Wararat Chiangjong

Altered plasma proteome during an early phase of peritonitis-induced sepsis

Sepsis is a systemic response to infection commonly found in critically ill patients and is associated with multi-organ failure and high mortality rate. Its pathophysiology and molecular mechanisms are complicated and remain poorly understood. In the present study, we performed a proteomics investigation to characterize early host responses to sepsis as determined by an altered plasma proteome in a porcine model of peritonitis-induced sepsis, which simulated several clinical characteristics of human sepsis syndrome. Haemodynamics, oxygen exchange, inflammatory responses, oxidative and nitrosative stress, and other laboratory parameters were closely monitored. Plasma samples were obtained from seven pigs before and 12 h after the induction of sepsis, and plasma proteins were resolved with two-dimensional gel electrophoresis (n=7 gels/group; before being compared with during sepsis). The resolved proteins were stained with the SYPRO Ruby fluorescence dye and subjected to quantitative and comparative analyses. From approx. 1500 protein spots visualized in each gel, levels of 36 protein spots were significantly altered in the plasma of animals with sepsis (sepsis/basal ratios or degrees of change ranged from 0.07 to 21.24). Q-TOF (quadrupole-time-of-flight) MS and MS/MS (tandem MS) identified 30 protein forms representing 22 unique proteins whose plasma levels were increased, whereas six forms of five unique proteins were significantly decreased during sepsis. The proteomic results could be related to the clinical features of this animal model, as most of these altered proteins have important roles in inflammatory responses and some of them play roles in oxidative and nitrosative stress. In conclusion, these findings may lead to a better understanding of the pathophysiology and molecular mechanisms underlying the sepsis syndrome.

Characterizations and proteome analysis of platelet-free plasma-derived microparticles in β-thalassemia/hemoglobin E patients.

Aggregatability and oxidative damage of red blood cells (RBCs), platelet activation and increased amount of blood cells-derived microparticles (MPs) are thought to be the etiologies for the thrombotic risk in thalassemia, but with unclear mechanisms. Here we report cellular origins and increases in number, oxidative stress status, and procoagulant activity, as well as altered proteome of MPs isolated from β-thal/HbE patients. Flow cytometric analysis revealed that β-thal/HbE patients had significantly higher levels of phosphatidylserine (PS)-bearing MPs in platelet-free plasma (PFP) as compared to normal subjects. The high levels of MPs correlated with not only the increased procoagulant activity but also the increased platelet counts. Additionally, these PS-bearing MPs were originated mostly from platelets and RBCs, both of which had increased levels of reactive oxygen species. Proteome analysis of MPs by 2-DE followed by Q-TOF MS and MS/MS analyses identified 29 proteins with significantly altered levels in MPs derived from β-thal/HbE patients (e.g. the increased levels of peroxiredoxin 6, apolipoprotein E, cyclophilin A and heat shock protein 90). These findings suggest that the oxidative damage in platelets and RBCs potentially induces production of MPs with altered proteome that may, in turn, facilitate thromboembolic complications, which are commonly found in thalassemic patients. This article is part of a Special Issue entitled: Integrated omics.

Should Urine pH Be Adjusted Prior to Gel-Based Proteome Analysis?

The urine has become one of the most widely used clinical samples for biomarker discovery. The pH of human urine may vary largely from 4.5 to 8.0. Previously, it was questionable whether the urine pH would affect proteome analysis and whether the urine pH needed to be adjusted prior to proteome analysis remained unclear. We therefore performed a systematic analysis of the effect of urine pH on proteome profile. Midstream second morning and random afternoon urine samples were collected from 5 males and 5 females who were healthy and had no recent medication. After removal of cells and debris by low-speed centrifugation, pH levels of individual samples were measured and urinary proteins were isolated by 75% ethanol precipitation. Equally loaded 100 microg of proteins from individual samples were resolved in 2-DE (linear pH 3-10) and visualized with SYPRO Ruby fluorescence stain. There was no significant correlation between difference in the morning versus afternoon urine pH (DeltapH) and %match of protein spots derived from morning versus afternoon urine samples in individual samples (Pearsons r = 0.074; p = 0.839). In parallel, all individual samples with equal volume were pooled. The pH of the pooled urine was adjusted to 4-10 and urinary proteome profiles were analyzed as for individual samples. ANOVA with Tukeys posthoc multiple comparisons showed no significant differences in total number of detected spots and %match among various pH levels. Our data suggest that the urine pH has no significant effects on urinary proteome profile and thus needs no adjustment prior to gel-based proteome analysis.

Peeling as a novel, simple, and effective method for isolation of apical membrane from intact polarized epithelial cells.

Apical membrane of polarized epithelial cells is generally isolated by physicochemical methods, that is, precipitation with polyethylene glycol (PEG) or MgCl(2) followed by differential centrifugation or sucrose density gradient centrifugation. However, these protocols are considerably sophisticated and frequently accompanied by impurities (e.g., contaminations of basolateral membrane and intracellular organelles), particularly by inexperienced investigators. We have developed a simple and effective method for isolation of apical membrane from intact polarized renal tubular epithelial cells. On the basis of hydrous affinity and/or ionic interaction, the apical membrane could be efficiently peeled from the cells by four different materials-Whatman filter paper, nitrocellulose membrane, cellophane, and glass coverslip-all of which are available in most research laboratories. Phase-contrast and laser-scanning confocal microscopic examinations using anti-ZO-1 antibody showed that other parts of the cells, particularly tight junction complex, remained intact after peeling by all four of these surfaces. Western blot analyses of gp135 (apical membrane marker) and of Na(+)/K(+)-ATPase, LAMP-2, COX-4, and calpain-1 (markers of basolateral membrane, lysosome, mitochondria, and cytosolic compartment, respectively) revealed that peeling with Whatman filter paper and glass coverslip was most and second-most effective, respectively, without any contaminations from basolateral membrane and other intracellular organelles that could be detected in the samples isolated by peeling with nitrocellulose membrane and cellophane and by conventional methods (i.e., precipitation with PEG or MgCl(2) followed by differential centrifugation or sucrose density gradient centrifugation). Our physical method is very simple, easy to follow (even by inexperienced investigators), time-saving, and cost-effective with a higher efficiency (as compared with conventional methods) for isolation of apical membrane from polarized epithelial cells.

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel‐based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non‐pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non‐pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.

Identification of Brugia malayi immunogens by an immunoproteomics approach

Filariasis remains a health problem in tropical countries. Identification of immunogens from its causative organism would lead to development of a better diagnostic test, as well as vaccine discovery to effectively prevent this disease. We applied immunoproteomics to define potential immunogens of adult Brugia malayi that were recognized by IgM, IgG1 and IgG4 in sera of patients with four distinct clinical spectra of filariasis, including endemic asymptomatic, lymphangitis, elephantiasis and microfilaremia (n=5/group). Sera of healthy individuals (n=5) from non-endemic area served as the negative control. Brugian proteins were resolved by 2-DE and subjected to 2-D Western blot analysis probed with these sera. A total of 30 immunoreactive proteins recognized by IgM, IgG1 and IgG4 in sera from all four filarial groups were identified by Q-TOF MS and MS/MS analyses. Interestingly, only three immunogens were recognized by IgM in lymphangitis, elephantiasis and microfilaremia, but not in endemic asymptomatic group. IgG1 recognized 20 immunogens in endemic asymptomatic, lymphangitis and microfilaremia (mostly in endemic asymptomatic group), but not in elephantiasis, whereas IgG4 recognized 28 immunogens in all four filarial groups (mostly in microfilaremia). This large data set is an important resource for further development of a new diagnostic test and/or vaccine for filariasis.

Calcium oxalate crystals increased enolase-1 secretion from renal tubular cells that subsequently enhanced crystal and monocyte invasion through renal interstitium

Calcium oxalate monohydrate (COM) crystals cause kidney stone disease by still unclear mechanisms. The present study aimed to characterize changes in secretion of proteins from basolateral compartment of renal tubular epithelial cells after exposure to COM crystals and then correlated them with the stone pathogenesis. Polarized MDCK cells were cultivated in serum-free medium with or without 100 μg/ml COM crystals for 20 h. Secreted proteins collected from the lower chamber (basolateral compartment) were then resolved in 2-D gels and visualized by Deep Purple stain (n = 5 gels/group). Spot matching and intensity analysis revealed six protein spots with significantly altered levels in COM-treated samples. These proteins were then identified by tandem mass spectrometry (Q-TOF MS/MS), including enolase-1, phosphoglycerate mutase-1, actinin, 14-3-3 protein epsilon, alpha-tubulin 2, and ubiquitin-activating enzyme E1. The increased enolase-1 level was confirmed by Western blot analysis. Functional analysis revealed that enolase-1 dramatically induced COM crystal invasion through ECM migrating chamber in a dose-dependent manner. Moreover, enolase-1 bound onto U937 monocytic cell surface markedly enhanced cell migration through the ECM migrating chamber. In summary, our data indicated that the increased secretory enolase-1 induced by COM crystals played an important role in crystal invasion and inflammatory process in renal interstitium.

Differential plasma proteome profiles of mild versus severe β-thalassemia/Hb E

The severity of thalassemia is currently classified based on clinical manifestations and multiple tests. In the present study, we performed a plasma proteome analysis to identify differentially expressed proteins compared between normal subjects and patients with mild and severe forms of β-thalassemia/hemoglobin E (Hb E). Plasma samples were collected from patients with mild (n = 8) and severe (n = 12) forms as well as healthy normal individuals (n = 12). Clinical chemistry revealed that several parameters, i.e., hematological indices, oxidative stress markers, antioxidant enzymes, and erythropoietic activity, had significant differences among these three groups. After removal of seven major abundant proteins, the plasma proteome profiles were compared using two-dimensional gel electrophoresis. Spot matching, quantitative intensity analysis, and statistics revealed differential levels of 32 and 9 proteins when comparing normal vs. patients and mild vs. severe forms, respectively. These proteins were successfully identified by quadrupole time-of-flight mass spectrometry and/or tandem mass spectrometry. The decreased level of ADP-ribosylation factor guanine nucleotide-exchange factor 2 in β-thalassemia/Hb E patients compared to healthy individuals and the decreased level of endothelin-converting enzyme 2 in severe form compared to the mild form of the disease were validated by Western blot analysis. Our data provide a number of proteins that may lead to better understanding of the pathophysiology of thalassemia or for novel biomarkers which can be used to simply differentiate mild and severe forms of β-thalassemia/Hb E without any need for multiple tests.

Marked changes in red cell membrane proteins in hereditary spherocytosis: a proteomics approach

Hereditary spherocytosis (HS) is the most common red cell membrane defect resulting from protein abnormalities. However, changes in red cell membrane proteins in HS remain under-investigated. We therefore evaluated red cell membrane proteome in non-splenectomized, mild-degree HS patients (n = 9) compared to healthy individuals (n = 5). Proteins derived from the red cell membranes of each subject were resolved in each two-dimensional gel and visualized by Deep Purple fluorescence staining. Spot matching and quantitative intensity analysis revealed 56 differentially expressed protein spots (41 increased and 15 decreased), which were then successfully identified by quadrupole time-of-flight mass spectrometry. Among these, seven isoforms/subunits of spectrin were markedly increased (up to 10.51 folds), whereas two isoforms/subunits of band-3 protein were decreased approximately 50% as compared to normal red cells. However, two isoforms/subunits of protein 4.1 were increased, while another isoform/subunit was decreased. All these significantly altered proteins were subjected to global protein network analysis using Ingenuity Pathways Analysis tool, which revealed three important networks related to HS, including Network I: Cell death, genetic and hematological disorders; Network II: Cell cycle, carbohydrate metabolism and molecular transport; and Network III: Genetic and hematological disorders, cell-to-cell signaling and interactions. These data offer many opportunities and new roadmaps for further functional studies to better understand the biology and pathogenic mechanisms of HS.

A comparative study of different dyes for the detection of proteomes derived from Escherichia coli and MDCK cells: Sensitivity and selectivity

We compared sensitivity and selectivity of five dyes for detection of 2D PAGE-resolved proteins derived from Escherichia coli and MDCK cells. The sensitivity of these dyes was in the following order: SYPRO Ruby>Deep Purple>CBB-G250>CBB-R250>Colloidal Gold. Also, we report herein for the first time the application of Colloidal Gold (which is commonly used for staining proteins on blotted membranes) for in-gel staining of proteins. For E. coli, most of the dyes preferably detected proteins with pI range of 4.0-6.9, whereas Deep Purple preferably detected proteins with less acidic range (pI 5.0-7.9). For MDCK cells, while other dyes preferably stained proteins at pI range of 5.0-7.9, Colloidal Gold preferably stained more basic proteins (pI 7.0-9.9). This preferential staining property of Colloidal Gold to basic proteins was confirmed in SDS-PAGE-separated lysozyme (pI 9.4), compared to calmodulin (pI 4.0) and albumin (pI 6.0). These data provide useful information to select appropriate dyes for gel-based proteomic analysis of individual samples.