Metallomics study in CSF for putative biomarkers to predict cerebral vasospasm

Cerebral vasospasm (CV) refers to physical narrowing of brain cerebral arteries due to over-contraction of the arterial wall, which often arises following a subarachnoid hemorrhage (SAH). CV is frequently associated with poorer outcomes in those patients. Between the ictus of SAH and its CV complication, there is a 3-7 days delay, which provides a time window to predict and possibly prevent the onset CV. Since the precise pathomechanism of CV is still unclear and approaches for predicting it are inefficient, more effective ways of predicting CV need to be developed. As a protective nourishing fluid flows through the subarachnoid space, cerebrospinal fluid (CSF) closely relates to the health states of the central nervous system (CNS). Analysis of CSF can provide invaluable information to diagnose, treat and prevent diseases of the CNS because of its relatively direct representation of events in the brain. Therefore, we assume that the components in CSF and their alterations may reflect the state of aneurismal SAH and the development of vasospasm. In this study, three types of CSF from healthy control, and patients who suffered SAH and its complication, CV, were investigated via two-dimensional separations in combination with elemental and molecular mass spectrometry detection for the identification of elemental species. Size exclusion chromatography (SEC) was initially used with selective metal detection by inductively coupled plasma mass spectrometry (ICPMS) for characterizing size distribution of metal species. Various molecular distribution patterns were exhibited at different metal detection points (Fe, Ni, Cu, Zn and Pb). Further identification of possible metallopeptides and metalloprotein in tryptic digested fractions from the three sample types were made via reverse phase (RP)-Chip and electrospray mass spectrometry (MS) in combination with the Spectrum Mill data base search engine accessing appropriate data bases. Comparisons were generated to show suggested protein similarities or differences across the three CSF sample types. Six protein families with possible protein markers were further identified, and may be considered as possible focus areas for discovering valuable biomarkers to preclude the debilitating or deadly vasospasm.     

Studying protein phosphorylation in low MW CSF fractions with capLC-ICPMS and nanoLC-CHIP-ITMS for identification of phosphoproteins

An initial study of protein phosphorylation in human cerebral spinal fluid (CSF) is described. CSF is an important body fluid for study of proteins and metabolites and may lead to the ultimate development of molecular markers to predict neurological diseases or their complications, such as in the case of hemorrhagic stroke. The use of capillary liquid chromatography coupled to inductively coupled plasma mass spectrometry (capLC-ICPMS) for screening using (31)P as the internal elemental tag atom at ultratrace levels, in combination with molecular mass spectrometry using Spectrum Mill and MASCOT database search engines for peptide identification, is a novel approach in its application to CSF relevant phosphopeptides and phosphorylated proteins. CapLC-ICPMS combined with nano liquid chromatography electrospray ionization, ion trap mass spectrometry (nanoLC-CHIP/ITMS), was utilized for initial experiments with CSF. Specific low-level screening for (31)P containing compounds is accomplished, and nanoLC-CHIP/ITMS provided the corresponding peptide information and subsequent protein identifications. The fractions containing (31)P from screening by the capLC-ICPMS were collected offline and analyzed separately with nanoLC-CHIP/ITMS. Synthetic phosphopeptides were used to test the method and to estimate lowest quantifiable limits for phosphorus. Tryptically digested beta-casein was then used to demonstrate the viability of the methodology for the complex CSF matrix from hemorrhagic stroke patients while also analyzing for native phosphopeptides in the CSF.     

Significant proteins affecting cerebral vasospasm using complementary ICPMS and MALDI-MS

Cerebral vasospasm (CV) following subarachnoid hemorrhagic stroke affects more than one million people each year. The etiology and prevention of CV is currently of great interest to researchers in various fields of medical science. More recently, the idea that selenium could be playing a major role in the onset of cerebral vasospasm has come into the spotlight. This study focused on using newly established metallomics techniques in order to explore the proteome associated with CV and if selenium might affect the discovered proteins. Size exclusion chromatography coupled to inductively coupled plasma mass spectrometry, along with LC-MALDI-TOF/TOF were both essential in determining protein identifications in three different sample types; a control (normal, healthy patient, CSF control), SAH stroke patients (no vasospasm, CSF C) and SAH CV patients (CSF V). The results of this study, although preliminary, indicate the current methods are applicable and warrant further application to these clinically important targets.     

A preliminary study of metalloproteins in CSF by CapLC-ICPMS and NanoLC-CHIP/ITMS

Cerebrospinal fluid (CSF) has frequently been studied to explore the total metal concentrations in patients with neurodegenerative diseases. Some examples of neurologic diseases include but are not limited to intracerebral hemorrhage, intraventricular hemorrhage, traumatic brain injury, subarachnoid hemorrhage and hydrocephalus. In this study, however, a comprehensive approach was begun using metallomics methods. First, two molecular weight cutoff filters were used to separate CSF constituents by molecular weight. The remaining CSF was then separated with capillary liquid chromatography/normal bore liquid chromatography and analyzed with inductively coupled mass spectrometry (ICPMS). With this ICPMS screening, a possible iron associated protein was suggested by nanoliquid chromatography-CHIP/ion trap mass spectrometry (nanoLC-CHIP/ITMS) identification in conjunction with a Spectrum Mill database search. In this preliminary study, three different types of pooled CSF were partially characterized by their metal (Pb, Mg, Zn, Fe and Cu) containing species with suggestions for fuller studies. Chemical 'differences' in the CSF and metal constituents suggests some utility in this analysis for understanding some of the complications observed following subarachnoid hemorrhage.     

CSF hypocretin-1/orexin-A concentrations in patients with subarachnoid hemorrhage (SAH)

The aim of this study was to examine the role of the hypothalamic hypocretin/orexin system in complications of delayed ischemic neuronal deficit (DIND) resulting from symptomatic vasospasm in patients with aneurysmal subarachnoid hemorrhage (SAH). CSF hypocretin-1/orexin-A levels were measured in 15 SAH patients. DIND complications occurred in seven patients with symptomatic vasospasm. Hypocretin-1/orexin-A levels were low in SAH patients during the 10 days following the SAH event. CSF hypocretin-1/orexin-A levels were lower in patients with DIND complications than in those who did not develop DIND. A significant transient decline in CSF hypocretin-1/orexin-A levels was also observed at the onset of DIND in all patients with symptomatic vasospasm. The reduced hypocretin/orexin production observed in SAH patients may reflect reduced brain function due to the decrease in cerebral blood flow. These results, taken together with recent experimental findings in rats that indicate hypocretin receptor 1 (orexin 1 receptor) mRNA and protein are elevated following middle cerebral artery occlusion, suggest that a reduction in hypocretin/orexin production in SAH and DIND patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia.     

Clinical-scale high-throughput human plasma proteome analysis: lung adenocarcinoma

Clinical proteomics requires the stable and reproducible analysis of a large number of human samples. We report a high-throughput comprehensive protein profiling system comprising a fully automated, on-line, two-dimensional microflow liquid chromatography/tandem mass spectrometry (2-D microLC-MS/MS) system for use in clinical proteomics. A linear ion-trap mass spectrometer (ITMS) also known as a 2-D ITMS instrument, which is characterized by high scan speed, was incorporated into the microLC-MS/MS system in order to obtain highly improved sensitivity and resolution in MS/MS acquisition. This system was used to evaluate bovine serum albumin and human 26S proteasome. Application of these high-throughput microLC conditions and the 2-D ITMS resulted in a 10-fold increase in sensitivity in protein identification. Additionally, peptide fragments from the 26S proteasome were identified three-fold more efficiently than by the conventional 3-D ITMS instrument. In this study, the 2-D microLC-MS/MS system that uses linear 2-D ITMS has been applied for the plasma proteome analysis of a few samples from healthy individuals and lung adenocarcinoma patients. Using the 2-D and 1-D microLC-MS/MS analyses, approximately 250 and 100 different proteins were detected, respectively, in each HSA- and IgG-depleted sample, which corresponds to only 0.4 microL of blood plasma. Automatic operation enabled the completion of a single run of the entire 1-D and 2-D microLC-MS/MS analyses within 11 h. Investigation of the data extracted from the protein identification datasets of both healthy and adenocarcinoma groups revealed that several of the group-specific proteins could be candidate protein disease markers expressed in the human blood plasma. Consequently, it was demonstrated that this high-throughput microLC-MS/MS protein profiling system would be practically applicable to the discovery of protein disease markers, which is the primary objective in clinical plasma proteome projects.     

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET_18–34 (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066 5nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET_18–34 were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.Endothelin-1 (ET-1)¹ is the most potent vasoconstrictor in the central nervous system. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) (1). However, it is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. ET-1 is produced upon the cleavage of its precursor BigET-1 by the highly specific metalloprotease Endothelin Converting Enzyme-1 (ECE-1). The rate-limiting step of ET-1 production is the expression and localization of ECE-1, whose catalytic activity is confined to the extracellular C-terminal domain. Previously, we have demonstrated that the catalytically active C terminus can be shed from the cell surface (2). This results in the release of a truncated but catalytically active form of ECE-1 into the extracellular milieu.Although the presence of a soluble form of ECE-1 has been demonstrated in vitro, it is yet to be shown in any human biological fluid. In this study, we have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of ECE-1 in CSF of SAH subjects.     

Identification of 5-Hydroxy Eicosatetraenoic Acid in Cerebrospinal Fluid After Subarachnoid Hemorrhage

Abstract: To detect and identify lipid peroxides in the CFS following subarachnoid hemorrhage (SAH), CSF samples were obtained sequentially from 10 patients who developed typical vasospasm and were analyzed by HPLC and gas chromatography-mass spectrometry. One of the peaks appearing on the 7th day after SAH was identified as 5-hydroxy eicosatetraenoic acid. On HPLC, an identical peak was detected in samples from other SAH patients. The results gave unequivocal evidence that peroxides of arachidonic acid are present in the CSF following SAH, and a correlation between them and the occurrence of vasospasm seemed likely. The hypothesis that lipid peroxides are involved in the genesis of vasospasm deserves further investigation.     

Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful. To identify ALS specific biomarkers, we compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). We identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects. Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS results for transthyretin and cystatin C by immunoblot and immunohistochemistry using commercially available antibodies. These findings identify a panel of CSF protein biomarkers for ALS.     

Comparative peptidome analyses of the profiles of the peptides ranging from 1­–10 KD in CSF samples pooled from probable sporadic CJD and non-CJD patients

The shotgun strategy applying tandem mass spectrometry has been widely used to identify the proteins that are differentially distributed among diseases for its high reliability and efficiency. To find out the potential difference of protein profiles in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, especially in the fraction ranging from 1–10 KD, the CSF samples of 40 probable sporadic CJD (sCJD) patients, 32 non-CJD cases with dementia and 17 non-CJD cases without dementia were separately pooled and enriched by the magnetic beads based weak cation exchange chromatography (MB-WCX). After trypsin digestion, each enriched CSF was separated and identified by RP-HPLC-ESI-QTOF MS/MS. In total, 42, 53 and 47 signals of proteins were identified in the pooled CSF fraction less than 10 KD of probable sCJD, non-CJD with dementia and non-CJD without dementia, respectively. Compared with that of probable sCJD, the similarity of CSF protein profiles of non-CJD with dementia (76.2%) were higher than that of non-CJD without dementia (57.1%). Nine CSF proteins were found to be specially observed in probable sCJD group. Those data may help to select the potential biomarkers for diagnosis of CJD. Additionally, further studies of the small segments of cellular proteins in CSF of CJD patients may also provide scientific clues for understanding the neuropathogenesis of TSEs.     

Increase of the IL-1 beta and IL-6 levels in CSF in patients with vasospasm following aneurysmal SAH

Cytokines play a key role in mutual influence of the immunological, endocrine and CNS systems. It has been proven that proinflammatory ILs may intensify the cascade of biochemical changes in ischemic brain damage. Vasospasm, which may accompany SAH and often coexists with symptoms of DINDs, is the cause of ischemic changes in the brain. It is thought that immunological mechanisms may be one of the causes of degenerative-productive changes in vessel walls, in delayed vasospasm following SAH, which lead to substantial vasospasm and in consequence too cerebral ischemia. In the randomly selected group of patients, who underwent surgical treatment after aneurysmal SAH, we determined the concentration of IL-1 beta and IL-6 in CSF in the periods between Days 0 to 3; 4 to 7; and 8 to 15 after the occurrence of SAH. The presence and dynamics of development of vasospasm were assessed on the basis of increasing DINDs as well as CT and cerebral angiography. We examined the concentrations of ILs in CSF using radioimmunological methods, applying commercially available tests for their assessment. We found that in the period between 8 and 15 days after SAH, in increasing delayed vasospasm and DINDs, here is a statistically significant increase concentration of IL-1 beta in CSF (105.4 +/- 46.9 pg x ml-1; p<0.005), and no significant changes in patients without vasospasm and neurological deficits. On the other hand, we noted a statistically significant increase concentration of IL-6 in CSF (4802 +/- 1170 ng x ml-1; p<0.05) only in the acute phase after SAH (Days 0-3) in patients in poor clinical condition, in whom delayed vasospasm and cerebral ischemia developed later. This increase of ILs level in CSF is probably related to the intensity of the SAH, and secondarily aggravates the vasospasm and ischemic changes in the brain.     

Corticotropin releasing hormone (CRH) increases beta-endorphin (beta-end like) concentration in cerebrospinal fluid of rats with vasospasm following subarachnoid hemorrhage.

The chronic stage of vasospasm occurring several days after subarachnoid hemorrhage (SAH) is characterized by the development of histopathologic changes in cerebral arteries causing cerebral ischemia. Numerous experimental data indicate the involvement of immune mechanisms in the angiopathy caused by SAH. Endogenous opioids play also an important role in the ischemic lesions of the brain. Corticotropin releasing hormone (CRH) induces the release of beta-endorphin (beta-END) from hypothalamic neurons and also from mononuclear white blood cells. The function of CRH and beta-END in vasospasm following SAH and the interrelationship between neuroendocrine and immune changes requires further elucidation. In the present study we investigated the influence of CRH injected into cerebral cisterna magna (CM) of rats on beta-END-like level in cerebrospinal fluid (CSF) in acute and chronic phase of cerebral vasospasm following artificial SAH. Acutely CRH induced a significant rise of beta-END-like in CSF both in SAH and sham SAH rats. However, in rats subjected to SAH, a single injection of CRH caused a prolonged rise of 5-END in CSF, which was also seen 2 days after SAH, during the chronic phase of vasospasm. The obtained results indicate that CRH increases neuroendocrine changes induced by SAH, probably by an activation of immune cells involved in the patomechanism of chronic vasospasm.     

Comparative peptidome analyses of the profiles of the peptides ranging from 1–10 KD in CSF samples pooled from probable sporadic CJD and non-CJD patients

The shotgun strategy applying tandem mass spectrometry has been widely used to identify the proteins that are differentially distributed among diseases for its high reliability and efficiency. To find out the potential difference of protein profiles in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, especially in the fraction ranging from 1–10 KD, the CSF samples of 40 probable sporadic CJD (sCJD) patients, 32 non-CJD cases with dementia and 17 non-CJD cases without dementia were separately pooled and enriched by the magnetic beads based weak cation exchange chromatography (MB-WCX). After trypsin digestion, each enriched CSF was separated and identified by RP-HPLC-ESI-QTOF MS/MS. In total, 42, 53 and 47 signals of proteins were identified in the pooled CSF fraction less than 10 KD of probable sCJD, non-CJD with dementia and non-CJD without dementia, respectively. Compared with that of probable sCJD, the similarity of CSF protein profiles of non-CJD with dementia (76.2%) were higher than that of non-CJD without dementia (57.1%). Nine CSF proteins were found to be specially observed in probable sCJD group. Those data may help to select the potential biomarkers for diagnosis of CJD. Additionally, further studies of the small segments of cellular proteins in CSF of CJD patients may also provide scientific clues for understanding the neuropathogenesis of TSEs.Key words: Creutzfeldt-Jakob disease, CSF, MB-HPLC-ESI-QTOF, 1–10 KD, comparative peptidome     

Identification of phosphorylation sites in the polo-like kinases Plx1 and Plk1 by a novel strategy based on element and electrospray high resolution mass spectrometry

A novel strategy for the determination of protein phosphorylation sites is described and applied to the polo-like kinases Plx1 (Xenopus laevis) and Plk1 (Homo sapiens). The strategy comprises the sequential application of the following techniques: proteolytic digestion, capillary liquid chromatography (LC)-inductively coupled plasma mass spectrometry with phosphorus detection, capillary LC-electrospray mass spectrometry and electrospray tandem mass spectrometry. In this approach, phosphopeptides are generated, their elution time in capillary LC is determined, candidate phosphopeptides at the corresponding elution times are identified, and positive identification and sequencing of phosphopeptides is performed in the last step of the analysis. Using this technique, Ser25/26, Ser326, and Ser340 were identified as phosphorylation sites in recombinant Plx1, and Ser340 was identified as the major phosphorylation site in a kinase-dead mutant of Plx1 expressed in okadaic acid-treated Sf9 insect cells. A site corresponding to Ser326 in Plx1 was also shown to be phosphorylated in the human polo-like kinase Plk1 (Ser335). Element mass spectrometry with phosphorus detection provides a quantitative phosphorylation profile of all phosphorylation sites accessible by LC.     

PhosphoBlast, a computational tool for comparing phosphoprotein signatures among large datasets

Identification of specific protein phosphorylation sites provides predicative signatures of cellular activity and specific disease states such as cancer, diabetes, Alzheimer disease, and rheumatoid arthritis. Recent progress in phosphopeptide isolation technology and tandem mass spectrometry has provided the means to identify thousands of phosphorylation sites from a single biological sample. These advances now make it possible to profile global changes in the phosphoproteome at an unprecedented level. However, although this technology is generating a wealth of information, there is currently no efficient means to identify phosphoprotein signatures shared among large phosphoprotein databases. Identification of common phosphoprotein signatures found in biologically relevant systems and their conservation throughout evolution would provide valuable insight into mechanisms of signal transduction and cell function. Here we describe the development of a computational program (PhosphoBlast) that can rapidly match thousands of phosphopeptides that share phosphorylation sites within and across species. PhosphoBlast analysis of several large phosphoprotein datasets from the literature revealed common phosphorylation signatures shared across diverse experimental platforms and species. Moreover PhosphoBlast is a powerful analysis tool to identify specific phosphosite mutations. Comparison of the mouse and human phosphoproteomes revealed more than 130 specific phosphoamino acid mutations, some of which are predicted to alter protein function. Further analysis revealed that known phosphorylated amino acids are more evolutionally conserved than the Ser/Thr/Tyr amino acids not known to be phosphorylated. Together our results demonstrate that PhosphoBlast is a versatile mining tool capable of identifying related phosphorylation signatures and phosphoamino acid mutations among complex proteomics datasets in a highly efficient and accurate manner. PhosphoBlast will aid in the informatics analysis of the phosphoproteome and the identification of phosphoprotein biomarkers of disease.     

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.     

Proteomic analysis of human ventricular cerebrospinal fluid from neurologically normal, elderly subjects using two-dimensional LC-MS/MS

A two-dimensional liquid chromatography separation scheme coupled to tandem mass spectrometry (2-D LC-MS/MS) was utilized to profile the proteome of human CSF. Ventricular CSF samples acquired post-mortem from 10 cognitively normal elderly subjects (mean +/- SEM Braak stage = 1.7 +/- 0.2) were analyzed to determine their protein composition. Raw CSF samples were subjected to an immunobased processing method to remove highly abundant albumin and immunoglobulin (Ig), allowing better detection of lower-abundance proteins. Samples were subjected to trypsin proteolysis followed by C18 solid-phase extraction. Tryptic CSF peptides were separated using a 2-D LC column, in which both strong cation exchange (SCX) and C18 phases were packed into a single capillary. MS/MS spectra of CSF peptides were searched against a human sub-database of the NBCI nonredundant database using the SEQUEST algorithm. Search results were further filtered using DTAselect, and individual samples were compared to one another using Contrast. Using this method, we were able to unambiguously identify 249 CSF proteins from 10 subjects. Of these proteins, 38% were unique to individual subjects, whereas only 6% were common to all 10 subjects. These results suggest considerable subject-to-subject variability in the CSF proteome.     

Lipopolysaccharide-responsive phosphoproteins in Nicotiana tabacum cells.

Mounting evidence is merging to affirm the effectiveness of bacterial lipopolysaccharides (LPS) as biological control agents, inducers of innate immunity, and to stimulate/potentiate the development of defense responses in plants through protein phosphorylation-mediated signal perception/transduction responses. In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins. Substantial differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. In this study, qualitative and quantitative changes in phosphoproteins of Nicotiana tabacum suspension cells during elicitation by LPS from the Gram-negative bacteria, Burkholderia cepacia, were analyzed using two-dimensional electrophoresis in combination with a phosphoprotein-specific gel stain. Trypsin digested phosphoproteins were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and nano-electrospray-ionization liquid chromatography tandem mass spectrometry (nano-ESI-LC/MS/MS). A total of 27 phosphoproteins were identified from 23 excised gel spots. The identified phosphoproteins indicate that LPS(B.cep)-induced signal perception/transduction involves G-protein coupled receptor signaling, Ca(2+)/calmodulin-dependent signaling pathways, H(+)-ATPase regulation of intracellular pH, thioredoxin-mediated signaling and phosphorylation of 14-3-3 regulatory proteins. Other targets of LPS(B.cep)-responsive phosphorylation included NTP pool maintenance, heat shock proteins, protein biosynthesis and chaperones as well as cytoskeletal tubulin. The results add novel insights into the biochemical process of LPS perception and resulting signal transduction.     

Increased levels of F2-isoprostanes following aneurysmal subarachnoid hemorrhage in humans

Subarachnoid hemorrhage (SAH) resulting from aneurysmal rupture is the major cause of nontraumatic SAH. We hypothesized that oxidative stress could be increased following aneurysmal SAH due to hemoglobin release and ischemia-reperfusion injury and that may further contribute to poor outcome. We collected plasma and cerebrospinal fluid (CSF) samples from 11 non-SAH controls and 15 aneurysmal SAH patients for up to 10 days after surgery and investigated status of oxidative stress in patients. Results showed that mean or peak levels of F(2)-isoprostanes (F(2)-IsoPs), a specific marker of lipid peroxidation, and total nitrate/nitrite, metabolites of nitric oxide and peroxynitrite, in CSF and plasma were significantly higher in SAH patients than in controls. First-day levels were also higher in CSF, but not in plasma, in SAH patients. Moreover, mean and peak levels of CSF F(2)-IsoPs were positively correlated with poor outcome or severity of clinical conditions in patients. Furthermore, levels of retinol, delta-tocopherol, beta+gamma-tocopherol, lutein, beta-carotene, and coenzyme Q(10) in plasma were significantly lower in SAH patients than in controls. Our results indicate that oxidative damage may play important roles in the severity and complications of aneurysmal SAH and suggest that means to suppress lipid peroxidation may be beneficial in improving the outcome of aneurysmal SAH.     

Rapid analysis of metabolic stability of dopamine receptor antagonists and detection of their metabolites by liquid chromatography/tandem mass spectrometry

In vitro metabolic stability of dopamine D(3)/D(4) receptor antagonists and identification of their metabolites by high-performance liquid chromatography (HPLC) coupled with ion-trap mass spectrometry (ITMS) were assessed in rat liver microsomes. The compounds were divided into three cassette groups for rapid quantitative analysis of multiple drugs and simultaneous detection of their metabolites. The samples from incubation with rat liver microsomes were pooled into designed cassette groups and analyzed by HPLC/electrospray ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and for 30min. The metabolic stability of the examined dopamine receptor antagonists was in the range of 9.9-84.4%. In addition, the present cassette analysis allowed the simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multistage tandem mass spectrometry on the ion-trap instrument allowed characterization of the structure of the detected metabolites. N,O-dealkylation and ring hydroxylation reactions were identified as major metabolic reactions in piperazinylalkylisoxazole derivatives. These results suggested that the present approach is useful for the rapid evaluation of metabolic stability and structural characterization of metabolites within a short period in new drug discovery.