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.publishedVersio

Effects of blood contamination and the rostro-caudal gradient on the human cerebrospinal fluid proteome

Over the last years there has been an increased focus on the importance of knowing the effect of pre-analytical influence on the proteomes under study, particularly in the field of biomarker discovery. We present three proteomics studies examining the effect of blood contamination and the rostro-caudal gradient (RCG) on the cerebrospinal fluid (CSF) proteome, in addition to plasma/CSF protein ratios. The studies showed that the central nervous system (CNS) derived proteins appeared to be unaffected by the RCG, while the plasma-derived proteins showed an increase in concentration towards the lumbar area. This implies that the concentration of the plasma-derived proteins in CSF will vary depending on the volume of CSF that is collected. In the CSF samples spiked with blood, 262 of 814 quantified proteins showed an abundance increase of more than 1.5 fold, while 403 proteins had a fold change of less than 1.2 and appeared to be unaffected by blood contamination. Proteins with a high plasma/CSF ratio appeared to give the largest effect on the CSF proteome upon blood contamination. The results give important background information on how factors like blood contamination, RCG and blood-CNS-barrier influences the CSF proteome. This information is particularly important in the field of biomarker discovery, but also for routine clinical measurements. The data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange with identifier PXD000401

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

Categorization of proteins based on fold change, R-squared values and major expected contributing source of origin.

<p>In order to explore how different proteins were affected by the rostro-caudal gradient, we used the fold change and R-squared values of the proteins quantified with SID-MRM and iTRAQ to categorize the proteins into the three categories: affected by the RCG, unaffected by the RCG and uncertain. The fold change was calculated between the 1-2^nd and 44-45^th mL of CSF (referred to as RCG point 1 and 7), and the classification was based on the criteria from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090429#pone-0090429-t001" target="_blank">Table 1</a>.The proteins are also categorized into groups based on the major expected contributing source of origin, with Uniprot as the primary reference unless otherwise stated. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090429#pone.0090429.s006" target="_blank">Table S5</a> for details. The asterisk (*) marks conflicting results: affected + unaffected  =  uncertain; affected + uncertain  =  affected; unaffected + uncertain  =  unaffected. Proteins only quantified in the SID-MRM study is marked with ^a.</p

Flow chart of the rostro-caudal gradient study.

<p>In the rostro-caudal gradient (RCG) study, we examined the seven following points of the RCG from a PSP patient: 1-2^nd, 10-11^th, 16-17^th, 24-25^th, 31-32^nd, 38-39^th and 44-45^th mL CSF, referred to as RCG point 1-7, respectively. Twelve samples were digested and iTRAQ labeled (114-117). A reference, (labeled with iTRAQ reagent 114) containing the same amount of each RCG point, was included in each experiment. The RCG points 1 and 7 were included twice. After digestion and iTRAQ-labeling, samples were combined as follows: Exp. 1 (reference, 44-45^th mL, 24-25^th mL and 1-2^nd mL), Exp. 2 (reference, 1-2^nd mL, 38-39^th mL and 16-17^th mL), and Exp. 3 (reference, 10-11^th mL, 44-45^th mL and the 31-32^nd mL). The three experiments were fractionated by mixed mode reversed phase-anion chromatography (MM (RP-AX)) and analyzed on an Orbitrap Velos Pro. The protein abundances were averaged for each protein in the duplicate samples.</p

Overview of the conducted studies.

<p>Blood contamination: in experiment A protein depleted CSF was separated by SDS-PAGE, in experiment B crude CSF was in-solution digested. Progenesis LC-MS was used for data analysis. In the rostro-caudal gradient study, we used iTRAQ-labeling with mixed mode reversed phase-anion chromatography (MM (RP-AX)) fractionation. The Spectrum Mill software was used for data analysis. For verification we used stable isotope dilution (SID) multiple reaction monitoring (MRM) to monitor 70 peptides. MM (RP-AX) chromatography was used for fractionation and the MultiQuant software was used for data analysis. In the plasma/CSF ratio study equal amount of corresponding CSF and plasma from five patients were compared using dimethyl labeling. Samples were fractionated using strong cation exchange (SCX) chromatography. Proteome discoverer was used for data analysis. For all discovery experiments the samples were analyzed on an Orbitrap Velos Pro and for SID-MRM the samples were analyzed on a Q-trap 5500. SIS = Stable Isotope Standards</p

Flow chart of the plasma/CSF study.

<p>We compared the cerebrospinal fluid (CSF) and plasma protein ratio of five patients (P1-P5) using dimethyl labeling. The reference sample was a mix of equal total amount of CSF and plasma, and was labeled by light reagents. The five CSF samples were labeled by intermediate (IM) reagents, and the plasma samples were labeled by the heavy reagents. The light, IM and heavy labeled samples were combined and fractionated into eight fractions by strong cation exchange chromatography and analyzed on an Orbitrap Velos Pro. The average (and standard deviation) protein concentration of CSF and plasma, age at sampling and ratio male/female of the five patients are presented in the figure.</p

Protein concentration measurement of along the rostro-caudal gradient.

<p>Protein concentration of the cerebrospinal fluid (CSF)-derived proteins from the seven points along the rostro-caudal gradient (RCG). The CSF was measured in triplicates with Qubit, and error bars of the standard deviation are included. R squared value 0.8931.</p

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

Cerebrospinal fluid proteome shows disrupted neuronal development in multiple sclerosis

Despite intensive research, the aetiology of multiple sclerosis (MS) remains unknown. Cerebrospinal fluid proteomics has the potential to reveal mechanisms of MS pathogenesis, but analyses must account for disease heterogeneity. We previously reported explorative multivariate analysis by hierarchical clustering of proteomics data of MS patients and controls, which resulted in two groups of individuals. Grouping reflected increased levels of intrathecal inflammatory response proteins and decreased levels of proteins involved in neural development in one group relative to the other group. MS patients and controls were present in both groups. Here we reanalysed these data and we also reanalysed data from an independent cohort of patients diagnosed with clinically isolated syndrome (CIS), who have symptoms of MS without evidence of dissemination in space and/or time. Some, but not all, CIS patients had intrathecal inflammation. The analyses reported here identified a common protein signature of MS/CIS that was not linked to elevated intrathecal inflammation. The signature included low levels of complement proteins, semaphorin-7A, reelin, neural cell adhesion molecules, inter-alpha-trypsin inhibitor heavy chain H2, transforming growth factor beta 1, follistatin-related protein 1, malate dehydrogenase 1 cytoplasmic, plasma retinol-binding protein, biotinidase, and transferrin, all known to play roles in neural development. Low levels of these proteins suggest that MS/CIS patients suffer from abnormally low oxidative capacity that results in disrupted neural development from an early stage of the disease