CSF-PR 2.0: An Interactive Literature Guide to Quantitative Cerebrospinal Fluid Mass Spectrometry Data from Neurodegenerative Disorders.

The rapidly growing number of biomedical studies supported by mass spectrometry based quantitative proteomics data has made it increasingly difficult to obtain an overview of the current status of the research field. A better way of organizing the biomedical proteomics information from these studies and making it available to the research community is therefore called for. In the presented work, we have investigated scientific publications describing the analysis of the cerebrospinal fluid proteome in relation to multiple sclerosis, Parkinson's disease and Alzheimer's disease. Based on a detailed set of filtering criteria we extracted 85 data sets containing quantitative information for close to 2000 proteins. This information was made available in CSF-PR 2.0 (http://probe.uib.no/csf-pr-2.0), which includes novel approaches for filtering, visualizing and comparing quantitative proteomics information in an interactive and user-friendly environment. CSF-PR 2.0 will be an invaluable resource for anyone interested in quantitative proteomics on cerebrospinal fluid.

Label-free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis.

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non-MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label-free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p-value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS-enriched proteins.

Quantitative proteomics suggests decrease in the secretogranin-1 cerebrospinal fluid levels during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing-remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase-3-like protein 1, secretogranin-1 (Sg1), cerebellin-1, neuroserpin, cell surface glycoprotein MUC18, testican-2 and glutamate receptor 4. An independent sample set of 13 early-MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early-MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.

Cerebrospinal fluid proteomics in multiple sclerosis.

Multiple sclerosis (MS) is an immune mediated chronic inflammatory disease of the central nervous system usually initiated during young adulthood, affecting approximately 2.5 million people worldwide. There is currently no cure for MS, but disease modifying treatment has become increasingly more effective, especially when started in the first phase of the disease. The disease course and prognosis are often unpredictable and it can be challenging to determine an early diagnosis. The detection of novel biomarkers to understand more of the disease mechanism, facilitate early diagnosis, predict disease progression, and find treatment targets would be very attractive. Over the last decade there has been an increasing effort toward finding such biomarker candidates. One promising strategy has been to use state-of-the-art quantitative proteomics approaches to compare the cerebrospinal fluid (CSF) proteome between MS and control patients or between different subgroups of MS. In this review we summarize and discuss the status of CSF proteomics in MS, including the latest findings with a focus on the last five years. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR).

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia.

BACKGROUND: The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. RESULTS: Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. CONCLUSIONS: Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.

Identification of dynamic changes in proteins associated with the cellular cytoskeleton after exposure to okadaic acid.

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100-500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.

Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid.

BACKGROUND: The mechanisms behind formation and filling of intracranial arachnoid cysts (AC) are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1) Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2) Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF) and plasma. METHODS: AC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA) to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM) initiated detection and sequence analysis). RESULTS: We did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC.In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that the majority of abundant proteins in AC fluid also can be found in CSF. Compared to plasma, as many as 104 proteins in AC were not found in the list of 3017 plasma proteins. CONCLUSIONS: Based on the protein content of AC fluid, our data indicate that temporal AC is a homogenous condition, pointing towards a similar AC filling mechanism for the 14 patients examined. Most of the proteins identified in AC fluid have been identified in CSF, indicating high similarity in the qualitative protein content of AC to CSF, whereas this was not the case between AC and plasma. This indicates that AC is filled with a liquid similar to CSF. As far as we know, this is the first proteomics study that explores the AC fluid proteome.

Increased interaction between DJ-1 and the Mi-2/ nucleosome remodelling and deacetylase complex during cellular stress.

DJ-1 was originally identified to be an oncogenic product, but has later been shown to be highly multifunctional. DJ-1 plays a role in oxidative stress response and transcriptional regulation, and loss of its function leads to an early onset of Parkinsonism. To further understand the mechanisms behind DJ-1's role in cell survival and death, we investigated alternations in endogenous DJ-1 protein-protein interaction in apoptotic cells exposed to the phosphatase inhibitor okadaic acid. By combining cellular stable isotopic labelling of amino acids in cell culture, sub-cellular fractionation, co-immunoprecipitation, and MS, we identified a novel group of DJ-1 interaction partners that increased their association to DJ-1 in okadaic acid-exposed cells. These proteins were integral components of the Mi-2/nucleosome remodelling and deacetylase (NuRD) complex. Knockdown of DJ-1 and MTA2, a core component of the NuRD complex, had a similar and pro-apoptotic effect on the transcriptional- and p53-dependent cell death induced by daunorubicin. On the other hand, MTA2 knockdown had no significant effect on the progression of p53-independent okadaic acid-induced apoptosis. Our data suggest that the increased DJ-1/NuRD interaction is a general anti-stress response regulated by okadaic acid-induced modifications of DJ-1. The observed interaction between DJ-1 and the NuRD complex may give new clues to how DJ-1 can protect cells from p53-dependent cell death.

Iodine concentration in Norwegian milk and dairy products.

The present study was conducted to determine the iodine concentration in Norwegian-produced milk and a selection of dairy products. The iodine concentration of eighty-five samples of milk and dairy products was analysed by inductively coupled plasma-MS. Low-fat milk and organic milk were sampled from nineteen and seven different locations in Norway, respectively, during the summer and winter season of 2000. Other milk and dairy products were chiefly collected during the summer season. Low-fat milk from the summer season had significantly lower median iodine concentration (88 microg/l, range 63-122 microg/l) compared with low-fat milk from the winter season (232 microg/l, range 103-272 microg/l). The median iodine concentration of organic summer milk (60 microg/l) was significantly lower than the iodine concentration of organic winter milk (127 microg/l). There were no significant differences in the low-fat-milk samples with regard to geographical sampling location. Whey cheese (Tine Gudbrandsdalsost) iodine concentration was significantly higher (803 microg/kg) than the median iodine concentration in casein cheeses such as Jarlsberg and Norvegia of 201 and 414 microg/kg, respectively. With a recommended iodine intake of 150 microg/d for adults, a daily intake of 0.4 litres milk meets the requirement with 25 % during the summer and more than 60 % during the winter season. Thus, milk and dairy products are important determinants of iodine intake in Norway.