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.     

Combinatorial peptide ligand library plasma treatment: Advantages for accessing low‐abundance proteins

Depletion of major blood proteins is one of the most promising approaches to accessing low‐abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low‐abundance proteins in plasma using 2‐DE in an acidic restricted pH range (4–7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI‐TOF related to their absence in the 2‐D gels from native or albumin and IgG‐depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low‐abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.     

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.     

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.     

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.     

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.     

Optimizing protein recovery yield from serum samples treated with beads technology

Proteomics studies are often complicated by the wide dynamic range of the biological fluids, in which few highly abundant proteins obscure the signal of low abundant ones. To overcome this problem, several techniques have been developed on the basis of "depletion principles," namely immuno-subtraction with specific antibodies against the most-abundant proteins. Unfortunately, the probability of codepletion is a noteworthy drawback associated with these strategies. The ProteoMiner (PM) technology is a novel approach, consisting of a combinatorial library of hexapeptide ligands coupled to beads, that allows the capture of all species present in a proteome, but at much reduced protein concentration differences, simultaneously enhancing the concentration of the most dilute species. In this study, we evaluated the compatibility of the PM kit's elution reagent with 2-DE analysis, comparing five different purification methods on serum samples eluted from the beads: the "ReadyPrep 2-D Clean-up kit" and precipitation with organic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol. Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitation with ACN offered the most promising approach, showing the best spot resolution in all regions of the pH gradient and the greatest number of protein spots visualized on 2-D gels.     

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.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

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.     

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.     

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.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

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.     

Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry

We identified several low-abundance proteins of bovine colostrum and mature milk using the immunoabsorption technique and two-dimensional electrophoresis (2-DE) followed by microsequencing and mass spectrometry. Two major milk proteins, beta-casein and immunoglobulin G (IgG), were effectively removed from the milk using immunoabsorbents. Milk samples before and after immunoabsorption were separated by 2-DE. Protein identification of the spots on 2-DE was performed by either gel comparison, microsequencing, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF-MS), peptide mass fingerprinting or peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight-MS (Q-TOF). Significant differences in protein patterns were observed between the low-abundance proteins of colostrum and mature milk. In addition, several low-abundance proteins including fibrinogen beta-chain, chitinase 3-like 1, alpha-antitrypsin, complement C3 alpha-chain, gelsolin and apolipoprotein H were observed only in colostrum. However, the level of beta-casein fragments increased significantly during this lactation period. alpha-Lactalbumin and beta-lactoglobulin as well as some low-abundance proteins including bovine serum albumin, serotransferrin and lactoferrin were identified in both colostral and mature milk. Low-abundance proteins in bovine colostrum may have special physiologic relevance to the health and development of calves early in lactation.     

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.     

血清中低分子量蛋白质的提取和鉴定

采用改进的圆盘凝胶电泳提取人血清中低分子量蛋白质, 去除了血清中分子量大于3×104的蛋白质, 将提取的低分子量蛋白质热变性后直接在溶液中酶解成肽, 经液相色谱-质谱分析, 并进行Mascot数据库检索, 确认出人血清中97种蛋白质.     

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.     

Effects of aprotic solvents on the stability of metal‐free superoxide dismutase probed by native electrospray ionization–ion mobility–mass spectrometry

Considering that aprotic solvents are often used as cosolvents in investigating the interactions between small molecules and proteins, we assessed the effects of five aprotic solvents represented by dimethylformamide (DMF) on the structure stabilities of metal‐free SOD1 (apo‐SOD1) by native electrospray ionization–ion mobility–mass spectrometry (ESI‐IM‐MS). These aprotic solvents include DMF, 1,3‐dimethyl‐2‐imidazolidinone (DMI), dimethyl sulfoxide (DMSO), acetonitrile (ACN), and tetrahydrofuran (THF). Results indicated that DMI, DMSO, and DMF at low percentage concentration could reduce the average charge and the dimer dissociation of apo‐SOD1. By contrast, ACN and THF at low concentration have no similar effect. DMF was selected as a representative solvent to further investigate the detailed effects on the structure stability of apo‐SOD1 by using collision‐induced dissociation and unfolding. The results reveal that the addition of minimal DMF to an aqueous protein solution can protect against the unfolding and dissociation of dimer, even under destabilizing conditions (such as low pH or high cone voltage). When the different percentage concentrations of DMF were added, the average collision cross section of apo‐SOD1 showed that apo‐SOD1 became compacted when the DMF concentration increased from 0% to 1% and eventually started extending when increased from 1% to 20%. The results indicated that DMF has similar effects to DMSO in native mass spectrometry (MS) and it can also be used as a cosolvent besides DMSO in investigating the stabilities of proteins and the interactions between small molecules and proteins.