Visualized heterooligomeric subunit structures of 817 human cellular proteins by correlating native protein 2D maps with protein interaction databases.

Previously, we have reported on the reconstruction of 4323 native protein 2D maps of human bronchial smooth muscle cells (HBSMC) by combining nondenaturing 2DE, grid gel-cutting, and quantitative LC-MS/MS. In this work, we report on the visualization of the heterooligomeric subunit structures of HBSMC proteins by correlating the native protein 2D maps with the information in protein interaction databases. Image analysis of the 2D maps was employed as the first approach. Each of the native protein 2D maps of 2327 proteins, which had three or more detected squares in the native protein 2D maps, was compared with the 2D maps of the remaining 2326 proteins scoring the degree of overlap in an area around the quantity peak and the protein partner which showed the best score was decided. The protein pairs were examined on their reported interactions referring to protein interaction databases. In order to consider the cases where a protein has multiple functions and the heterooligomeric subunit structures might not be detected from the image analysis, prior database search was employed as the second approach. Each of the 1689 HBSMC proteins, which had five or more detected squares in the native protein 2D maps, was examined on its interactor proteins described in the databases, then the native 2D map was compared with the maps of the interactor proteins to find the overlap which reasonably supported the interaction. Summarizing these examinations, 215 heterooligomeric subunit structures of 817 human cellular proteins could be visualized on the native protein 2D maps.

Simultaneous speculation of 401 monomeric or homo-oligomeric subunit structures of human cellular proteins, mining the information in 1901 native 2D protein maps reconstructed from one nondenaturing 2DE gel.

Subunit structures of proteins are essential for their properties and functions, but there is a lack of method for global detection of the status of proteins being monomers or homo-oligomers. In this work, we report on a new method to simultaneously speculate hundreds of monomeric or homo-oligomeric subunit structures of cellular proteins, based on in-depth analysis of native 2D protein maps. Previously we have reported on the analysis of soluble proteins of human bronchial muscle cells (HBSMC) by combining nondenaturing 2DE, grid gel-cutting and quantitative LC-MS/MS. Totally 4323 proteins were detected and for each protein the quantity distribution on the gel was reconstructed as a native 2D map. In this work, this large dataset of maps were further mined with bioinformatic analysis. The native 2D maps of 1901 HBSMC proteins that were detected in at least five out of the grid-cut 972 gel squares were examined and 658 proteins that showed one major quantity-peak distribution were subjected to further analysis. After excluding those that mainly formed hetero-oligomeric structures, the monomeric or homo-oligomeric subunit structures of 505 proteins were speculated. The quotient of the apparent molecular mass of the quantity-peak position on the native 2D map divided by the theoretical molecular mass was calculated for each protein, to speculate the number of monomers which constituted its subunit structure. The suggested composition was then compared with the "Subunit structure" record of the protein in UniProtKB. When the database record included possible interactions with other proteins, their native 2D maps were extracted from the native map dataset, presented together and compared to confirm the prominent subunit structure. With this new approach, the monomeric or homo-oligomeric subunit structures of 401 proteins were speculated. Among them, 162 proteins had the speculated subunit structures coinciding with their database records, and 91 proteins with matched database records as being monomers or homo-oligomers but mismatched at the numbers of the composing monomers. For 148 proteins that did not have database record, their subunit structures were newly speculated. We expect this method, combining nondenaturing 2DE separation with in-depth proteomic and bioinformatic analysis, would suggest a means to achieve large-scale information on monomeric and homo-oligomeric subunit structures of cellular proteins.

Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS.

The supernatant and precipitate protein fractions from human bronchial smooth muscle cells (HBSMC) were analyzed using 1D SDS-PAGE-LC-MS/MS and the performance of the method was compared with that of nondenaturing 2DE-LC-MS/MS applied to the supernatant fraction, the methods and the results have been reported previously. The 1D gel lanes were cut into pieces of the same size, in order to enable the reproduction of digital 1D images of the individual MS-assigned proteins. When the obtained information on individual HBSMC proteins was compared between the two methods, SDS-PAGE is advantageous in visualizing the quantity differences between differently treated samples, whereas nondenatuing 2DE is advantageous in visualizing protein interactions. SDS-PAGE-MS of the supernatant fraction provided the assignment of 2552 proteins and their percent abundance ranged from 3.5% to 2 × 10-4%. 2DE-MS of the supernatant fraction provided 4323 proteins with percent abundance ranged from 3.6% to 1 × 10-5%, suggesting that the step of isoelectric focusing served to raise the sensitivity of the method. The proteins in the precipitate fraction, which could not be analyzed by 2DE-MS, were characterized by the abundance of proteins allocated to "membrane" within the category "Cellular component" of UniProtKB database and especially those allocated to "transmembrane" within the subcategory "membrane." On the other hand, there were about 600 "membrane" proteins which showed more than two-fold higher percent abundance in 2DE-MS than in SDS-PAGE-MS. These results showed that 1D SDS-PAGE and nondenaturing 2DE would provide complementary information on the analysis of proteins and protein interactions in cells.

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole-gel slicing and quantitative LC-MS/MS: Utilizing native MS-electropherograms in proteomic analysis for discovering structure and interaction-correlated differences.

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.

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.

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 1. Preparation of more than 4000 native protein maps.

Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm × 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm × 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [1]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6% to 1 × 10(-5) % of the total protein quantity in the grid area. Each protein map was characterized by several features, including the position of quantity peak square, number of detected squares, and degree of concentration (focused or dispersed). About 4% of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with a defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2D gel.

Native protein mapping and visualization of protein interactions in the area of human plasma high-density lipoprotein by combining nondenaturing micro 2DE and quantitative LC-MS/MS.

A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high-density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions.

Performance of nondenaturing micro 2-DE followed by third-dimension SDS-PAGE in the analysis of Escherichia coli soluble proteins.

In a previous paper, we reported on the analysis of Escherichia coli (strain K-12) soluble proteins by nondenaturing micro 2-DE/3-DE and MALDI-MS-PMF [Manabe, T., Jin, Y., Electrophoresis 2010, 31, 2740-2748]. To evaluate the performance of the 2-DE/3-DE technique, a nondenaturing 2-DE gel just after the second-dimension run was cut into 12 vertical strips, each 2 mm-wide strip was set on a micro slab gel, and third-dimension SDS-PAGE was run in parallel. Each of the twelve 3-DE gels showed about 150-200 CBB-stained spots. Two of the 3-DE gels were selected for the assignment of polypeptides using MALDI-MS-PMF and totally 161 polypeptides were assigned on the two 3-DE gels, in which 81 have been assigned on the nondenaturing micro 2-DE gel and 80 were newly assigned. Most of the newly assigned polypeptides resided in faintly stained spots on the 3-DE gels, which indicates that the polypeptides were purified in the process of the third-dimension separation. The comparisons of the apparent mass values estimated from the second-dimension (nondenaturing pore-gradient PAGE) mobility with those estimated from the third-dimension (SDS-PAGE) mobility suggested the oligomer structures of the assigned polypeptides and they matched well with those described in a database (UniProtKnowledgebase). The technique of nondenaturing micro 2-DE/3-DE, combined with MALDI-MS-PMF, could become an efficient method to obtain information on the quaternary structures of hundreds of cellular soluble proteins simultaneously because of its high efficiency in protein/polypeptide separation and assignment.

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.

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus.

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA(3) gene (WT*). The DeltaPsb30 mutant appears more susceptible to photodamage, has a cytochrome b(559) that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b(559) into the low potential form. Structural reasons for such effects are discussed.

Analysis of PEG-fractionated high-molecular-mass proteins in human plasma by non-denaturing micro 2-DE and MALDI-MS PMF.

The process of 0-4% PEG precipitation of high-molecular-mass proteins (around and above 1x10(3) kDa) in human plasma was analyzed by non-denaturing micro 2-DE employing agarose IEF gels in the first dimension, and by MALDI-MS PMF. The PEG 0-2% precipitate could be mostly re-dissolved (fraction PS2%), but some part remained as precipitate (fraction PP2%). Non-denaturing 2-DE followed by MALDI-MS PMF showed that PS2% contained fibrinogen (Fb), fibronectin (FN), and their oligomers as the major components, and von Willebrand factor and coagulation factor XIII as the minor components. PP2% was directly subjected to MALDI-MS PMF and FN and Fb were assigned, suggesting it was formed by co-precipitation of the two proteins. The PEG 2-4% precipitate could be totally dissolved, but after freezing and thawing the solution, a small amount of precipitate appeared. MALDI-MS PMF analysis of the precipitate (PP4%) suggested that its major constituent is complement C1. The soluble fraction of the PEG 2-4% precipitate (PS4%), analyzed by non-denaturing 2-DE and MALDI-MS PMF, contained C4b-binding protein and its complex with complement C4, low-density lipoproteins, IgM, and complement C1 subcomponent q, together with Fb, FN, and their oligomers. When the PS4% fraction was analyzed by a Type-II 2-DE technique (non-denaturing IEF followed by SDS-PAGE, no reducing agent being used), several proteins in molecular mass range from 7x10(1) to 2x10(2) kDa appeared, suggesting that these proteins interacted with the high-molecular-mass proteins, then dissociated in the presence of SDS.

Differences in protein distribution between human plasma preparations, EDTA-plasma and heparin-plasma, analyzed by non-denaturing micro-2-DE and MALDI-MS PMF.

The effects of plasma preparation methods on the status of human plasma proteins were analyzed by non-denaturing micro-2-DE followed with polypeptide assignment with MALDI-TOF MS and PMF. In order to facilitate the separation of high-molecular-mass plasma proteins (up to ca. 2x10(3) kDa) in short separation time, agarose micro-IEF gels were employed. The comparisons of the 2-DE patterns between EDTA-plasma (1.0 mg EDTA/mL blood) and heparin-plasma (0.025 mg heparin/mL blood) revealed the differences in protein distribution around pI 5.4-6.5 and apparent molecular mass of ca. 1x10(3) kDa, which were mainly attributed to the presence of the complexes of complement C4b and C4b-binding protein in heparin-plasma and their absence in EDTA-plasma. The distribution of several spots around pI 5.0-5.6 and apparent molecular mass 1.2-1.5x10(2) kDa was also found to be different; the fragments of complement C3 and C4 were detected in heparin-plasma but not in EDTA-plasma. The 2-DE pattern of high-heparin-plasma (0.50 mg heparin/mL blood) showed pI changes of three plasma proteins, fibronectin, complement factor B, and pre-alpha-inhibitor when compared with that of heparin-plasma, suggesting the interactions between heparin and the proteins. These results demonstrated that subtle changes in plasma proteins, caused by the different plasma preparation procedures, can be analyzed by non-denaturing agarose-IEF/micro-2-DE followed by MALDI-MS and PMF.

Performance of agarose IEF gels as the first dimension support for non-denaturing micro-2-DE in the separation of high-molecular-mass plasma proteins and protein complexes.

Agarose micro-column gels (1% w/v agarose, diameter 1.4 mm and length 35 mm) were prepared as the first-dimension IEF support for non-denaturing 2-DE and the performance was compared with that of polyacrylamide gels (4.2% T and 4.8% C, the same gel size) using a human plasma sample. Sorbitol was not added in the agarose IEF gels, since its presence not only delayed the focusing of the proteins but also deteriorated the protein resolution. The optimum IEF time of the agarose gels for separation of 2 microL plasma sample (ca. 120 microg proteins) was decided to be 46 min, which is much shorter than that of the polyacrylamide gels (75 min). MALDI-MS and PMF assignment of the spots on the micro-2-DE gels at apparent molecular mass above ca. 5x10(2) kDa and pI from 4 to 8 revealed that when polyacrylamide IEF gels were used, many of the high-molecular-mass proteins resided at the sample loading edge or in basic pI regions as smear bands. When agarose IEF gels were used, most of the high-molecular-mass proteins moved to more acidic pI positions and were better focused, and their apparent pI values matched well with those previously reported for purified proteins. These results demonstrated the advantages of agarose-IEF/2-DE for the separation of high-molecular-mass proteins and protein complexes under non-denaturing conditions.

Varus-valgus laxity correlates with pain in osteoarthritis of the knee.

Pain during osteoarthritis (OA) of the knee does not necessarily correlate with the severity of the radiographic grade, and the mechanism of pain has not been completely clarified. The purpose of this study was to evaluate risk factors for pain in the knee OA using epidemiologic analyses. We evaluated 518 out of 4183 people over the age of 40 (156 males and 362 females) from Shinyoshitomi village, Japan. Mean ages were 63.8 years for men and 60.7 years for women. Screening included a physical examination of the knee and a standing AP roentgenogram of the bilateral knee. Radiographic OA was defined as a Kellgren-Lawrence grade 2 or higher. All data were coded and pain risk factors were evaluated using a multiple logistic regression model. Radiographic OA was observed in 18.4% of men and 26% of women. Of these subjects with OA, 10.9% of men and 32.5% of women complained of knee pain. Seven factors-age, gender, BMI, radiographic grade, varus-valgus laxity, torque of quadriceps muscles, and varus-valgus alignment-were evaluated as potential risk factors for pain. A significant increase in the odds ratio was observed with varus-valgus laxity (p=0.005; odds ratio, 3.04). Our results suggest that varus-valgus laxity is a risk factor for pain during knee OA.

Two subtypes of radiographic osteoarthritis in the distal interphalangeal joint of the hand.

BACKGROUND: The etiology and pathogenesis of hand osteoarthritis (OA) are not completely clarified, and several factors may cooperate in a multifactorial fashion in its development. The purpose of this study was to clarify the effects of the dominant hand that contribute to the development of distal interphalangeal (DIP) joint OA using epidemiological analyses. METHODS: A total of 518 subjects (156 men, 362 women) in a rural community were analyzed. Their mean age was 63.8 years for men and 60.7 years for women. Anteroposterior (AP) standing radiographs of bilateral knees, lateral views of the lumbar spine, and AP views of bilateral hands were obtained. Furthermore, a survey of their life patterns was conducted using self-administered questionnaires. Radiographic osteoarthritis was defined as Kellgren and Lawrence grade 2 or higher. Hand OA was limited to Heberden's nodes. Generalized OA (GOA) was defined as bilateral knee OA plus lumbar spine OA. RESULTS: GOA was observed in 13.0% of the subjects. The incidence of DIP joint OA was significantly higher in the GOA group than that in the non-GOA group. In the GOA group, the incidence of right-hand DIP joint OA in right-handed and left-handed subjects was 37.5% and 40.0%, respectively, without a significant difference. In the non-GOA group, however, the incidence of right-hand DIP joint OA in right-handed and left-handed subjects was 16.4% and 3.2%, respectively, with a significant difference. With a multiple logistic regression model, the P value of the handedness was marginal (0.060), but a clear tendency of increase in the odds ratio (7.129) was observed in the dominant hand for the non- GOA group. In contrast, there was no effect of the handedness on right-hand DIP joint OA in the GOA group. CONCLUSIONS: There are two subtypes of hand DIP joint OA in terms of the etiology. One is environmental, and the other is genetic.

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 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.

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.

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.