No abstract

Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.

Preeclapsia (PE) is a severe disorder that occurs during pregnancy, leading to maternal and fetal morbidity and mortality. PE affects about 3-8% of all pregnancies. In this study, we conducted liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS) to analyze serum samples depleted of the six most abundant proteins from normal and PE-affected pregnancies to profile serum proteins. A total of 237 proteins were confidently identified with ＜ 1% false discovery rate from the two groups of duplicate analysis. The expression levels of those identified proteins were compared semiquantitatively by spectral counting. To further validate the candidate proteins with a quantitative mass spectrometric method, selective reaction monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant women with severe PE (n = 8) or normal pregnant women (n = 5) was conducted. α2-HS-glycoprotein (AHSG), retinol binding protein 4 (RBP4) and α-1-microglobulin/bikunin (AMBP) and Insulin like growth factor binding protein, acid labile subunit (IGFBP-ALS) were confirmed to be differentially expressed in PE using SRM (P ＜ 0.05). Among these proteins, AHSG was verified by ELISA and showed a statistically significant increase in PE samples when compared to controls.

Research on the Bonghan system has recently prompted great interest in the theory proposed by Bong Han Kimin in the early 1960s. In order to study the biochemical characteristics of the Bonghan system, we analyzed Bonghan ducts (BHD) on the surface of rabbit intestines and characterized the liquid in the BHD at the level of the proteome. Proteomic analysis was performed using nano LC-ESI MS/MS. Using a solution digestion technique, we identified 70 different proteins in the liquid of the BHD. We used gel-based digestion to analyze the BHD itself and our results showed the presence of 207 proteins. We used these proteins to analyze gene ontology (GO) to yield insights into biological processes, molecular functions and cellular compartmentalization. Remarkably, GO clustering showed high concentrations of proteins involved in metabolism. These proteins are not usually found in blood, lymph or blood vessels, and thus can be useful for characterizing BHD. It is worth studying their association with stem cells, especially mesenchymal stem cells, cancer cells and myeloid cells.

^Äëíê~Åí= In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in bëÅÜÉêáÅÜá~=Åçäá. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LC-MS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system. © KSBB hÉóïçêÇëW=ìåå~íìê~ä= êÉÅçãÄáå~åí= éêçíÉáåI= ÉñéêÉëëáçå= ëóëíÉãI= êÉëáÇìÉ= ëéÉÅáÑáÅ= ìåå~íìê~ä=~ãáåç=~ÅáÇ= áåÅçêéçê~íáçåI= iJ Üçãçéêçé~êÖóäÖäóÅáåÉ=