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MS. We (ms + we)
Selected AbstractsCharacterization of sialylated and fucosylated glycopeptides of ,2-glycoprotein I by a combination of HILIC LC and MALDI MS/MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Akira Kondo Abstract Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N -glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human ,2-glycoprotein I (,2-GPI, apolipoprotein H), which contains multiple N -glycosylation sites. Conditions for rapid proteolytic digestion of ,2-GPI using low-specificity proteases were optimized to detect ,2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N -glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of ,2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation. [source] Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 5 2006Akira Shimizu Abstract Some variant proteins cause diseases, and some diseases result in increases of proteins with abnormally modified structures. The detection, characterization, and estimation of the relative amounts of protein variants and abnormally modified proteins are important for clinical diagnosis and for elucidation of the mechanisms of the pathogenesis of diseases. Analysis of the covalent structures of proteins using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization MS (LC-ESI-MS), which had been developed by the early 1990s, have largely replaced analyses by conventional protein chemistry. Here, we review the detection and characterization of hemoglobin variants, HbA1c measurement, detection of carbohydrate-deficient transferrin, and identification of variants of transthyretin (TTR) and Cu/Zn-superoxide dismutase (SOD-1) using soft ionization MS. We also propose the diagnostic application of the signals of modified forms of TTR, that is, S-sulfonated TTR and S-homocysteinyl TTR. The relative peak height ratio of the abnormal/normal components gives valuable information about the instability of variants and enables the detection of unstable Hb subunits or thalassemia heterozygotes. We found unique modified structures of TTR that suggested changes in amyloid fibrils. © 2006 Wiley Periodicals, Inc. [source] Review: Mitochondria and disease progression in multiple sclerosisNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 6 2008D. Mahad Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Recent evidence suggests that dysfunction of surviving demyelinated axons and axonal degeneration contribute to the progression of MS. We review the evidence for and potential mechanisms of degeneration as well as dysfunction of chronically demyelinated axons in MS with particular reference to mitochondria, the main source of adenosine-5,-triphosphate in axons. Besides adenosine-5,-triphosphate production, mitochondria play an important role in calcium handling and produce reactive oxygen species. The mitochondrial changes in axons lacking healthy myelin sheaths as well as redistribution of sodium channels suggest that demyelinated axons would be more vulnerable to energy deficit than myelinated axons. A dysfunction of mitochondria in lesions as well as in the normal-appearing white and grey matter is increasingly recognized in MS and could be an important determinant of axonal dysfunction and degeneration. Mitochondria are a potential therapeutic target in MS. [source] Application of quantitative immunoprecipitation combined with knockdown and cross-linking to Chlamydomonas reveals the presence of vesicle-inducing protein in plastids 1 in a common complex with chloroplast HSP90CPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2009Heinrich Heide Abstract Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein,protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein,protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle-inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross-linking step (-X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross-linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions. [source] Proteomic patterns for classification of ovarian cancer and CTCL serum samples utilizing peak pairs indicative of post-translational modificationsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2007Chenwei Liu Abstract Proteomic patterns as a potential diagnostic technology has been well established for several cancer conditions and other diseases. The use of machine learning techniques such as decision trees, neural networks, genetic algorithms, and other methods has been the basis for pattern determination. Cancer is known to involve signaling pathways that are regulated through PTM of proteins. These modifications are also detectable with high confidence using high-resolution MS. We generated data using a prOTOFÔ mass spectrometer on two sets of patient samples: ovarian cancer and cutaneous t-cell lymphoma (CTCL) with matched normal samples for each disease. Using the knowledge of mass shifts caused by common modifications, we built models using peak pairs and compared this to a conventional technique using individual peaks. The results for each disease showed that a small number of peak pairs gave classification equal to or better than the conventional technique that used multiple individual peaks. This simple peak picking technique could be used to guide identification of important peak pairs involved in the disease process. [source] Construction of quantitative proteome reference maps of mouse spleen and lymph node based on two-dimensional gel electrophoresisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2006Yayoi Kimura Abstract Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000,spots on the 2-DE images and consequently were able to determine that 919,spots were derived from 328,different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform. [source] The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacteriumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2006Jeong Wook Lee Abstract Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement. [source] Potential protein markers for nutritional health effects on colorectal cancer in the mouse as revealed by proteomics analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2006Githa Breikers Abstract It is suggested that colorectal cancer might be prevented by changes in diet, and vegetable consumption has been demonstrated to have a protective effect. Until now, little is known about the effects of vegetable consumption at the proteome level. Therefore, the effect of increased vegetable intake on the protein expression in the colonic mucosa of healthy mice was studied. Aim was to identify the proteins that are differentially expressed by increased vegetable consumption and to discriminate their possible role in the protection against colorectal cancer. Mice were fed four different vegetable diets, which was followed by analysis of total cellular protein from colonic mucosal cells by a combination of 2-DE and MS. We found 30 proteins that were differentially expressed in one or more diets as compared to the control diet. Six could be identified by MALDI-TOF MS: myosin regulatory light chain 2, carbonic anhydrase I, high-mobility group protein 1, pancreatitis-associated protein 3, glyceraldehyde-3-phosphate dehydrogenase and ATP synthase oligomycin sensitivity conferral protein. Alterations in the levels of these proteins agree with a role in the protection against colon cancer. We conclude that these proteins are suitable markers for the health effect of food on cancer. The observed altered protein levels therefore provide support for the protective effects of vegetables against colorectal cancer. [source] Markers of the Hepatic Component of the Metabolic Syndrome as Predictors of Mortality in Renal Transplant RecipientsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2010D. M. Zelle Cardiovascular disease (CVD) is a leading cause of mortality in renal transplant recipients (RTRs). Metabolic syndrome (MS) is highly prevalent in RTRs. Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic component of MS. We investigated associations of NAFLD markers with MS and mortality. RTRs were investigated between 2001 and 2003. NAFLD markers, alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and alkaline phosphatase (AP) were measured. Bone and nonbone fractions of AP were also determined. Death was recorded until August 2007. Six hundred and two RTRs were studied (age 52 ± 12 years, 55% men). At baseline 388 RTRs had MS. Prevalence of MS was positively associated with liver enzymes. During follow-up for 5.3[4.5,5.7] years, 95 recipients died (49 cardiovascular). In univariate Cox regression analyses, GGT (HR = 1.43[1.21,1.69], p < 0.001) and AP (HR = 1.34[1.11,1.63], p = 0.003) were associated with mortality, whereas ALT was not. Similar associations were found for cardiovascular mortality. Adjustment for potential confounders, including MS, diabetes and traditional risk factors did not materially change these associations. Results for nonbone AP mirrored that for total AP. ALT, GGT and AP are associated with MS. Of these three enzymes, GGT and AP are associated with mortality, independent of MS. These findings suggest that GGT and AP are independently related to mortality in RTRs. [source] Impact of exposure to war stress on exacerbations of multiple sclerosisANNALS OF NEUROLOGY, Issue 2 2008Daniel Golan MD Objective To assess the relation between stress caused by the perils of rocket attack on civilian centers in northern Israel during the 2006 war between Hezbollah and Israel and multiple sclerosis (MS) exacerbations. Methods Participants were 156 patients with relapsing-remitting MS. We compared the number of relapses during and after the war with similar time periods at the preceding year. Exposure to war events and resulting subjective stress were evaluated by means of structured interviews using questionnaires previously validated. Results During the 33 days of the war, there were 18 relapses among our patients, compared with 1 to 6 relapses in similar time periods over the 12 months before the war (p < 0.001,0.02). There was no increase in relapse rate during the 3 months that followed the war (p = 0.58). The percentage of patients reporting the experience of intense subjective stress during the hostilities was significantly greater among patients with wartime relapse compared with the rest of the patients (44 vs 20%). The proportion of patients reporting high levels of distress associated with exposure to rocket attacks, displacement from home, and perceived life threat was greater in relapsing patients compared with those in remission (67 vs 42%, p = 0.05; 33 vs 11%, p = 0.02; and 33 vs 15%, p = 0.08, respectively). Interpretation Our data suggest that civilian exposure to war stress is associated with increased risk for MS relapse. These findings provide insight to stress-related risk factors associated with relapses of MS. Ann Neurol 2008 [source] The value of conventional high-field MRI in MS in the light of the McDonald criteria: a literature reviewACTA NEUROLOGICA SCANDINAVICA, Issue 3 2010L. S. Lunde Larsen Lunde Larsen LS, Larsson HBW, Frederiksen JL. The value of conventional high-field MRI in MS in the light of the McDonald criteria: a literature review. Acta Neurol Scand: 122: 149,158. © 2010 John Wiley & Sons A/S. The diagnosis of MS is based on the revised McDonald criteria and is multidisciplinary. Both clinical and paraclinical measures are included. High-field magnetic resonance imaging (MRI) is becoming increasingly available and it is therefore necessary to clarify possible advantages of high-field MRI in MS. The aim of this paper was to review MRI studies in MS where a direct comparison of MRI at high field with MRI at 1,1.5 tesla (T) had been performed. The studies evaluated were found by searching Pubmed with relevant terms including MeSH terms. The reviewed studies all found the conspicuity of lesions to be better at high field. Of the seven studies, six found more and bigger lesions at high-field MRI. In the present paper, the relevant MRI sequences are evaluated in detail. The detection of more lesions at high-field strength did not seem to lead to earlier diagnosis of clinically definite multiple sclerosis. Further larger studies of patients with clinically isolated syndromes are needed to settle the question of a diagnostic consequence of high-field imaging in MS. We suggest that the next revision of the McDonald diagnostic criteria include a recommendation of field strength. [source] Recovery and refractoriness of auditory evoked fields after gaps in click trainsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004Alexander Gutschalk Abstract When clicks are presented in a train at a rate above ,5 Hz, they evoke a sustained field in human auditory cortex that can be recorded by magnetoencephalography. In this study we evaluated how this sustained field continues when a click train is interrupted by a silent gap. The stimuli were click trains with interclick intervals of either 12 or 24 ms, which produce pitches of 83.3 or 41.7 Hz, respectively. The click trains were 996 ms in duration with a gap of 12, 24, 48, 96, or 192 ms beginning 504 ms post-stimulus onset. The sustained field for click trains with short gaps was similar to the one evoked by a continuous click train. Subtraction of the response evoked by a solitary click train of 504 ms enabled estimation of the sustained field in the interval after the gap. The comparison revealed that the sustained field amplitude after the gap was larger than that at the onset of the initial click train in the interval from 150 to 350 ms after onset, and the difference decreased with gap duration. In contrast, the transient P1m was refractory for gaps up to 48 ms, but had nearly recovered its initial amplitude for gaps of 192 ms. We discuss how these results might relate to the perception, i.e. if an interrupted click train is perceived as one continuous sound with a transient gap or as two successive events. [source] Loudness summation and the mismatch negativity event-related brain potential in humansPSYCHOPHYSIOLOGY, Issue 1 2006Attila Oceák Abstract Infrequently omitting a sound from a repetitive sequence elicits the mismatch negativity (MMN) ERP response when the stimulus onset asynchrony (SOA) is less than 200 ms. We contrasted two alternative explanations of omission MMN. (1) Each sound starts a separate temporal integration process. Omissions violate the constancy of the temporal structure within the integration window. (2) Sounds preceding an omission are perceived to be louder than those followed by a sound within the integration period, because omissions allow the full stimulus aftereffect to be included in perceived loudness. We varied the SOA between 117 and 217 ms. For this case, the temporal structure explanation predicts that no MMN will be elicited, whereas the loudness summation explanation predicts that MMN will be elicited. MMN was elicited by tone omissions with random SOA, suggesting that loudness summation plays an important role in the elicitation of omission MMN. [source] Local recovery of Ca2+ release in rat ventricular myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 2 2005Eric A. Sobie Excitation,contraction coupling in the heart depends on the positive feedback process of Ca2+ -induced Ca2+ release (CICR). While CICR provides for robust triggering of Ca2+ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca2+ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca2+ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca2+ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca2+ spark sites. Examination of these repetitive Ca2+ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by ,80 ms. We conclude that restitution of Ca2+ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca2+ to SR regulatory proteins. [source] | ||||||||||||||||