Objectives
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.
Methods
...We studied an isogenic daptomycin-susceptible (DAPS) and daptomycin-resistant (DAPR) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques.
Results
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 DAPR 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 DAPS/DAPR clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAPR 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.
Conclusions
Compatible with previous in vitro observations, in vivo-acquired DAPR 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.
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.
Prenatal hydronephrosis is a common condition that may spontaneously resolve after birth. However, this condition can result in renal damage and requires surgical correction in a number of cases. ...Preventing renal damage is paramount, but existing diagnostic technology is invasive, exposes infants to radiation, is costly, and is often indeterminate. A better understanding of the pathophysiology of renal obstruction as reflected in the urinary proteome may provide new insights into the disease that could potentially alter the clinical management of hydronephrosis. We performed a quantitative proteomics study of urine that was surgically obtained from eight clinically significant, unilaterally obstructed infants versus eight healthy controls, with the goal of identifying quantitatively varying proteins and the biological networks associated with them. Notably, urine was obtained from both the obstructed kidney and the bladder. Over 1100 proteins were identified, and a total of 76 quantitatively varying proteins were identified. Proteins involved in oxidative stress, inflammation, and renal disease pathways showed the most significant abundance differences. This study gives a deeper understanding of the critical proteomic changes associated with renal obstruction and represents the deepest proteomic profile of renal obstruction to date.
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.
•We analyze 12 adult and infant urine samples to >1500 proteins using MS.•Using spectral counting, we determine over 200 quantitatively varying proteins.•We identify functional differences associated with each cohort.•Differences include metabolism, cellular growth, immune response and adhesion.•These findings are highly relevant for future clinical work on urinary proteomics.
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.
Purpose 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. Materials and Methods 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. Results 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. Conclusions Proteomics offers new approaches to the study of genitourinary tract diseases, and the potential to identify clinically relevant biomarkers and new therapeutic targets.
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.
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.
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.
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