On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.     

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.     

Studies on the Basic Conformation of Lysozyme by Electrospray Ionization-Mass Spectrometry (ESI- MS)

In the present work, 2-mercaptoethanol and dithiothreitol were used to hydrogenise the internal disulfide bond in lysozyme. The experimental results indicate that the charge distribution of the proteins are different in the reaction process. From the calculated molecular weight, the reduction process of the disulfide bond in the molecules can be described, and the number of the disulfide bonds in the molecule can also be determined.     

Two-dimensional electrophoresis of the fatty acid binding protein from human heart: evidence for a thiol group which can form an intermolecular disulfide bond

A 100,000 g supernatant from human heart muscle, containing cytosolic proteins with some contaminating plasma proteins, was analyzed for fatty acid binding protein (FABP) by two-dimensional electrophoresis (2-DE) using isoelectric focusing under nondenaturing conditions in the first dimension. FABP purified from human heart muscle was found to comigrate with a major spot in 2-DE gels of the supernatant. This spot was comparable with those of the myoglobins in staining intensity. When purified FABP was charged with [3H]palmitate and subjected to nondenaturing 2-DE, radioactivity always comigrated with this protein. Under denaturing and reducing conditions in the second dimension, FABP was found to have a pI of 5.3 and an apparent molecular weight of 15,000. Isoforms of FABP, reported here for the first time to occur in human heart muscle, were observed as minor spots focusing at pH 5.1 and 5.7. When electrophoresis in the second dimension was carried out under denaturing but nonreducing conditions, an additional protein appeared at pH 5.3 with an apparent molecular weight of about 30,000. This protein was identified as a dimer of FABP and evidence for the involvement of an intermolecular disulfide bond in this dimerization is presented.     

Assessment of the Effects of Glycosylation on the Pattern and Kinetics of Degradation of Lenograstim in Comparison to Filgrastim Using a Stability-Indicating Orthogonal Testing Protocol

In the biopharmaceutical industry, protein aggregation and/or degradation has profound pathological implications and is encountered routinely during production, shipping, storage and administration. Lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) was subjected to stress conditions, namely, oxidation, pH, temperature, agitation and repeated freeze–thaw to generate all possible degradation products. An orthogonal stability-indicating testing protocol (RP-HPLC, SE-HPLC, ELISA and SDS-PAGE) was developed and validated for assessment of the pattern and kinetics of aggregation/degradation, under the studied experimental conditions. Results indicated clearly that Lenograstim is susceptible to degradation induced by the studied stress conditions. However, Lenograstim was found relatively more stable than Filgrastim (non-glycosylated recombinant human granulocyte colony-stimulating factor) which was attributed to the effect of glycosylation. Oxidized forms and high molecular weight aggregates of Lenograstim and Filgrastim were detected in all samples subjected to stress conditions to different degrees. ELISA assay and SDS-PAGE results were generally in agreement to those obtained using SE-HPLC assay which confirmed its selectivity to the intact drug. However, formation of soluble aggregates of both drugs was found to occur via physical adsorption and formation of intermolecular disulfide bonds. Results confirmed the need for an orthogonal testing protocol since it was impossible to reveal all types of degradation products using a single technique. Results raised a concern about the efficacy and safety of such sensitive products and highlighted the need for simple tools to inspect biologics for soluble aggregates and sub-visible particles before administration.

     

Analysis of plasma protein adsorption onto polystyrene particles by two-dimensional electrophoresis: comparison of sample application and isoelectric focusing techniques

Two-dimensional electrophoresis (2-DE) was previously established for analysis of plasma protein adsorption patterns on particulate carriers for intravenous drug targeting. This study addresses a possible effect of polymeric particles on protein separation in the first dimension, e.g., hindrance of protein entry into the gel or interaction of particles with the gel matrix. Polystyrene beads of mean diameter 100, 200 and 1000 nm were used as model carriers. Two different separation techniques were performed in the first dimension of 2-DE to study possible interactions of the beads with the different gel matrices, i.e., carrier ampholytes (CA) and immobilized pH gradients (IPGs). Comparison of gels obtained from samples including the particles from samples separated from the polystyrene beads showed no noteworthy differences. Therefore, a negative effect of the particles can be excluded, and particle separation from the sample is not necessary. Another goal of this study was the transfer of analytical protocols for isoelectric focusing from CA to IPGs with regard to enhanced reproducibility, faster sample processing, and easier handling. Transfer from CA to IPGs was carried out successfully and showed improved resolution of basic proteins. In contrast to that, lower amounts of a few high molecular mass proteins were detected, especially when sample application cups were employed. A qualitative change in the obtained protein pattern was not observed. Increased entry of high molecular weight proteins was achieved by in-sample rehydration instead of using sample cups.     

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.     

Molecular Dynamics Simulations of Proteins with Chemically Modified Disulfide Bonds

Proteins that are used as therapeutic drugs act in the extracellular microenvironment. They usually have a small number of intramolecular disulfide bonds to help maintain their tertiary structure in the vascular circulation. In general, most cysteine residues are part of a disulfide bond with free sulfhydrals being uncommon. We have studied whether the site-specific chemical reduction of disulfides and the incorporation of a 3-carbon methylene bridge between the cysteines in interferon-α 2a would change the structure of this protein. Bridging of both of the disulfide bonds of interferon-α 2a was studied using two different molecular simulation protocols: (1) molecular dynamics, and (2) stochastic dynamics. We have shown that the disulfide bonds in interferon-α 2a can be reduced and chemically modified without significantly altering the tertiary structure of the protein. This offers the novel possibility of chemically modifying therapeutically important proteins without affecting their biological properties.     

PDI-Regulated Disulfide Bond Formation in Protein Folding and Biomolecular Assembly

Disulfide bonds play a pivotal role in maintaining the natural structures of proteins to ensure their performance of normal biological functions. Moreover, biological molecular assembly, such as the gluten network, is also largely dependent on the intermolecular crosslinking via disulfide bonds. In eukaryotes, the formation and rearrangement of most intra- and intermolecular disulfide bonds in the endoplasmic reticulum (ER) are mediated by protein disulfide isomerases (PDIs), which consist of multiple thioredoxin-like domains. These domains assist correct folding of proteins, as well as effectively prevent the aggregation of misfolded ones. Protein misfolding often leads to the formation of pathological protein aggregations that cause many diseases. On the other hand, glutenin aggregation and subsequent crosslinking are required for the formation of a rheologically dominating gluten network. Herein, the mechanism of PDI-regulated disulfide bond formation is important for understanding not only protein folding and associated diseases, but also the formation of functional biomolecular assembly. This review systematically illustrated the process of human protein disulfide isomerase (hPDI) mediated disulfide bond formation and complemented this with the current mechanism of wheat protein disulfide isomerase (wPDI) catalyzed formation of gluten networks.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

A small-molecule catalyst of protein folding in vitro and in vivo

BACKGROUND: The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (Eo'). RESULTS: A synthetic small-molecule dithiol, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an Eo' value of -0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo. CONCLUSIONS: A small-molecule dithiol with a low thiol pKa value and high disulfide Eo' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo.     

A comparison of depletion versus equalization for reducing high-abundance proteins in human serum

In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner^? (PM) have been assessed by 1‐D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa.     

Characterizing closely spaced, complex disulfide bond patterns in peptides and proteins by liquid chromatography/electrospray ionization tandem mass spectrometry

Identifying the Cys residues involved in disulfide linkages of peptides and proteins that contain complex disulfide bond patterns is a significant analytical challenge. This is especially true when the Cys residues involved in the disulfide bonds are closely spaced in the primary sequence. Peptides and proteins that contain free Cys residues located near disulfide bonds present the additional problem of disulfide shuffling via the thiol-disulfide exchange reaction. In this paper, we report a convenient method to identify complex disulfide patterns in peptides and proteins using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with partial reduction by tris(2-carboxyethyl)phosphine (TCEP). The method was validated using well-characterized peptides and proteins including endothelin, insulin, alpha-conotoxin SI and immunoglobulin G (IgG2a, mouse). Peptide or protein digests were treated with TCEP in the presence of an alkylation reagent, maleimide-biotin (M-biotin) or N-ethylmaleimide (NEM), followed by complete reduction with dithiothreitol and alkylation by iodoacetamide (IAM). Subsequently, peptides that contained alkylated Cys were analyzed by capillary LC/ESI-MS/MS to determine which Cys residues were modified with M-biotin/NEM or IAM. The presence of the alkylating reagent (M-biotin or NEM) during TCEP reduction was found to minimize the occurrence of the thiol-disulfide exchange reaction. A critical feature of the method is the stepwise reduction of the disulfide bonds and the orderly, sequential use of specific alkylating reagents.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

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.     

Alkaline cleavage of covalent bonds in chicken insulin and bovine alpha-lactalbumin analyzed by matrix-assisted laser desorption/ionization-mass spectrometry

In the course of searching methods to extract proteins from Coomassie blue-stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine alpha-lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C-terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.     

Self-assembly of a diblock copolymer with pendant disulfide bonds and chromophore groups: a new platform for fast release

An amphiphilic block copolymer comprising poly(ethylene glycol) (PEG) and poly(2-(methacryloyl)oxyethyl-2'-hydroxyethyl disulfide) (PMAOHD) blocks was synthesized by atom transfer radical polymerization (ATRP). Pyrenebutyric acid was conjugated to the block copolymer by esterification, and a block copolymer with pendant disulfide bonds and pyrenyl groups (PEG-b-P(MAOHD-g-Py)) was obtained. (1)H NMR and gel permeation chromatography (GPC) results demonstrated the successful synthesis of the block copolymer. The cleavage of the disulfide bonds and the degrafting of the pyrenyl groups were investigated in THF and a THF/methanol mixture. Fluorescence spectroscopy, GPC, and (1)H NMR results demonstrated fast cleavage of the disulfide bonds by Bu(3)P in THF. Fluorescence results showed the ratio of the intensity of the excimer peak to the monomer peak decreased rapidly within 20 min. GPC traces of the block copolymer moved to a long retention time region after addition of Bu(3)P, indicating the cleavage of the disulfide bonds and the degrafting of the pyrenyl groups. PEG-b-P(MAOHD-g-Py) can self-assemble into micelles with poly(MAOHD-g-Py) cores and PEG coronae in a mixture of methanol and THF (9:1 by volume). The dissociation of the micelles in the presence of Bu(3)P was investigated. After cleavage of the disulfide bonds in the micellar cores, a pyrene-containing small molecular compound and a block copolymer with pendant thiol groups were produced. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and (1)H NMR were employed to track the dissociation of the polymeric micelles. All the techniques demonstrated the dissociation of the micelles and the fast release of pyrenyl groups from the micelles.     

Analysis of high molecular mass proteins larger than 150 kDa using cyanogen bromide cleavage and conventional 2-DE

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.     

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.     

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains β-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS). BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds. For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H]⁺, of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.