The phosphoproteome and its physiological dynamics in Staphylococcus aureus

Phosphorylation events on proteins during growth and stress/starvation can represent crucial regulation processes inside the bacterial cell. Therefore, serine, threonine and tyrosine phosphorylation patterns were analyzed by two powerful complementary proteomic methods for the human pathogen Staphylococcus aureus. Using 2D-gel analysis with a phosphosensitive stain (Pro-Q Diamond) and gel-free titanium dioxide based phosphopeptide enrichment, 103 putative phosphorylated proteins with successfully mapped 68 different phosphorylation sites were found in the soluble proteome of S. aureus. Additionally, in a proof of concept study, 8 proteins phosphorylated on arginine residues have been identified. Most important for functional analyses of S. aureus, proteins related to pathogenicity and virulence were found to be phosphorylated: the virulence regulator SarA, the potential antimicrobial target FbaA and the elastin-binding protein EbpS. Besides newly identified phosphorylation sites we compared our dataset with existing data from literature and subsequent experiments revealed additional phosphorylation events on highly conserved localizations in FbaA. Differential analysis of phosphorylation signals on the 2D-gels showed significant changes in phosphorylation under different physiological conditions for 10 proteins. Among these, we were able to detect newly appearing signals for phosphorylated isoforms of FdaB and HchA under nitrosative stress conditions.     

Monoliths with chiral surface functionalization for enantioselective capillary electrochromatography

The state-of-the-art in CEC enantiomer separations with monolithic capillary columns is comprehensively reviewed. The various types of monolithic columns comprising in situ organic polymer monoliths, molecularly imprinted polymer (MIP) monoliths, silica monoliths and monoliths made from particles are discussed with a focus on materials’ synthesis, chemistry and properties as well as column aspects. Monolithic MIP-type porous layer open-tubular (PLOT) columns are treated herein as well. From this survey of the literature, the authors come to the conclusion that monolithic silica capillaries appear to become the preferred column type for CEC enantiomer separations of low-molecular drugs and other chiral pharmaceuticals or chemicals.     

阳离子色谱法测定奶粉中三聚氰胺的含量

目的:建立婴儿配方奶粉中三聚氰胺含量的高效液相色谱分析方法。方法:采用强阳离子交换色谱柱,以乙睛—磷酸盐缓冲液(用磷酸调节pH至3.0)=25:75,流速为1.20mL/min,波长为240nm。结果:三聚氰胺在0.1673μg～0.8360μg浓度范围内线性良好,相关系数为0.9999,平均回收率为100.6%,RSD为0.91%。结论:本方法操作简便可靠,结果准确,重现性好,可作为该产品质量控制方法。     

iTRAQ-based proteomic analysis of combination therapy with taurine,epigallocatechin gallate,and genistein on carbon tetrachloride-induced liver fibrosis in rats

Combination therapy with taurine, epigallocatechin gallate, and genistein was effective in alleviating the progression of liver fibrosis in our previous study. To better understand the anti-fibrotic mechanisms of combination therapy, an iTRAQ-based proteomics approach was used to study the expression profiles of proteins in carbon tetrachloride-induced liver fibrosis rats following combination therapy. The anti-fibrotic effects of combination therapy were assessed directly by liver histology, and indirectly by measurement of serum biochemical markers and antioxidant enzymes. The results showed that combination therapy could significantly improve the liver function, as indicated by decreasing levels of alanine aminotransferase (ALT), aspartate transaminase (AST), transforming growth factor-β1 (TGF-β1), and collagen I, increasing levels of total antioxidative capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), and reducing the pathological tissue damage. A total of 89 differential expressed proteins in response to combination therapy were identified by iTRAQ, which were interacted with each other and involved in different biological processes and pathways. Four differentially expressed proteins (Tpi1, Txn1, Fgb, and F7) involved in antioxidant defense system, glycolysis pathway and coagulation cascade pathway were validated by enzyme-linked immunosorbent assay. Our work provided valuable insights into the molecular mechanism of combination therapy against liver fibrosis, and the identified targets may be useful for treatment of liver fibrosis in future.     

Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N = 3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 ± 11.0 (mean ± S.D.) in 10⁵ tyrosine and 4.4 ± 3.9 (mean ± S.D.) in 10³ tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R² = 0.55, p = 0.0065, n = 23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.