Proteomics Analysis of Thermoplasma Quinone Droplets.

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin-N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D-SDS-PAGE/LC-MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods-column chromatography, immunoprecipitation, and LD ultracentrifugation-are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole-gel slicing and quantitative LC-MS/MS: Utilizing native MS-electropherograms in proteomic analysis for discovering structure and interaction-correlated differences.

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

Interaction of TiO2 nanoparticles with proteins from aquatic organisms: the case of gill mucus from blue mussel.

To better understand the mechanisms of TiO2 nanoparticle (NP) uptake and toxicity in aquatic organisms, we investigated the interaction of NPs with the proteins found in gill mucus from blue mussels. Mucus is secreted by many aquatic organisms and is often their first line of defense against pathogens, xenobiotics, and other sources of environmental stress. Here, five TiO2 NPs and one SiO2 NP were incubated with gill mucus and run out on a one-dimensional polyacrylamide gel for a comparative qualitative analysis of the free proteins in the mucosal solution and the proteins bound to NPs. We then used nanoscale liquid chromatography coupled with tandem mass spectrometry to identify proteins of interest. Our data demonstrated dissimilar protein profiles between the crude mucosal solution and proteins adsorbed on NPs. In particular, extrapallial protein (EP), one of the most abundant mucus proteins, was absent from the adsorbed proteins. After thermal denaturation experiments, this absence was attributed to the EP content in aromatic amino acids that prevents protein unfolding and thus adsorption on the NP. Moreover, although the majority of the protein corona was qualitatively similar across the NPs tested here (SiO2 and TiO2), a few proteins in the corona showed a specific recruitment pattern according to the NP oxide (TiO2 vs SiO2) or crystal structure (anatase TiO2 vs rutile TiO2). Therefore, protein adsorption may vary with the type of NP. Graphical abstract Proteins with adsorption selectivity as identified from isolated bands.

Differentiation of mucinous from non-mucinous pancreatic cyst fluid using dual-stained, 1 dimensional polyacrylamide gel electrophoresis.

BACKGROUND: Pancreatic cysts are being increasingly identified in patients. Mucinous cysts have malignant potential whereas non-mucinous cysts do not. Distinguishing potentially malignant cysts from harmless ones by the characterization of cyst fluid contents remains a difficult problem. This study was undertaken to determine whether cyst fluid mucin glycoprotein analysis could differentiate mucinous from non-mucinous pancreatic cysts. METHODS: Cyst fluid from 28 patients who underwent resection of a pancreatic cyst was used for the study. In each case the type of cyst was histologically identified. One dimensional SDS polyacrylamide gel electrophoresis (1D-SDS PAGE) was performed on cyst fluid samples. For the detection of the separated proteins, we employed a novel dual staining technique. The gel was first stained with periodic acid Schiff (PAS), a mucin histochemical stain followed by a secondary protein staining with Simply Blue Safestain (Invitrogen). RESULTS: Visual scoring (based on the presence of mucins) gave a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 88% for prediction of mucinous histology. CONCLUSIONS: One dimensional SDS polyacrylamide gel electrophoresis of pancreatic cyst fluid, followed by mucin (PAS) and protein (Simply Blue Safestain) staining, provides a means of concentrating and visualizing mucins, which allows the accurate differentiation of mucinous from non-mucinous histology in pancreatic cysts.