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.     

Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.     

Analysis of two-dimensional electrophoretic patterns of proteins obtained from the sera of normal and tumor-bearing nude rats

To classify two-dimensional electrophoresis (2-DE) patterns of normal and abnormal samples, a new parameter, named shift value, is introduced. The shift value is defined by the product of three indices; differences in density, differences in area, and the Euclid distance between peaks of matched glycoprotein spots in the 2-DE patterns. Shift values obtained from the differences between the 2-DE patterns of tumor-bearing and normal sera (control) were always found to be larger than those obtained from two control patterns. The shift value obtained from glycoprotein spots could be more effectively used to distinguish normal and abnormal patterns than that obtained from simple proteins. In our earlier attempts to compare serum proteins of normal and tumor patients, we could not discriminate differences caused by genetic background. In order to circumvent this difficulty, we employed an inbred strain of nude rats into which various types of human cancer had been implanted. Reliability of the analysis is enhanced by using a polyacrylamide gel backed with a silanized glass support and an automatic 2-DE apparatus.     

Expression of high-abundance proteins in sera of patients with endometrial and cervical cancers: analysis using 2-DE with silver staining and lectin detection methods

The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in‐gel electrophoresis

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor‐specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non‐cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in‐gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine‐rich α2 glycoprotein‐1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.     

A proteome database of human primary T helper cells

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Noncovalent interactions in human plasma proteins analyzed by the comparison of nondenaturing and denaturing micro-2-D gel electrophoresis patterns after polypeptide assignment using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass fingerprinting

Previously, we reported the analysis of human plasma proteins by 2-DE under nondenaturing conditions (Type-I 2-DE) followed by the assignment of stained spots using MALDI-MS and PMF [1]. Here, we employ 2-DE conditions modified only in the second-dimensional separation; SDS was added in the gradient slab gel aiming to dissociate noncovalently bound proteins/polypeptides (Type-II 2-DE). Totally 169 CBB-stained spots on a micro-2-DE gel were numbered and subjected to polypeptide assignment using MALDI-MS-PMF. One hundred sixty spots out of the 169 provided significant match (p <0.05) with polypeptides in databases. Comparisons of the results of polypeptide assignment on the two 2-DE patterns indicated that 10 polypeptides in 20 stained spots on the Type-I 2-DE pattern [1] shifted toward low-molecular-weight positions on the Type-II 2-DE pattern, demonstrating the presence of noncovalent interactions. Seventeen polypeptides in 38 stained spots were only assigned on the Type-II 2-DE gel, which could mostly be accounted for by the disruption of noncovalent protein-protein interactions in the presence of SDS, i.e., protein/polypeptide complexes which might form smear bands on the Type-I 2-DE gel dissociate to form clear spots on the Type-II 2-DE gel. The method employed here, comparisons of nondenaturing and denaturing 2-DE maps with polypeptide assignment by MALDI-MS-PMF, would enable the simultaneous detection of multiple noncovalent interactions in complex protein/polypeptide systems.     

Analysis of mouse beta-haptoglobin chain by lectin affinoblotting detection

Two different human mammary carcinoma cell lines were xenotransplanted into nude mice. Serum samples were obtained prior to and after transplantation and investigated by two-dimensional electrophoresis (2-DE). By comparison of these silver-stained patterns additional protein spots were detected resulting either from proteins secreted or shed by the tumor itself or from mouse proteins induced by the tumor or the transplantation procedure. One group of spots detectable in post-transplantation serum as well as in control serum after mock-transplantation but not in pretransplantation serum was microsequenced and identified as mouse beta-haptoglobin. The carbohydrate structures of beta-haptoglobin were characterized by two different immunochemical glycoprotein staining procedures to detect differential terminal glycan modifications.     

Proteome database of unsensitized CD4 positive T lymphocytes in T cell receptor transgenic mice

We established a two-dimensional electrophoresis (2-DE) mapping database of splenic CD4 T cells prepared from I-A(d)-restricted ovalbumin (OVA)(323-339) specific T cell receptor (TCR) transgenic mice (OVA23-3). First we examined the purification of CD4 T cells and found that the high purity of cells produced more accurate protein maps. The first dimension utilized narrow-range immobilized pH gradients (IPGs), pH 4.0-5.0, pH 4.5-5.5, pH 5.0-6.0, and pH 5.5-6.7. Approximately 1300 spots were detected by silver staining. Detection was performed by in-gel tryptic digestion of the spots, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) technology and database searches via the world wide web (WWW). We have so far identified 255 proteins on 2-DE gels of whole cell lysates. This is the first construction of a proteome database for murine unsensitized CD4 T lymphocytes. To examine this further, 2-DE mapping was utilized for splenic CD4 T cells from another TCR transgenic mouse strain (DO11.10 TCR transgenic mice). Mapping patterns were found to be almost identical to those from CD4 T cells from OVA23-3 mice. These results indicated that the 2-DE maps in this study could be used for mouse CD4 T cells to examine protein changes in cells given certain stimuli.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker’s Respiratory Allergy

Wheat allergens are responsible for symptoms in 60–70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21–27 kDa that were recognized by the pooled baker’s IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.     

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.     

Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

High-resolution two-dimensional gel electrophoresis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differences in protein expression. In the gels of pH 5–8 immobilized pH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocardial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identified by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease. __________ Translated from Chemical Journal of Chinese Universities, 2006, 8(27): 1467–1471 [译自: 高等学校化学学报]     

2-D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins

Most lung cancers are diagnosed too late for curative treatment to be possible, therefore early detection is crucial. Serum proteins are a rich source of biomarkers and have the potential to be used as diagnostic and prognostic indicators for lung cancer. In order to examine differences in serum levels of specific proteins associated with human lung squamous carcinoma, immunodepletion of albumin and five other high-abundant serum proteins followed by 2-D difference gel electrophoresis (DIGE) analysis and subsequent MS was used to generate a panel of proteins found to be differentially expressed between the cancer and normal samples. Proteins found to have increased abundance levels in squamous cell carcinoma sera compared to normal sera included apolipoprotein A-IV precursor, chain F; human complement component C3c, haptoglobin, serum amyloid A protein precursor and Ras-related protein Rab-7b. Proteins found to have lower abundance levels in squamous cell carcinoma sera compared to normal sera included alpha-2-HS glycoprotein, hemopexin precursor, proapolipoprotein, antithrombin III and SP40; 40. The data presented here demonstrate that high-abundant protein removal combined with 2-D DIGE is a powerful strategy for the discovery of potential biomarkers. The identification of lung cancer-specific biomarkers is crucial to early detection, which in turn could lead to a dramatic increase in survival rates.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.     

Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.     

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.