Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.     

Comparison of SYPRO Ruby and Deep Purple using commonly available UV transilluminator: wide-scale application in proteomic research

Fluorescent stains are becoming increasingly useful in proteomics research involving protein expression as well as post-translational modification studies and are particularly useful for samples which are expensive and scarce. The fluorescent dyes Deep Purple and SYPRO Ruby are widely used in protein expression studies. Using UV transillumination and Charged Coupled Device (CCD) based imaging system, their relative sensitivity to detect proteins separated by two-dimensional polyacrylamide gel electrophoresis and downstream protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was compared. Using mouse liver homogenate, we detected a greater number of spots using SYPRO Ruby over Deep Purple stain. However, the number of matched peptides and the percentage of amino acid residues identified for 21 different proteins were comparable suggesting their equivalency for LC-MS/MS identification. In spite of comparable MS compatibility, we recommend the use of SYPRO Ruby for expression proteomics due to its higher sensitivity in detecting protein spots.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

Identification of Neuron-Specific Enolase and Nonneuronal Enolase in Human and Rat Brain on Two-Dimensional Polyacrylamide Gels

The location of the enzymes neuron-specific enolase and nonneuronal enolase on two-dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: comigration on polyacrylamide gels with the appropriate purified protein and staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron-specific enolase from rat and human brain is highly pure, as only one spot is obtained on two-dimensional gels. Further, the antiserum to neuron-specific enolase is highly specific, as it reacts only with neuron-specific enolase on nitrocellulose blots derived from two-dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two-dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two-dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.     

The instantaneous monitoring of polyacrylamide gels during electrophoresis.

The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.     

Identification of protein bands in polyacrylamide gel by protein printing

Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.     

Binding mitochondria to cryogel monoliths allows detection of proteins specifically released following permeability transition

Following proapoptotic signals such as calcium-induced mitochondrial permeability transition or translocation of proapoptotic proteins, mitochondria induce cell death through release of apoptogenic proteins. The mechanism of release and the identity of the released proteins are currently debated. Earlier attempts at identification of the apoptogenic proteins have been hampered by a high nonspecific background. Our aim was to develop a novel method where background release was eliminated, allowing proteins specifically released from mitochondria following proapoptotic stimulation to be identified. Liver mitochondria were immobilized and washed on cryogel monoliths prior to induction of protein release (calcium or Bid/Bax). Immobilized mitochondria exhibited normal morphology and swelling response and retained respiratory activity. The released proteins were collected, concentrated, separated on polyacrylamide gels which were cut into pieces, trypsin-digested, and analyzed using liquid chromatography-tandem mass spectrometry. Control samples contained no protein, and stimulation with calcium and Bid/Bax resulted in identification of 68 and 82 proteins, respectively. We conclude that, in combination with the robust proteomic approach, immobilization on cryogel monoliths is a fruitful approach for studying specific protein release from isolated mitochondria. We propose that this method is a powerful tool to further characterize the role of mitochondria in cell death induction.     

A new two-dimensional gel electrophoresis system for the analysis of complex protein mixtures: Application to the ribosome of E. coli

A new method for two-dimensional polyacrylamide gel electrophoresis of proteins is described. The method, illustrated here by its application for the analysis of ribosomal proteins of E. coli, has a high resolving power. The proteins S15 and S16 can be resolved either following alkylation or under reducing conditions. This was not possible with urea gel systems previously employed. The method should be advantageous in the identification of the components of dimers formed with the reagent methyl 4-mercaptobutyrimidate. An additional advantage of the new method is that both dimensions are run at an acidic pH. For ribosomal proteins it is therefore unnecessary to either polymerize the protein sample in the middle of the first dimension disc gel or to electrophorese two samples with opposite polarity.     

Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.     

Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. This enables identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE can be used to compare quantities of proteins in related samples, such as those from altered environments or from mutant and wild type, thus allowing the response of classes of proteins to be determined. Those proteins showing a correlated difference in expression may participate in related processes, and this subsequently helps to define protein function. Although there are many limitations of the 2D gel technology that mean it will never be comprehensive in protein coverage, its use for the identification of relatively abundant proteins is now widespread. However, there are still surprisingly few examples of quantitative analysis of changes in protein abundance. In this review we highlight recent advances towards true quantitative analysis of 2D gels that will lead to better prediction of protein function. Despite the development of promising alternatives, 2D PAGE is likely to remain in extensive use for the foreseeable future, because the technology is now simple and readily available to many laboratories.     

Identification of Proteins by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry Following In-Gel Digestion in Low-Salt, Nonvolatile Buffer and Simplified Peptide Recovery

Matrix-assisted laser desorption ionization–mass spectrometry is an efficient analytical method for large-scale identification of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Following in-gel digestion, the salt present in the peptide extracts is usually removed by chromatography prior to analysis. Desalting is a labor-intensive and time-consuming step, limiting the total number of samples that can be processed daily. We improved the daily sample output by performing the in-gel protein digestion in low-salt, nonvolatile buffer and simplifying the recovery of the generated peptides, collecting them in a small volume by sonication. This technique is routinely used for identification of proteins ofHaemophilus influenzaeand human brain. The methodology described facilitates the analytical process and allows the analysis of hundreds of proteins per day. Furthermore, it represents an essential step toward process automation.     

Towards a standardized human proteome database: quantitative proteome profiling of living cells

Comparative proteome profiling, performed by two-dimensional polyacrylamide gel electrophoresis or multidimensional protein identification technology, usually relies on the relative comparison of samples of interest with respect to a reference. Currently, no standardized quantitative protein expression database of human cells, facilitating data comparisons between different laboratories, exists. Recently, we have published two-dimensional polyacrylamide gel electrophoresis-based techniques to assess absolute protein data comprising protein amounts, synthesis rates and biological half-lives (Mol. Cell. Proteomics 2002, 1, 528-537). Determination of protein amounts by fluorography of two-dimensional gels was followed by the exact quantification of the amount of incorporated (35)S radiolabel. Here we demonstrate an application of this highly standardized method to quiescent human T cells, phythaemagglutinin-stimulated T cells and Jurkat cells, a human T lymphoblast cell line. While the protein composition of quiescent T cells differed significantly compared to that of Jurkat cells, it was only slightly different compared to the activated T cells. Synthesis profile analyses demonstrated that activated T cells clearly differed from the quiescent cells, performing apparently almost like lymphoblast cells. The great sensitivity of this approach was further demonstrated with human umbilical vein endothelial cells treated for six hours with vascular endothelial growth factor. While no significant alteration of protein amounts was detected at all upon activation, the synthesis rate of several proteins was found to be more than doubled.     

Horizontal carryover of proteins on one-dimensional polyacrylamide gels may jeopardize gel-enhanced liquid chromatography mass spectrometry proteomic interpretations

Mass spectrometric identification of gel-separated proteins is a cornerstone of many proteomic efforts. Often the protein compositions of two neighboring lanes (typically representing the experiment and control) are compared assuming that proteins are separated only in a vertical dimension and do not spread horizontally. However, we noticed that horizontal protein spreading commonly occurs on one-dimensional polyacrylamide gels and might lead to a misleading judgment regarding the presence or absence of particular proteins even in the distantly spaced lanes. Therefore, we suggest that experimental and control samples should always be loaded on separate gels.     

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.     

EBP, a program for protein identification using multiple tandem mass spectrometry datasets

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.     

Identification of covalently bound fatty acids on acylated proteins immobilized on nitrocellulose paper

A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described. As demonstrated for [3H]palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices. Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography. The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system.     

A sensitive immunotransfer method for the detection of oligodendroglial plasma membrane antigens

Efficient transfer of a wide molecular weight range of plasma membrane proteins from polyacrylamide gels to nitrocellulose paper has been achieved. Using published immunodetection procedures (sensitivity: 1 ng protein), two major antigens of molecular weights of 71,000 and 82,000 have been identified in oligodendroglial plasma membranes. This immunotransfer method will be extremely useful for the detection of low levels of antigen in complex membrane protein mixtures.     

尿液蛋白富集方法的比较

人尿液中蛋白含量低,在进行质谱分析时易被高丰度蛋白掩盖。因此,发展高效和高选择性的富集方法,是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法,等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本,富集尿液蛋白,SDS-PAGE分离尿蛋白,比较不同方法纯化的效率;通过质谱分析,比较不同纯化方法的肽段鉴定效果,确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法,饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测,在保证回收蛋白质量的前提下,可减少高丰度白蛋白的干扰,富集更多低丰度蛋白,提高了质谱鉴定的灵敏度。综上所述,饱和硫酸铵提取尿蛋白的效果较好,该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。     

Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI Sepsityper(TM) Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3) -10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (10(4) cfu mL(-1) ), bacterial identification could be performed after initial incubation at 37°C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.