distribution of peptides per fraction, based on theoretical and experimental data. The "pICarver" software tool also increases the throughput of the approach by reducing the number of fractions and merging the peptide-poor regions. A set of isoelectric point fluorescent peptide markers was also developed in combination with the pICarver program to calibrate the pH gradient of commercially available strips. These markers enhanced the precision of pICarver predications. The overall strategy allowed detecting false positive identification and post-translational modifications. The software tool is freely available on
    No preview · Article · Oct 2008 · Journal of Proteome Research
  • [Show abstract] [Hide abstract] ABSTRACT: Imaging Mass Spectrometry (IMS) has emerged as a powerful technique in the field of proteomics. The use of Immobilized pH Gradient-IsoElectric Focusing (IPG-IEF) is also a new trend, as the first dimension of separation, in shotgun proteomics. We report a combination of these two outstanding technologies. This approach is based on the separation of shotgun-produced peptides by IPG-IEF. The peptides are then transferred by capillarity to a capture membrane, which is then scanned by the mass spectrometer to generate MS images. This high-throughput methodology allows a preview of the sample to be obtained in a single day. We report the application of this new pipeline for differential comparison of the membrane proteome of two different strains of Staphylococcus aureus bacteria in a proof-of-principle experiment.
    No preview · Article · Sep 2008 · Rapid Communications in Mass Spectrometry
  • [Show abstract] [Hide abstract] ABSTRACT: IntroductionBackground Isoelectric FocusingShotgun ProteomicsShotgun IEFShotgun IEF WorkflowApplicationsDiscussion and Outlook Isoelectric FocusingShotgun ProteomicsShotgun IEF
    No preview · Chapter · Apr 2008
  • [Show abstract] [Hide abstract] ABSTRACT: Acquisition of tandem mass spectra from peptides or other analytes deposited on non-conductive membranes is inhibited on instruments combining matrix-assisted laser desorption/ionization with tandem time-of-flight analyzers (MALDI-TOF/TOF™) due to a charging effect. A thin layer of gold renders the membrane conductive. This allows adequate data acquisition on MALDI-TOF/TOF™ systems. Therefore, this methodology extends the capacity of the molecular scanner concept to tandem mass spectrometry. Copyright
    No preview · Article · Feb 2005 · Rapid Communications in Mass Spectrometry