Capillary electrophoresis on-line hyphenated with mass spectrometry for analysis of insulin-like growth factor-1 in pharmaceutical preparations.

Insulin-like growth factor-1 (IGF-1) is a 70-amino acid single-chain polypeptide, which has found application in diagnostics as a biomarker of growth hormone disorders and as a therapy for growth failure in children and adolescents. Due to its strong anabolic effects, it is often abused by athletes for doping purposes. Here, we developed an on-line hyphenated method based on capillary zone electrophoresis (CZE) and triple quadrupole mass spectrometry (MS) detection with electrospray ionization (CZE-electrospray ionization source-MS [CZE-ESI-MS]) for the determination of IGF-1 in pharmaceutical matrices. We achieved a highly efficient, accurate, repeatable, sensitive, and selective analysis of IGF-1 with favorable migration times (<15 min). Optimized and validated CZE-ESI-MS method was successfully applied for the determination of IGF-1 in injectable solutions (Increlex®), and its presence was also confirmed in nutritional preparations (tablets and liquid colostrum). This is the first validated CZE-ESI-MS method for the determination of IGF-1 in pharmaceutical matrices revealing the potential of capillary electrophoresis for its use in drug quality control laboratories with benefits, such as high separation efficiency, high-speed analysis, low sample consumption, as well as environmental and cost aspects.

Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens.

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species (E. faecium E745, K. pneumoniae KP257, A. baumannii AYE, and E. cloacae S11) as well as E. faecalis V583 (a close relative of E. faecium) and a clinical isolate of A. baumannii AC02 using an optimized solvent sampling system. In each case, top-down LESA MS/MS was employed for protein identification. In total, 24 proteins were identified from 37 MS/MS spectra by searching against protein databases for the individual species. The MS/MS spectra for the identified proteins were subsequently searched against multiple databases from multiple species in an automated data analysis workflow with a view to determining the accuracy of identification of unknowns. Out of 24 proteins, 19 were correctly assigned at the protein and species level, corresponding to an identification success rate of 79%.

Direct identification of bacterial and human proteins from infected wounds in living 3D skin models.

Trauma is one of the leading causes of death in people under the age of 49 and complications due to wound infection are the primary cause of death in the first few days after injury. The ESKAPE pathogens are a group of bacteria that are a leading cause of hospital-acquired infections and a major concern in terms of antibiotic resistance. Here, we demonstrate a novel and highly accurate approach for the rapid identification of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) directly from infected wounds in 3D in vitro skin models. Wounded skin models were inoculated with bacteria and left to incubate. Bacterial proteins were identified within minutes, directly from the wound, by liquid extraction surface analysis mass spectrometry. This approach was able to distinguish closely related strains and, unlike genomic approaches, can be modified to provide dynamic information about pathogen behaviour at the wound site. In addition, since human skin proteins were also identified, this method offers the opportunity to analyse both host and pathogen biomarkers during wound infection in near real-time.

Electroporation and Mass Spectrometry: A New Paradigm for In Situ Analysis of Intact Proteins Direct from Living Yeast Colonies.

Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging. Their thick, chitin-reinforced cell walls render cell lysis difficult, complicating their analysis and identification by methods routinely used for bacteria, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Liquid extraction surface analysis mass spectrometry (LESA-MS) has previously been applied to the analysis of intact proteins from Gram-positive and Gram-negative bacterial colonies sampled directly on solid nutrient media. To date, a similar analysis of yeast colonies has not proved possible. Here we demonstrate the rapid release of intact yeast proteins for LESA-MS by electroporation using a home-built high-voltage device designed to lyse cells grown in colonies on agar media. Detection and identification of previously inaccessible proteins from baker's yeast Saccharomyces cerevisiae, as well as two clinically relevant yeast species (Candida glabrata and Cryptococcus neoformans), is shown. The electroporation approach also has the potential to be translated to other mass spectrometric analysis techniques, including MALDI and various ambient ionization methods.

Quantitative Imaging of Proteins in Tissue by Stable Isotope Labeled Mimetic Liquid Extraction Surface Analysis Mass Spectrometry.

Absolute quantification of proteins in tissue is important for numerous fields of study. Liquid chromatography-mass spectrometry (LC-MS) methods are the norm but typically involve lengthy sample preparation including tissue homogenization, which results in the loss of information relating to spatial distribution. Here, we propose liquid extraction surface analysis (LESA) mass spectrometry (MS) of stable isotope labeled mimetic tissue models for the spatially resolved quantification of intact ubiquitin in rat and mouse brain tissue. Measured ubiquitin concentrations are in agreement with values found in the literature. Images of rat and mouse brain tissue demonstrate spatial variation in the concentration of ubiquitin and demonstrate the utility of spatially resolved quantitative measurement of proteins in tissue. Although we have focused on ubiquitin, the method has the potential for broader application to the absolute quantitation of any endogenous protein or protein-based drug in tissue.

Innovative surface modification of Ti-6Al-4V alloy with a positive effect on osteoblast proliferation and fatigue performance.

A novel approach of surface treatment of orthopaedic implants combining electric discharge machining (EDM), chemical milling (etching) and shot peening is presented in this study. Each of the three techniques have been used or proposed to be used as a favourable surface treatment of biomedical titanium alloys. But to our knowledge, the three techniques have not yet been used in combination. Surface morphology and chemistry were studied by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Fatigue life of the material was determined and finally several in-vitro biocompatibility tests have been performed. EDM and subsequent chemical milling leads to a significant improvement of osteoblast proliferation and viability thanks to favourable surface morphology and increased oxygen content on the surface. Subsequent shot-peening significantly improves the fatigue endurance of the material. Material after proposed combined surface treatment possesses favourable mechanical properties and enhanced osteoblast proliferation. EDM treatment and EDM with shot peening also supported early osteogenic cell differentiation, manifested by a higher expression of collagen type I. The combined surface treatment is therefore promising for a range of applications in orthopaedics.

The gene expression of human endothelial cells is modulated by subendothelial extracellular matrix proteins: short-term response to laminar shear stress.

Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Tissue engineering strives to produce bio-artificial conduits to provide resistance to thrombosis. The objectives of our study were to culture endothelial cells (EC) on composite assemblies of extracellular matrix proteins, and to evaluate the cellular phenotype under flow. Cell-adhesive assemblies were fabricated on glass slides as combinations of collagen (Co), laminin (LM), and fibronectin (FN), resulting in three samples: Co, Co/LM, and Co/FN. Surface topography, roughness, and wettability were determined. Human saphenous vein EC were harvested from cardiac patients, cultured on the assemblies and submitted to laminar shear stress (SS) of 12 dyn/cm(2) for 40, 80, and 120 min. Cell retention was assessed and qRT-PCR of adhesion genes (VE-cadherin, vinculin, KDR, CD-31 or PECAM-1, ß1-integrins) and metabolic genes (t-PA, NF-κB, eNOS and MMP-1) was performed. Quantitative immunofluorescence of VE cadherin, vinculin, KDR, and vonWillebrand factor was performed after 2 and 6 h of flow. Static samples were excluded from shearing. The cells reached confluence with similar growth curves. The cells on Co/LM and Co/FN were resistant to flow up to 120 min but minor desquamation occurred on Co corresponding with temporary downregulation of VE cadherin and vinculin-mRNA and decreased fluorescence of vinculin. The cells seeded on Co/LM initially more upregulated vinculin-mRNA and also the inflammatory factor NF-κB, and the cells plated on Co/FN changed the expression profile minimally in comparison with the static control. Fluorescence of VE cadherin and vonWillebrand factor was enhanced on Co/FN. The cells cultured on Co/LM and Co/FN increased the vinculin fluorescence and expressed more VE cadherin and KDR-mRNA than the cells on Co. The cells plated on Co/FN upregulated the mRNA of VE cadherin, CD-31, and MMP 1 to a greater extent than the cells on Co/LM and they enhanced the fluorescence of VE cadherin, KDR, and vonWillebrand factor. Some of these changes sustained up to 6 h of flow, as confirmed by immunofluorescence. Combined matrices Co/LM and Co/FN seem to be more suitable for EC seeding and retention under flow. Moreover, Co/FN matrix promoted slightly more favorable cellular phenotype than Co/LM under SS of 2-6 h.

Two aspartic proteinases secreted by the pathogenic yeast Candida parapsilosis differ in expression pattern and catalytic properties.

Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1, SAPP2, and SAPP3. We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1 p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1 p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.