Carol L. Nilsson

Proteome analysis of cerebrospinal fluid proteins in Alzheimer patients

&NA; By comparing the CSF proteome between Alzheimer disease (AD) patients and controls it may be possible to identify proteins that play a role in the disease process and thus to study the pathogenesis of AD. We used mini‐gel technology in a two‐dimensional electrophoresis procedure, sensitive SYPRO Ruby staining and mass spectrometry for clinical screening of disease‐influenced CSF proteins in 15 AD patients and 12 controls. The levels of six proteins and their isoforms, including proapolipoprotein, apolipoprotein E, &bgr;‐2 microglobulin, retinol‐binding protein, transthyretin, and ubiquitin, were significantly altered in CSF of AD patients. The most prominently altered proteins were the apolipoproteins, especially proapolipoprotein.

Comparative genome- and proteome analysis of cerebral cortex from MK-801-treated rats

cDNA microarrays and two‐dimensional gel‐electrophoresis in combination with mass spectrometry, were used to screen alterations in mRNA and protein levels, respectively, in cerebral cortex of MK‐801‐treated rats. The rats were divided in two groups; group 1 (short‐term treated) and group 2 (long‐term treated). In group 1, four genes were up‐regulated and five down‐regulated. In group 2, seven genes were up‐regulated and six down‐regulated. In group 1, the levels of one protein was increased and eight proteins reduced. In group 2, the levels of two proteins were increased and four proteins reduced. Several of the altered genes (casein kinase 2, glutamic acid decarboxylase, synaptotagmin, gamma aminobutyric acid [GABA] transporter, creatine kinase, and cytochrome c oxidase) and proteins (superoxide dismutase, hsp 60, hsp 72 and γ‐enolase) have previously been connected to schizophrenia. Alterations of the genes (microglobulin, c‐jun proto‐oncogene, 40S ribosomal protein S19, adenosine diphosphate (ADP)‐ribosylation factors, platelet‐derived growth factor, fructose‐bisphophate aldolase A, and myelin proteolipid) and the proteins (stathmin, H+‐transp. Adenosine triphosphate (ATP) synthase, pyruvate dehydrogenase, β‐actin and α‐enolase), have not, to our knowledge, earlier been implicated in schizophrenia pathology. Overall, these results with a combined approach of genomics and proteomics add to the validity of subchronic N‐methyl‐D‐aspartate (NMDA)‐receptor antagonist treatment as an animal model of schizophrenia.

Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1α signaling network: An initial study in GSC11 glioblastoma stem cells

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

Structural and Quantitative Comparison of Cerebrospinal Fluid Glycoproteins in Alzheimer’s Disease Patients and Healthy Individuals

Glycoproteins in cerebrospinal fluid (CSF) are altered in Alzheimer’s Disease (AD) patients compared to control individuals. We have utilized albumin depletion prior to 2D gel electrophoresis to enhance glycoprotein concentration for image analysis as well as structural glycoprotein determination without glycan release using mass spectrometry (MS). The benefits of a direct glycoprotein analysis approach include minimal sample manipulation and retention of structural details. A quantitative comparison of gel-separated glycoprotein isoforms from twelve AD patients and twelve control subjects was performed with glycoprotein-specific and total protein stains. We have also compared glycoforms in pooled CSF obtained from AD patients and control subjects with mass spectrometry. One isoform of α1-antitrypsin showed decreased glycosylation in AD patients while another glycosylated isoform of an unassigned protein was up-regulated. Protein expression levels of α1-antitrypsin were decreased, while the protein levels of apolipoprotein E and clusterin were increased in AD. No specific glycoform could be specifically assigned to AD.

Advances in Quantitative Phosphoproteomics

Sample Preparation 735 Chromatographic Methods for Phosphoproteomics 735 Combined Chromatographic Approaches 736 Metal-Based Enrichment Strategies 737 Phosphotyrosine Enrichment 737 Mass Spectrometry Analysis and Data Processing 737 Quantitation 739 From Data Analysis to Signaling Pathways 740 New Biological Insights Derived from Quantitative Phosphoproteomic Applications 741 Perspectives 742 Author Information 743 Biography 743 Acknowledgment 743 References 743

Fingerprinting of Helicobacter pylori strains by matrix-assisted laser desorption/ionization mass spectrometric analysis.

Helicobacter pylori is an important human gastrointestinal pathogenic bacterium which is believed to colonize approximately one-half of the worlds population. Different strains of H. pylori possess virulence proteins for tissue colonization, host evasion and tissue damage. The bacteria display genomic instabilities that include gene rearrangements and gene exchange. Recently, methods for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have been established for monitoring biomarkers in bacterial extracts. In order to establish a set of H. pylori specific biomarkers as well as a set of strain-specific biomarkers, we examined lysates and extracts from six different strains of this bacterium. Three different MALDI matrices, alpha-cyano-4-hydroxycinnamic acid, sinapinic acid, and ferulic acid were tested for sensitivity of analysis. Also, the effects of solubilizing analytes with the detergent n-octyl-beta-D-glucopyranoside were explored. It was found that a set of H. pylori specific, and a probable set of strain-specific, biomarkers could be established using MALDI-TOFMS. The use of H. pylori fingerprinting by MALDI may be useful for typing of these bacteria, or for studying genetic drift at the phenotypic level in specific strains.

HpaA is essential for Helicobacter pylori colonization in mice.

ABSTRACT Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice.

Differences in the neuropeptide Y-like immunoreactivity of the plasma and platelets of human volunteers and depressed patients

In this study, patients with recurrent major depression were found to have significantly lower neuropeptide Y-like immunoreactivity (NPY-LI) in platelet-poor plasma (p < 0.01) and significantly higher NPY-LI in platelets (p < 0.001) compared to controls. Also, qualitative differences in the NPY-LI in platelet-poor plasma (PPP) and platelets were detected when the samples were analyzed by HPLC followed by RIA of the collected fractions.

Overexpression of ST6GalNAcV, a ganglioside-specific α2,6-sialyltransferase, inhibits glioma growth in vivo

Aberrant cell-surface glycosylation patterns are present on virtually all tumors and have been linked to tumor progression, metastasis, and invasivity. We have shown that expressing a normally quiescent, glycoprotein-specific α2,6-sialyltransferase (ST6Gal1) gene in gliomas inhibited invasivity in vitro and tumor formation in vivo. To identify other glycogene targets with therapeutic potential, we created a focused 45-mer oligonucleotide microarray platform representing all of the cloned human glycotranscriptome and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Among the many significant changes in glycogene expression observed, of particular interest was the observation that an additional α2,6-sialyltransferase, ST6 (α-N-acetyl-neuraminyl-2,3-β-galactosyl-1,3)-N-acetylgalactosaminide α2,6-sialyltransferase 5 (ST6GalNAcV), was expressed at very low levels in all glioma and glioma cell lines examined compared with normal brain. ST6GalNAcV catalyzes the formation of the terminal α2,6-sialic acid linkages on gangliosides. Stable transfection of ST6GalNAcV into U373MG glioma cells produced (i) no change in α2,6-linked sialic acid-containing glycoproteins, (ii) increased expression of GM2α and GM3 gangliosides and decreased expression of GM1b, Gb3, and Gb4, (iii) marked inhibition of in vitro invasivity, (iv) modified cellular adhesion to fibronectin and laminin, (v) increased adhesion-mediated protein tyrosine phosphorylation of HSPA8, and (vi) inhibition of tumor growth in vivo. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters invasivity in a manner that may have significant therapeutic potential.

The cerebral hemorrhage-producing cystatin C variant (L68Q) in extracellular fluids

A variant of the normal extracellular cysteine protease inhibitor cystatin C (L68Q-cystatin C), is the amyloid precursor in hereditary cystatin C amyloid angiopathy (HCCAA). It has been suggested that the mutation causes cellular entrapment ofL68Q-cystatin C in vivo and that the variant protein is not secreted to extracellular fluids. In order to test this hypothesis, we used matrix-assisted laser desorption ionization time-of-flight mass spectrometry in an effort to demonstrate the presence ofL68Q- along with wildtype cystatin C in plasma and cerebrospinal fluid (CSF) of HCCAA-patients. Plasma from all five investigated HCCAA-patients contained both L68Q- and wildtype cystatin C. The presence of approximately equal amounts of cystatin C dimers and monomers was demonstrated in plasma from HCCAA-patients, whereas only monomers could be found in normal plasma. L68Q-wildtype-cystatin C heterodimers seem to be present in the dimeric cystatin C population. CSF from six HCCAA-patients also contained cystatin C-dimers and monomers, but the dimeric fraction was minute. CSF from control patients did not contain dimeric cystatin C These results suggest that the milieu of L68Q-cystatin C is important for its stability and dimerization status and that certain milieus might hinder its further development into oligomers, amyloid fibrils and other precipiting aggregates.