Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.     

Enterobacterial 38-kDa outer membrane protein is an age-dependent molecular marker of innate immunity and immunoglobulin deficiency as results from its reactivity with IgG and IgA antibody

In earlier studies on an animal model we observed protective properties of outer membrane proteins (OMPs) of Shigella, Hafnia, and Escherichia coli strains. In order to investigate human sera for reactivity with OMPs we subjected these proteins to immunoblotting with umbilical cord plasma and sera from children and adults. The IgG and IgA antibodies interacted primarily with a 38-kDa protein, in similar way for several enterobacterial strains, but different for Pseudomonas aeruginosa. This observation prompted us to determine the reactivity with the purified 38-kDa OMP in the sera of several groups of children. The reactivity of the protein from Shigella flexneri serotype 3a with sera in ELISA was age dependent, increasing from low reactivity in infants to the adult antibody level. The IgG and IgA antibody specific response thus revealed the normal pattern of immunity. The level of IgA and IgG antibody was significantly low in child patients with IgA and/or IgG immunoglobulin deficiencies, but was at the healthy control level in children with recurrent respiratory tract inflammation. These data correlated with total IgA and IgG levels in immunoglobulin-deficient children. The results indicate that this protein may serve as an immunodiagnostic marker, but also as an antigen carrier in vaccines.     

Identification and characterization of diadenosine 5',5"'-P1,P2 -diphosphate and diadenosine 5',5"'-P1,P3-triphosphate in human myocardial tissue.

We examined whether human cardiac tissue contains diadenosine polyphosphates and investigated their physiological role. Extracts from human cardiac tissue from transplant recipients were fractionated by size exclusion-, affinity-, anion exchange- and reversed-phase chromatography. MALDI-MS analysis of two absorbing fractions revealed molecular masses of 676.2 Da and 756.0 Da. The UV spectra of both fractions were identical to that of adenosine. Postsource decay MALDI mass spectrometry indicated that the molecules with a mass of 676.2 Da and 757.0 Da contained AMP and ATP, respectively. As shown by enzymatic cleavage, both molecules consist of two adenosines interconnected by either two or three phosphates in 5'-positions of the riboses. Two substances can be identified as 5',5"'-P1,P2-diphosphate (Ap2A) and 5',5"'-P1, P3-triphosphate (Ap3A). Ap2A and Ap3A, together with ATP and ADP, are stored in myocardial-specific granules in biologically active concentrations. In the isolated perfused rat heart, Ap2A and Ap3A caused dose-dependent coronary vasodilations. In myocardial preparations, Ap2A and Ap3A attenuated the effect of isoproterenol, exerting a negative inotropic effect. The calcium current of guinea pig ventricular myocytes, stimulated by isoproterenol, was also attenuated by Ap2A and Ap3A. The presence of Ap2A and Ap3A in cardiac-specific granules and the actions of these substances on the myocardium and coronary vessels indicate a role for these substances as endogenous modulators of myocardial functions and coronary perfusion.     

Impaired bactericidal activity of serum of a child suffering from focal proliferative glomerulonephritis.

The serum of a child with focal proliferative glomerulonephritis was found to exhibit a weaker bactericidal activity against Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis and Escherichia coli strains as compared with sera of the child's parents. The child's serum showed a low haemolytical activity of complement as well as a low C3 concentration. The authors believe that the abnormal complement concentration could cause the impaired bactericidal activity of the patient serum.     

Phagocytosis and killing of Staphylococcus aureus by granulocytes in mice after granulocyte-macrophage colony stimulating factor (GM-CSF) injection

GM-CSF is a hematopoietic growth factor. In vitro it stimulates the proliferation of myeloid progenitors and formation of granulocyte and macrophage colonies. It was found that GM-CSF in vitro is also stimulated the function of mature granulocytes, but we have no information about such influence in vivo. The purpose of this investigation was the evaluation in vivo of the GM-CSF effect on phagocytosis, bactericidal activity, and lysosome enzyme activities in granulocytes. GM-CSF was injected into mice subcutaneously during 5 consecutive days in the dose of 1 microgram/kg/d. The examination of the percent of cell phagocytizing bacteria (Staphylococcus aureus), NBT test, bactericidal activity and activation of acid phosphatase, alkaline phosphatase, peroxidase and esterase was performed every day and an evident increase of the tested parameters was found. These results prove in vivo activation of granulocytes by GM-CSF.     

Usefulness of ELISA and radial immunodiffusion tests for evaluation of the degree of purification of influenza diagnostic and vaccine preparations

It is necessary to use new diagnostic tests for careful and rapid evaluation of a degree of purification and immunogenicity of vaccine anti-influenza preparations. In this study in order to obtain this purpose a radial immunodiffusion++ test and immunoenzymatic test (ELISA) were used Recommended by WHO radial immunodiffusion++ test enable to determine a level of haemagglutinin of particular types and subtypes of influenza virus in polyvalent preparations. However, this test is time consuming therefore for hemagglutinin level determination ELISA test was adapted. This test is hundred times more sensitive and can be applied with success for determination of hemagglutinin level of influenza virus A or B. For evaluation of a degree of purification of vaccine preparations ELISA was elaborated, in which as an index of purification of preparation a level of ovalbumin is determined. This test is specific and extremely sensitive, and it is possible to determine ovalbumin level with accuracy of 1ng in 1 ml of preparation.     

Sensitivity of Shigella strains to bactericidal activity of human serum. I. Participation of two independently active mechanisms in bactericidal action against Shigella sonnei phase II.

Normal human serum is strongly bactericidal for all studied Shigella sonnei phase II (10 strains). The studied bacteria were sensitive to two alternative mechanisms of the bactericidal activity of serum factors. The first mechanism involves the action of serum in which complement (C) is activated by the studied bacteria via the classical pathway. Lysozyme did not participate in this reaction. The second mechanism involves the combined action of two factors: C activated via the alternative pathway and lysozyme.