Improved solubilization of surface proteins from Listeria monocytogenes for 2-DE

Solubilization of bacterial surface (cell wall and membrane-associated) proteins for 2-DE is challenging, particularly in the case of Gram-positive bacteria. This is primarily due to strong protein association with the cell wall peptidoglycan and protein hydrophobicity. We solubilized surface proteins for 2-DE from the Gram-positive pathogen Listeria monocytogenes using mutanolysin, which digests cell wall peptidoglycan, and one of three different mixtures of zwitterionic detergent and chaotropes: (i) CHAPS/urea, (ii) amidosulfobetaine-14 (ASB-14)/urea/thiourea (iii) N-decyl-N,N'-dimethyl-3-ammonio-1-propanesulfonate/urea/thiourea. Cell lysis with mutanolysin followed by solubilization with ASB-14/urea/thiourea gave the highest overall protein yield with the best 2-DE resolution. Protein spot identification by MALDI-TOF/TOF-MS analysis revealed 29 characterized surface proteins of L. monocytogenes, 17 of which have not previously been reported on the surface proteome map. This is the first report describing the successful solubilization and 2-DE of L. monocytogenes proteins bound to the cell surface via an LPXTG motif or by a hydrophobic tail. The increase in surface proteome coverage obtained by mutanolysin and ASB-14/urea/thiourea solubilization suggests the utility of this method for future analytical and comparative studies of surface proteins from Listeria, and possibly other Gram-positive bacteria, using 2-DE proteomic analysis. An updated 2-DE reference map of L. monocytogenes surface proteins is presented.     

High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma

A 2-DE system has been devised in which proteins are first separated in their native state followed by separation according to mass under denaturing conditions (Nat/SDS-PAGE). Hydrophilic properties of the gel and the presence of dihydroxybisacrylamide in the first dimension allowed a good resolution for high-molecular-weight proteins and maintained interactions. With this method 252 plasma spots have been resolved and 140 have been characterized by MS as isoforms of 60 proteins, a relevant part of which (12) were not detected by traditional 2-D gels or by other nondenaturing 2-D techniques. The list includes complement factors (C4d, C7), coagulation factors (coagulation factor II, fibrin beta), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin beta, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and alpha2-macroglobulin and showed the presence of atypical albumin adducts corresponding to post-translational and oxidation products. Our system provides therefore new tools for resolving proteins, protein aggregates and complexes and amplifies the potentiality of traditional electrophoretic analysis.     

Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS

Previously, we have reported a high-efficiency method of protein extraction from CBB-stained polyacrylamide gels for molecular mass measurement with MALDI-TOF MS [1]. In the present work, the alkaline extraction method was applied to CBB-stained 2-DE gels on which human plasma proteins were separated in the absence of denaturant. In order to examine the performance of the method, ten spots with apparent molecular masses (MMapp) in the range of 65 to 1000 kDa were selected and the proteins were extracted from the gel pieces. The extracts were subjected to whole-mass measurement by MALDI-TOF MS, with and without DTT treatment. In addition, the extracts were subjected to in-solution trypsin digestion followed by MALDI-TOF MS and PMF analysis. Successful extraction of proteins from the ten spots, up to MMapp 1000 kDa, has been ascertained by the significant PMF assignment (MASCOT) with high sequence coverage of the respective proteins or polypeptides. When direct mass measurement of the extracted proteins was attempted, three spots in MMapp range 65-100 kDa provided mass peaks. Five spots in MMapp range 150-400 kDa did not give mass peaks of the intact proteins, but showed those of the constituent polypeptides after the DTT treatment. Extraction of proteins prior to trypsin digestion enabled the procedure of PMF analysis to be much simpler than the conventional in-gel digestion method, providing comparable protein scores and sequence coverage. The technique presented here suggests a new strategy for the characterization of proteins separated by nondenaturing 2-DE.     

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.     

Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue

Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris‐acetate polyacrylamide gels

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3–15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris‐acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris‐Tricine‐SDS‐sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS‐PAGE such as CBB staining and immunoblot. Thus, by using Tris‐acetate 3–15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10–500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), β‐actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 μg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris‐acetate‐PAGE system a very helpful tool for protein analysis.     

Improved resolution in the acidic and basic region of 2‐DE of insect antennae proteins using hydroxyethyl disulfide

Olfaction is essential for processing chemical signals in insects, but characterizing the proteins implicated in this process has proved challenging. We optimized 2‐DE gel resolution of insect proteins by using a buffer containing two reducing agents, DTT and hydroxyethyl disulfide. This buffer clearly improved resolution and decreased spot streaking and spot trains of 2‐DE in comparison to DTT alone. We described for the first time that the buffer with DTT and hydroxyethyl disulfide further to reducing streaking in the basic part of the gel eliminates false spots in the acidic gel regions that appeared when only DTT was used as reducing agent.     

High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification

The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.     

Analysis of E. coli soluble proteins by non‐denaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF

Escherichia coli (strain K‐12)‐soluble proteins were analyzed by nondenaturing micro 2‐DE and MALDI‐MS‐PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939–948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB‐stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2‐DE gels (one for wide mass range and one for low‐molecular‐mass range) were numbered and subjected to MALDI‐MS‐PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p<0.05) with polypeptides in Swiss‐Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2‐DE gels and the locations of the activity spots matched well with those of the MS‐assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss‐Prot (119 polypeptides as homo‐multimers and 25 as hetero‐multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2‐DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third‐dimension micro SDS‐PAGE (micro 3‐DE), about 190 CBB‐stained spots were detected. The assignment of the polypeptides separated on the 3‐DE gel would further provide information on protein/polypeptide interactions.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.     

Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC‐MS/MS

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB‐stained spots were numbered and subjected to in‐gel digestion and quantitative LC‐MS/MS. The analysis provided the assignment of 1–25 (average eight) non‐redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC‐MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well‐known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis.     

Investigation and reduction of sub‐microgram peptide loss using molecular weight cut‐off fractionation prior to mass spectrometric analysis

This work investigates the introduction of methanol and a salt modifier to molecular weight cut‐off membrane‐based centrifugal filters (MWCO) to enrich sub‐microgram peptide quantities. Using a neuropeptide standard, bradykinin, sample loss was reduced over two orders of magnitude with and without undigested protein present. Additionally, a bovine serum albumin tryptic digestion was investigated. Twenty‐seven tryptic peptides were identified from MALDI mass spectra after enriching with methanol while only two tryptic peptides were identified after MWCO separation using H₂O. The strategy presented here enhances recovery from MWCO separation for sub‐µg peptide samples. Copyright © 2012 John Wiley & Sons, Ltd.     

A comparison of 2-D chromatography separations using UV and (18)O quantification of proteins in similar proteomes

Proteins present in two mitochondria preparations were separated by 2-D chromatography using the ProteomeLab PF-2D Protein Fractional System, protein fractionation in two dimensions (PF-2D). The proteins in each first-dimension fraction were determined by trypsinization and LC-MS/MS. Chromatography peaks were quantified by UV detection using the "Mapping Tools" software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF-2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for (18)O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using (18)O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF-2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.     

Rapid characterization of covalent modifications to rat brain mitochondrial proteins after ex vivo exposure to 4-hydroxy-2-nonenal by liquid chromatography-tandem mass spectrometry using data-dependent and neutral loss-driven MS3 acquisition

The modification of mitochondrial proteins enriched from rat forebrain by the major lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was investigated using high performance liquid chromatography (HPLC) and tandem mass spectrometry. Subcellular fractionation in conjunction with a 'shotgun-based' approach that involved both conventional data-dependent and neutral loss (NL)-driven MS(3) data acquisition on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT) was utilized. Using a relatively rapid linear HPLC gradient (1 h) for complex mixture analysis, 24 sites of HNE modification on 15 unique proteins were identified which corresponded exclusively to Michael adduct formation on histidine residues. Since a number of HNE-modified peptides produced a predominant HNE NL fragment-ion signal upon collision-induced dissociation (CID), NL-driven MS(3) data-dependent acquisition was a valuable method to enhance fragmentation information for these particular modified peptides. Of the 24 HNE modification sites identified, approximately 25% were determined from the MS(3) spectra alone. We envision the reported methodology as an efficient screening approach for HNE modification site selectivity that could ultimately provide a foundation for the development of targeted schemes for the characterization of in vivo HNE-protein adducts.     

Revealing binding selectivity of inhibitors toward bromodomain-containing proteins 2 and 4 using multiple short molecular dynamics simulations and free energy analyses

ABSTRACT

Emerging evidences indicate bromodomain-containing proteins 2 and 4 (BRD2 and BRD4) play critical roles in cancers, inflammations, cardiovascular diseases and other pathologies. Multiple short molecular dynamics (MSMD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method were applied to investigate the binding selectivity of three inhibitors 87D, 88M and 89G towards BRD2 over BRD4. The root-mean-square fluctuation (RMSF) analysis indicates that the structural flexibility of BRD4 is stronger than that of BRD2. Moreover the calculated distances between the C_α atoms in the centres of the ZA_loop and BC_loop of BRD4 are also bigger than that of BRD2. The rank of binding free energies calculated using MM-GBSA method agrees well with that determined by experimental data. The results show that 87D can bind more favourably to BRD2 than BRD4, while 88M has better selectivity on BRD4 over BRD2. Residue-based free-energy decomposition method was utilized to estimate the inhibitor-residue interaction spectrum and the results not only identify the hot interaction spots of inhibitors with BRD2 and BRD4, but also demonstrate that several common residues, including (W370, W374), (P371, P375), (V376, V380) and (L381, L385) belonging to (BRD2, BRD4), generate significant binding difference of inhibitors to BRD2 and BRD4.     

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC‐MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an “overlap score,” (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 “overlap factors,” (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.     

Analysis of allergens in tubeimu saponin extracts by using rat basophilic leukemia 2H3 cell‐based affinity chromatography coupled to liquid chromatography and mass spectrometry

An affinity two‐dimensional chromatography method was developed for the recognition, separation, and identification of allergic components from tubeimu saponin extracts, a preparation often injected to treat various conditions as indicated by traditional Chinese medicine. Rat basophilic leukemia‐2H3 cell membranes were used as the stationary phase of a membrane affinity chromatography column to capture components with affinity for mast cells that could be involved in a degranulation reaction. The retained components were enriched and analyzed by membrane affinity chromatography with liquid chromatography and mass spectrometry via a port switch valve. Suitability and reliability of the method was investigated using appropriate standards, and then, the method was applied to identify components retained from tubeimu saponin extracts. Tubeimoside A was identified in this way as a potential allergen, and degranulation assays confirmed that tubeimoside A induces RBL‐2H3 cell degranulation in a dose‐dependent manner. An increase in Ca²⁺ influx indicated that degranulation induced by tubeimoside A is likely Ca²⁺ dependent. Coupled with the degranulation assay, RBL‐2H3 cell‐based affinity chromatography coupled with liquid chromatography and mass spectrometry is an effective method for screening and identifying allergic components from tubeimu saponin extracts.     

The purification and identification of human blood serum proteins with affinity to the antitumor active RL2 lactaptin using magnetic microparticles

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa‐casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein–protein interactions, which can affect its activity.