Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients

Allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma are prevalent airway diseases that can have a substantial impact on a patient's quality of life. MS analyses of biological fluids can effectively screen for proteins associated with disease processes, however, initial detection of diagnostic proteins is difficult due to protein complexity and dynamic range. To enhance the detection of lower abundance proteins, intact nasal lavage fluid (NLF) proteins from nonpolypoid AR and from asthmatic CRS patients were extensively fractionated prior to LC/MS/MS analysis. Pooled NLF samples were processed to remove low molecular weight molecules and high abundance plasma proteins. Anion exchange (AX) chromatography followed by RP‐LC further separated the remaining intact NLF proteins. The resulting fractions were digested with trypsin and the peptides analyzed by LC/MS/MS. Spectra were searched with MASCOT, SEQUEST, and X!Tandem to obtain peptide identifications and subsequently analyzed by Scaffold software to identify parent proteins with at least 99% confidence. The 197 identified proteins are compared to those previously cited in the literature and the workflow evaluated to determine the usefulness for the detection of lower abundance proteins. This is the first extensive list of NLF proteins generated from CRS patients with coexisting asthma.     

Molecular mechanism of the susceptibility difference between HLA-B*27:02/04/05 and HLA-B*27:06/09 to ankylosing spondylitis: substitution analysis,MD simulation,QSAR modelling,and in vitro assay

The human leukocyte antigen HLA-B27 is directly involved in the disease pathogenesis of ankylosing spondylitis (AS). HLA-B27 has a high degree of genetic polymorphism, with 105 currently known subtypes; the presence of aspartic acid at residue 116 (Asp116) has been found to play an essential role in AS susceptibility. Here, we systematically investigated the molecular mechanism of the susceptibility difference between the AS-associated subtypes HLA-B*27:02/04/05 and AS-unassociated subtypes HLA-B*27:06/09 to AS at sequence, structure, energetic and dynamic levels. In total seven variable residues were identified among the five studied HLA-B27 subtypes, in which Asp116 can be largely stabilized by a spatially vicinal, positively charged His114 through a salt bridge, while five other variable residues seem to have only a marginal effect on AS susceptibility. We also employed a quantitative structure–activity relationship approach to model the statistical correlation between peptide structure and affinity to HLA-B*27:05, a genetic ancestor of all other HLA-B27 subtypes and associated strongly with AS. The built regression predictor was verified rigorously through both internal cross-validation and external blind validation, and was then employed to identify potential HLA-B*27:05 binders from >20,000 cartilage-derived self-peptides. Subsequently, the binding potency of the top five antigenic peptides to HLA-B*27:05 was assayed in vitro using a FACS-based MHC stabilization experiment. Consequently, two (QRVGSDEFK and LRGAGTNEK) out of the five peptides were determined to have high affinity (BL₅₀ = 5.5 and 15.8 nM, respectively) and, as expected, both of them possess positively charged Lys at the C-terminus.     

Boc和Fmoc保护氨基酸与M树脂和王氏树脂结合率的比较

M树脂与Boc保护的亮氨酸、甘氨酸或苯丙氨酸的铯盐成酯反应摩尔比1:1.2,时间31h,其产物用重量法和水杨醛法测得的结合率均值分别为87.3%、89.1%和89.6%。王氏树脂与Fmoc保护的亮氨酸、甘氨酸或苯丙氨酸反应48h,树脂:Fmoc氨基酸:DCC:DMAP摩尔比为1:3:4:1,其产物结合率的均值分别为44.2%、30.4%和56.2%。结果表明,前者产物结合率高于后者。另外,重量法与水杨醛法测得的结合率相差不超过2.7%,但重量法更方便,易行。     

Dynamic and magnetic susceptibility effects on the MAS NMR linewidth of a tetrapeptide bound to different resins

Under magic angle spinning, the NMR spectrum of the tetrapeptide Ala‐Ile‐Gly‐Met bound to a Wang resin, and swollen in DMF, exhibits proton and carbon linewidths that are sharp enough to allow the complete characterization of the peptide using classical liquid‐state NMR methods. The proton linewidths of the bound peptide remain, however, about three times larger than those of the free peptide in solution. The residual NMR linewidth originates essentially from incompletely averaged magnetic susceptibility effects due to the Wang resin. Replacing the aromatic Wang resin with a PEGA or POEPOP resin removes this effect. To investigate the contribution to line broadening of the peptide dynamics, relaxation studies were performed on the peptide bound to Wang and POEPOP resins. Copyright © 2001 John Wiley & Sons, Ltd.     

Use of statistical methods to find the polysaccharide structural characteristics and the relationships between monosaccharide composition ratio and macrophage stimulatory activity of regionally different strains of Lentinula edodes

Multiple linear regression analysis was used to deduce the correlation between the monosaccharide composition ratios of 10 regionally different strains of Lentinula edodes and their in vitro macrophage stimulatory activities. Arabinose, xylose, mannose and galactose were identified as the monosaccharides that could be related to macrophage stimulatory activities. Additional principal component analysis and factor analysis methods were used to treat the same monosaccharide composition ratio data and the compositions of arabinose, xylose, mannose and galactose were found to be important. Interestingly, glucose, although presented in large compositions in all strains presumably forms the backbone of the polysaccharide structures, is not selected as the determinant factor for either structural characteristics or that of the in vitro macrophage stimulatory activities.