Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual

We test the ability of state-of-the-art two-dimensional electrophoresis (2-DE) technology to enable the proteome mapping of ante mortem cerebrospinal fluid (CSF) from a single individual. Using the sensitive technologies of a fluorescent protein stain and fluorescence laser scanning of 2-DE gels, combined with matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry (MALDI-TOF/TOF-MS) for protein identification, a highly detailed 2-DE map of the CSF proteome was created. The 2-DE map contains 600 identified spots representing 82 different proteins. Of the 82 proteins identified, 25 have not appeared in any previously published 2-DE map of CSF, and 11 have not been previously reported to exist in CSF. Most of the identifications originate from an ante mortem CSF sample collected from a single hydrocephalus patient. A supplemental map created from neurologically normal patients is also presented. A webpage with protein identification and scoring information from both maps is available at http://www.leelab.org/csfmap.     

Quantification of beta-trace protein and detection of transferrin isoforms in mixtures of cerebrospinal fluid and blood serum as models of rhinorrhea and otorrhea diagnosis

Different mixtures from a serum pool and a cerebrospinal fluid (CSF) pool were used as models to study CSF contamination in secretions by determining two CSF specific proteins: beta-trace protein (beta-TP) and the asialo-transferrin (a-Tf) band which was detected by isoelectric focusing (IEF) with Tf specific immunofixation. Beta-TP and total Tf were measured immunonephelometrically. Secretion/serum ratios of beta-TP content > 2.0 indicated CSF contaminations with > or = 5% (v/v) CSF; this was confirmed by detecting the a-Tf band by IEF. Reliable a-Tf bands were only revealed with secretion/serum rations of Tf contents < 0.1, indicating an interference of major sialo-Tf fractions with the a-TF band detection in the sample. For CSF detection in rhinorrhea and otorrhea, complementary use of beta-TP assay and a-Tf assay is recommended. Preanalytically, dilution or concentration of the sample as well as denaturation of Tf and beta-TP should be prevented by optimizing sample collection.     

Analysis of cerebrospinal fluid from patients with psychiatric and neurological disorders by two-dimensional electrophoresis: identification of disease-associated polypeptides as fibrin fragments

Two-dimensional electrophoresis (2-DE) of cerebrospinal fluid (CSF) samples--from 347 patients with various psychiatric and neurological disorders--and subsequent silver staining revealed two additional polypeptides (Mr 40,000) in 49% of 111 schizophrenics, 46% of 43 schizoaffective patients, 36% of 41 patients with affective disorders, 43% of 28 patients with multiple sclerosis, but not in 25 patients without neurological symptomatology, nor in 9 patients with Lues, and in only 2 of 25 patients with AIDS. The two polypeptides, as detected by 2-DE, eluted after size exclusion chromatography in fractions containing proteins with Mr greater than 200,000. After 2-DE of CSF samples, enriched by gel chromatography, the polypeptides were immobilized by blotting onto glass-fiber membranes and subjected to N-terminal sequencing. Polypeptide A was identified as beta-chain remnant (beta 2), derived from plasmin cleavage of fibrin(ogen). After size exclusion chromatography, 2-DE, and Western blotting, polypeptide A and B, as well as several other spots, reacted with fibrinogen antibodies, suggesting that the polypeptides are subunits of a fibrin degradation complex.     

Quantification of β-trace protein and detection of transferrin isoforms in mixtures of cerebrospinal fluid and blood serum as models of rhinorrhea and otorrhea diagnosis

Different mixtures from a serum pool and a cerebrospinal fluid (CSF) pool were used as models to study CSF contamination in secretions by determining two CSF specific proteins: β-trace protein (β-TP) and the asialo-transferrin (a-Tf) band which was detected by isoelectric focusing (IEF) with Tf specific immunofixation. β-TP and total Tf were measured immunonephelometrically. Secretion/serum ratios of β-TP content > 2.0 indicated CSF contaminations with ≥ 5% (v/v) CSF; this was confirmed by detecting the a-Tf band by IEF. Reliable ¶a-Tf bands were only revealed with secretion/serum rations of Tf contents < 0.1, indicating an interference of major sialo-Tf fractions with the a-TF band detection in the sample. For CSF detection in rhinorrhea and otorrhea, complementary use of β-TP assay and a-Tf assay is recommended. Preanalytically, dilution or concentration of the sample as well as denaturation of Tf and β-TP should be prevented by optimizing sample collection.     

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.     

Prostaglandin D2 synthase and its post-translational modifications in neurological disorders

Prostaglandin D2 synthase (PGDS) (beta-trace protein) is a highly abundant cerebrospinal fluid (CSF) glycoprotein. A number of studies have been performed to determine the potential value of this protein for the diagnosis of various neurological disorders. The measurement of total PGDS levels in CSF has proved marginally useful for this purpose, but promising results were obtained while investigating changes in the posttranslational modifications (PTM) pattern. Using 2-DE analysis, we previously showed that PGDS is differentially expressed in ante- and post mortem CSF samples. In the present study, we examined whether the PGDS isoforms may help to distinguish stroke and neurodegenerative disease patients from healthy subjects. The pattern of PGDS PTM was analyzed in CSF from patients with various neurological disorders (n = 44) using IEF/immunoblotting techniques. Strong alterations of this pattern were detected in patients with different forms of degenerative dementia. These findings are consistent with PGDS being altered in some neurological diseases and provide new opportunities for clinical applications.     

Study of human cerebrospinal fluid proteins by size exclusion-high performance liquid chromatography and two-dimensional gel electrophoresis

Cerebrospinal fluid (CSF) proteins were separated into three main fractions by size exclusion-high performance liquid chromatography (SE-HPLC). Subsequent analysis of each fraction by two-dimensional gel electrophoresis (2-DE) facilitated the detection of trace components in CSF and additionally provided more information about the native properties of various proteins. Certain proteins are present in a polymeric form and appear in the high molecular weight SE-HPLC fraction. In the middle molecular weight SE-HPLC fraction we found a CSF-specific transthyretin-related protein by immunoblotting with polyclonal antibodies to transthyretin. Possible interpolypeptide disulfide bonds of such polymeric proteins were studied using a nonreducing 2-DE system. This procedure revealed that all apolipoprotein E monomers in CSF, which are synthesized in astrocytes, are linked by disulfide bonds. In the CSF from a patient with clinically definite multiple sclerosis (MS), novel proteins appeared in the high molecular weight SE-HPLC fraction, which are obscured by other proteins if total CSF is analyzed.     

Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight.

We have analyzed protein patterns of human nasal lavage fluid (NLF) with two-dimensional gel electrophoresis (2-DE) and identified several proteins (such as transthyretin, Clara Cell protein 16, lipocalin-1, cystatin S, cystatin SN, immunoglobulin binding factor, statherin, calgranulin B, prolactin-inducible protein, and zinc-alpha2-glycoprotein) by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry. To investigate whether airway irritation causes alterations in NLF 2-DE patterns, we compared epoxy workers with airway irritation (n=8) and healthy controls (n=6) before and after 2 h exposure to the epoxy chemical, dimethylbenzylamine (DMBA, 100 microg/m3) in an exposure chamber. A 25 kDa protein with pI 5.5 was found to be altered in the NLF 2-DE patterns; a trypsin digest of the 2-DE spot analyzed by MALDI-TOF and expressed sequence tags (ESTs) determined after post-source decay (PSD) identified the protein as palate lung and nasal epithelial clone (PLUNC). In controls, the levels of NLF-PLUNC were generally lower after 2 h exposure, whereas in epoxy workers, the levels were increased three- to twentyfold after exposure. The human gene sequence for PLUNC was just recently reported and so far no biofunctional data are available. Our results suggest that PLUNC is involved in the airway inflammatory response after exposure to irritants.     

Identification of the apolipoprotein E4 isoform in cerebrospinal fluid with preparative two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

Apolipoprotein E (apoE) was isolated from human cerebrospinal fluid (CSF) from control individuals and patients with Alzheimer's disease (AD). The purification was performed with preparative two-dimensional electrophoresis (2-DE), involving liquid-phase isoelectric focusing (IEF) in the Rotofor cell in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution in the Mini Whole Gel Eluter. ApoE was characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known change of Cys to Arg in position 112 of the apoE4 isoform was identified. This was detected in CSF from AD patients, reflecting the increased frequency of the apoE4 allele in this population. This peptide was not detected in CSF samples from healty control individuals. The use of this rapid electrophoretic separation in proteomic studies of CSF proteins provides single proteins, such as apoE, of high purity in yields sufficient for characterization by MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a useful tool in the investigation of the pathophysiology of brain disorders such as AD.     

Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung diseases

Recently, we published an analytical two-dimensional electrophoresis (2-DE) protein map of human bronchoalveolar lavage fluid (BALF) using a pool of BALFs from various patients. In this report, the effect of lung disorders on the protein composition of the lung epithelial lining fluid was investigated by 2-DE of BALFs from individual patients with well-defined interstitial lung diseases: sarcoidosis, idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP), using improved experimental conditions. On these gels, about 600-1000 stained protein spots could be identified in a BALF sample containing 25 microg of protein, and our original human BALF protein database has, therefore, been considerably extended. Altogether, 429 protein spots corresponding to 66 different proteins (including isoforms, protein subunits and fragments) were identified by microsequence analysis and by matching with a human blood plasma 2-DE protein map available in the SWISS-2DPAGE database. A human 2-DE BALF database was established and is available on the World Wide Web (http://www.umh.ac.be/-biochim/proteomic.htm+ ++). The significance of the modifications observed between the different lung pathologies is discussed with the aim of understanding the mechanistic bases of lung disease pathogenesis and finding new potential lung markers of disorders.     

Protein expression patterns associated with advanced stage ovarian cancer

This is a comparative proteomic study on biopsies from patients with ovarian cancer to identify potential diagnostic/prognostic biomarkers in both healthy and tumor tissue, interstitial fluid (normal interstitial fluid and tumoral interstitial fluid and peritoneal effusion. Protein expression/identification was evaluated by 2-DE and MS analysis: six proteins showed differential expression in tumoral interstitial fluid and tumor tissue compared to normal interstitial fluid and healthy tissue: five were found to be downregulated and identified as galectin 3, glutathione S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin, was significantly upregulated in all pathological samples, including the ascitic fluid. Validation of S100A8 overexpression in carcinoma tissue was obtained by immunohistochemistry. To our knowledge, this is the first study to report an over-expression of calgranulin by 2-DE associated with MS/MS analysis on surgical biopsy. The reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways. Moreover, this study suggests that calgranulin expression may be used as a diagnostic and/or prognostic biomarker.     

Intranasal phototherapy is more effective than fexofenadine hydrochloride in the treatment of seasonal allergic rhinitis: results of a pilot study

We recently showed that intranasal phototherapy represents an efficient therapeutic modality for the treatment of patients with seasonal allergic rhinitis (SAR). The aim of this pilot study was to compare the efficacy of intranasal phototherapy with that of the new generation antihistamine fexofenadine HCl in SAR. A randomized open study was conducted in patients with a history of moderate-to-severe ragweed-induced SAR. Thirty-one patients were randomly assigned to receive either intranasal irradiation three times a week for 2 weeks, or 180 mg fexofenadine HCl per day for 2 weeks. Each patient kept a diary of symptoms for nasal obstruction, nasal itching, rhinorrhea, sneezing and palate itching. Total nasal score (TNS), a sum of scores for nasal symptoms, was also calculated. In the rhinophototherapy group the individual scores significantly decreased compared with baseline for all of the parameters. In the fexofenadine HCl group none of the scores improved significantly at the end of the treatment except sneezing. TNS was significantly decreased in the rhinophototherapy group, but no significant change was observed in the fexofenadine HCl group after 2 weeks of treatment. In conclusion, we found that intranasal phototherapy is more efficient than fexofenadine HCl in reducing clinical symptoms for SAR.     

Apolipoprotein E and other cerebrospinal fluid proteins differentiate ante mortem variant Creutzfeldt-Jakob disease from ante mortem sporadic Creutzfeldt-Jakob disease

The ability to perform an ante mortem differential diagnosis of Creutzfeld-Jakob disease (CJD) is aided by several clinical and molecular tests. There is a need for molecular tests which can reliably distinguish ante mortem variant CJD (vCJD) from ante mortem sporadic CJD (spCJD). A proteomics approach employing two-dimensional protein electrophoresis is applied to the study of ante mortem CSF samples obtained in collaboration with the CJD Surveillance Unit and the National Hospital for Neurology and Neurosurgery. The sample set includes two cases of vCJD, three cases of spCJD and three neurologic controls. Preliminary data using a panel of seven molecular markers is able to distinguish vCJD from spCJD using a heuristic clustering algorithm. One of the molecular markers has been identified as apolipoprotein E which appears to be upregulated in the cerebrospiral fluid (CSF) of patients with vCJD as compared to spCJD. Analysis of ante mortem CSF may help to differentiate patients with vCJD from those patients with spCJD.     

The transport of a drug to the cerebrospinal fluid directly from the nasal cavity: the relation to the lipophilicity of the drug.

The objective of the present study was to clarify the relation between drug transport to the cerebrospinal fluid (CSF) from the nasal cavity and the lipophilicity of the drug using hydrophilic sulfonamides as model drugs. The nasal cavity of the rat was perfused in a single pass system and the concentrations of sulfonamides in plasma and CSF were measured. The drug concentrations in CSF and plasma after nasal perfusion were compared with those after intravenous (i.v.) administration. The drug concentrations in the CSF were remarkably high after nasal perfusion in comparison with those after i.v. administration, though the time course of the plasma concentration was not much different from that after i.v. administration. These results suggested the existence of a direct transport pathway of the sulfonamides from the nose to the CSF. In addition, the drug concentrations in the CSF increased with increasing the lipophilicity of the drugs (the partition coefficient (Pc) of the drugs between isoamyl alcohol and pH 7.4 phosphate buffer). A significant correlation was observed between the drug concentrations in CSF and Pc. In conclusion, the direct transport pathway of the sulfonamides from the nose to the CSF was confirmed and, with regard to drugs with comparatively low lipophilicity, the degree of the transport depended on its Pc.     

Proteomics-driven progress in neurodegeneration research

Proteomics technologies have been widely used in the investigation of neurodegenerative and psychiatric disorders, and in particular in the detection of differences between healthy individuals and patients suffering from such diseases. Thus, brain and cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease, Down syndrome, Pick's disease, Parkinson's disease, schizophrenia, and other disorders as well as brain and CSF from animals serving as models of neurological disorders have been analyzed by proteomics. 2-DE followed by MALDI-TOF-MS has been mainly applied as this proteomics approach provides the possibility of convenient quantification of protein levels and detection of post-translational modifications. About 330 unique proteins with deranged levels and modifications have been detected by proteomics approaches to be related to neurodegeneration and psychiatric disorders. They are mainly involved in metabolism pathways, cytoskeleton formation, signal transduction, guidance, detoxification, transport, and conformational changes. In this article, we provide a summary of the major contributions of proteomics technologies in the study of neurodegenerative and psychiatric diseases, in particular, in the detection of changes in protein levels and modifications related to these disorders.     

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.     

Newly identified proteins in human nasal and bronchoalveolar lavage fluids: potential biomedical and clinical applications

Protein patterns of nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) were analyzed with two-dimensional gel electrophoresis (2-DE) and a number of previously unidentified proteins (lipocalin-1, cystatin S, transthyretin, immunoglobulin binding factor and an 11 kDa fragment of albumin) were identified by N-terminal amino acid sequencing. Lipocalin-1 was shown to be a dominant protein in NLF from healthy subjects but was almost undetectable in NLF from a patient with asthma. It further appeared that lipocalin-1 in NLF consists of eight forms with pIs between 5.2 and 5.5: three with the expected Mr of 17500, two with increased Mr (18000), and three truncated variants with Mr of 17000. Two forms of cystatin S were identified both in NLF and BALF: one with pI 5.1 and Mr 13000, and the other with pI4.9 and Mr 13500. The distribution of the two forms was clearly different in NLF and BALF from healthy subjects with the 4.9/13500 form constituting only about 13% in NLF but 69% in BALF. In NLF from subjects with upper airway irritation a twofold increased proportion of the 4.9/13500 form was detected. Amino acid sequence data and the spot position indicate that the 4.9/13500 form might be a phosphorylated variant of cystatin S. Lower levels of both forms of cystatin S were found in BALF from smokers than nonsmokers. The levels of transthyretin in NLF were decreased in subjects exposed to irritating chemicals. Finally, higher levels of IgBF were found in BALF from smokers than nonsmokers. Taken together, these results illustrate the potential biomedical and clinical applications of identifying proteins in 2-DE patterns of human BALF and NLF. The possibility to describe and monitor airway disorders at the molecular level is inferred.     

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P < 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.     

Analysis of the sinusitis nasal lavage fluid proteome using capillary liquid chromatography interfaced to electrospray ionization-quadrupole time of flight- tandem mass spectrometry

The nasal lavage fluids (NLFs) from four subjects with acute sinusitis were analyzed to investigate the amount of proteins expressed in this pathology at the beginning of the event (day 1) and after 6 days of treatment with antibiotics and a nasal steroid spray. The protein identification was performed with capillary liquid chromatography-electrospray-quadrupole time of flight-(LC-ESI-Q-TOF)-mass spectrometry. The samples collected on the first day contained high-abundant plasma proteins, such as albumin and immunoglobulins, glandular serous cell proteins (lysozyme, lactoferrin, and polymeric immunoglobulin receptor), epithelial keratins, and inflammatory cell proteins (myeloperoxidase, IL-16, and IL-17E). After six days of therapy, the complexity of the proteome was reduced to plasma proteins and lysozyme with no inflammatory markers. The presence of hemoglobin, however, suggested that significant squamous metaplasia with breaches in the epithelial barrier, or nasal steroid-related bleeding, had occurred. The proteomic approach presented here allowed us to identify, in the high complexity of acute sinusitis nasal secretions, the proteins that respond to a pharmacological treatment and that could be suitable as markers of this pathology.     

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2-DE profile after silver staining. These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42 degrees C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S. thermophilus.