A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyvomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.     

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Island clustering analysis for the comparison of the membrane and the soluble protein fractions of human brain proteome

A protein identified in multiple separate bands of a 1-D gel reflects variation in the molecular weight caused by alternative splicing, endoproteolytic cleavage, or PTMs, such as glycosylation or ubiquitination. To characterize such a protein distribution over the bands, we defined an entity called an 'island' as the band region including the bands of the same protein identified sequentially. We quantified the island distribution using a new variable called an Iscore. Previously, as described in Park et al.. (Proteomics 2006, 6, 4978-4986.), we analyzed human brain tissue using a multidimensional MS/MS separation method. Here, the new method of island analysis was applied to the previous proteome data. The soluble and membrane protein fractions of human brain tissue were reanalyzed using the island distribution. The proteome of the soluble fraction exhibited more variation in island positions than that of the membrane fraction. Through the island analysis, we identified protein modifications and protein complexes over the 1-D gel bands.     

用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

Effects of amylopectin structure and molecular weight on microstructural and rheological properties of mixed beta-lactoglobulin gels

Nongelling amylopectin fractions from potato and barley have been used to form mixed beta-lactoglobulin gels. The amylopectin fractions were produced by varying the time of alpha-amylase hydrolysis followed by sequential ethanol precipitation. The molecular weights, radius of gyration, chain length distribution, and viscosity of the fractions were established. The mixed gels were analyzed rheologically with dynamic mechanical analysis in shear and microstructurally with light microscopy, transmission electron microscopy, and nuclear magnetic resonance spectroscopy. The result of the gel studies clearly showed that small differences in the molecular weight of amylopectins have a significant influence on the kinetics of protein aggregation and thereby on the gel microstructure and the rheological behavior of the gel. Both an increase in the molecular weight and a higher concentration of amylopectins resulted in a more open protein network structure, with thicker strands of larger and more close-packed beta-lactoglobulin clusters, which showed a larger storage modulus. The transmission electron micrographs revealed that degraded amylopectins were enclosed inside the protein clusters in the mixed gels, whereas nondegraded amylopectin was only found outside the protein clusters. The volume-weighted mean value of the molecular weight of the amylopectins was found to vary between 3.2 x 10(4) and 5.0 x 10(7) Da and the ratio of gyration between 14 and 61 nm. The maximum in chain length distribution was generally somewhat distributed toward longer chain lengths for potato compared to barley, but the differences in chain length distribution were minor compared to those seen in the molecular weight and ratio of gyration between the fractions.     

Actin in ejaculated human sperm cells

Previous studies have identified an "actin-like" protein in human sperm by indirect immunofluorescence microscopy with various probes, but no real biochemical confirmation of actin was made. In this study, two-dimensional (2-D) gels of Nonidet P-40 (NP-40) extracts of purified human sperm cells revealed a protein with appropriate pI and molecular weight coordinates for actin. When excised from a 2-D gel, cleaved with N-chlorosuccinimide, and separated on a sodium dodecyl sulfate slab gel, this putative actin showed a cleavage pattern identical to those from known actin. Furthermore, when sperm were immediately purified from whole semen and extracted with 0.3% NP-40, actin was detected almost entirely in the soluble fraction, indicative of unpolymerized actin. Conclusions from these experiments support those implied by direct immunofluorescence microscopy: actin is present in human sperm and appears to be mainly unpolymerized.     

Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry

A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.     

Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry

The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and phosphorylation.     

Thiourea enhances mapping of the proteome from murine white adipose tissue

Adipose tissue imposes problems in two-dimensional (2-D) analysis due to its extremely high content of fat. To improve protein separation detergents and chaotropes were varied in the IEF step. The most important factor for obtaining distinct spots in the 2-D gel was whether thiourea was included or not. Many high molecular weight spots became resolved by using thiourea, while no spots disappeared or showed inferior characteristics, thus approximately twice as many spots were possible to quantify. Hydrophobic indices were compared for a set of proteins that gave rise to sharper spots with proteins that were not improved on the use of thiourea. The comparison did not give any statistically significant difference between the two groups of proteins. One of the effects obtained by inclusion of thiourea was that the dominating protein, serum albumin, appeared as more condensed spots allowing other minor proteins to be detected. This work resulted in a protocol which greatly enhances the resolution of proteins in adipose tissue. A 2-D map of mouse white adipose tissue from epididymal fat pads was constructed in which 140 spots were identified by mass spectrometry. This work lays the ground for our further studies on white adipose tissue in metabolic diseases such as obesity and dyslipidemia.     

Purification of an Auxin-Binding Protein from Etiolated Mung Bean Seedlings by Affinity Chromatography

An auxin-binding protein with high affinity for 2,4-D and IAAwas purified from the extract of etiolated mung bean seedlingsby affinity chromatography on 2,4-D-linked Sepharose 4B andby gel nitration on Sepharose 4B. Its molecular weight was estimatedto be about 390,000 by gel nitration on Sepharose 4B and itconsisted of two different subunits with molecular weights ofabout 47,000 and 15,000. This protein had no ribulose-l,5-bisphosphatecarboxylase activity. Its dissociation constants for 2,4-D andIAA were 9.3 x 10^–6 M and 3.2 x 10^–6 M, respectively,as determined by Scatchard's method. (Received December 21, 1982; Accepted March 23, 1983)     

Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis. I. Detection of four new proteins

Kaltschmidt and Wittmann's two dimensional gel electrophoresis was improved in the following points. Preruns using radical scavengers were carried out to eliminate free radicals remaining in gels. Gelation of sample solutions was not performed to avoid immobilization of proteins in the sample gels. Instead, for preparing sample gels, prior to the first dimension (1-D) electrophoresis, another electrophoresis was performed to charge proteins into gel pieces polymerized previously. Proteins migrated together with charged reductants to avoid formation of artificial disulfide bridges during migration. The second dimension (2-D) electrophoresis was carried out at a more acidic pH, 3.6, to get better separation of very small and basic proteins. With these modifications, quantitative yield and reproducibility became better, and many faint spots disappeared not only at the high molecular weight side but also in the region containing primary spots of ribosomal proteins. The proportionality of the migration distance to the logarithm of molecular weight was also increased. On the improved 2-D electrophoretogram of Escherichia coli ribosomal proteins, four new spots, called protein A, B, C, and D, were found in the basic region. Proteins A, B, and C belong to 50S subunits but D to 30S. Their molecular weights were determined electrophoretically as 6,400, 4,900, 8,200, and 5,900, respectively. Their copy numbers in crude ribosomes were estimated to be 0.6, 0.4, 0.3, and 0.1, respectively, by using 14C-labeling.     

Preservation of proteins in mummified tissues

Protein material was extracted from the dessicated tissues of several Egyptian mummies and a frozen Eskimo. The distribution and degree of preservation of high molecular weight protein was analyzed by gel filtration, protein assays, amino acid analysis, and polyacrylamide gel electrophoresis. The protein has undergone considerable degradation although some high molecular weight protein (C. 130,000 daltons) remains intact. Amino acid analysis of the extracted protein indicates the basic amino acids have undergone a chemical modification and may represent a point of preferential breakdown in the polypeptide chain. Atomic absorption spectrophotometry of tissue cations suggests a correlation between degree of preservation of mummified tissue and levels of sodium salts (natron) in the tissue.     

Identification of the MT3 molecule using two-dimensional gel electrophoresis and alloantisera

The MT3 antigen is defined serologically as a DR supertypic specificity and is strongly associated with DR4, DR7, and DRw9. To determine whether the MT3 molecule is distinct from the DR molecule, DR4 and MT3 antigens were immunoprecipitated from 125I-labeled plasma membrane glycoproteins of a DR4-homozygous, MT3-homozygous B lymphoid cell line, Wa, and compared by two-dimensional (2-D) gel electrophoresis. The precipitates with two different anti-DR4 alloantisera and with three different mouse antibodies against human Ia monomorphic determinants gave the same 2-D gel pattern consisting of one heavy chain with a molecular weight of 34 000 and a set of light chains with a molecular weight of 30 000, indicating that these polypeptides are the components of the DR4 molecule. On the other hand, all three anti-MT3 alloantisera used precipitated an identical set of anti-MT3 alloantisera specific light chains with a molecular weight of 30 000, and one heavy chain with a molecular weight of 34 000. The pI of the MT3 light chain was more acidic than that of the DR4 light chain. The amount of MT3 light chains was much smaller than that of DR4 light chains in unlabeled plasma membrane glycoproteins. Thus, we have demonstrated directly using 2-D gel electrophoresis and anti-MT3 alloantisera that the MT3 antigen is a new human Ia molecule distinct from DR4.     

Alpha 1-antitrypsin: apparent molecular weight heterogeneity shown by two-dimensional electrophoresis.

Two-dimensional (2-D) gel electrophoresis was used to examine charge and molecular weight variability of alpha 1-antitrypsin. Two-D electrophoresis resolved distinctive differences among individual phenotypes. Microheterogeneity of charge was seen for the different alleles that corresponded to the charge variability observed on isoelectric focusing gels. The molecular weights of the major components of each allele appeared to differ from each other by approximately 1,000, suggesting, that in addition to sialic acid, there may be differences in neutral sugar composition between the individual components. In comparison to the M allele components, the corresponding S and Z components had higher molecular weights. The MZ and MS phenotypes showed characteristic patterns of protein spot doublets. Computerized quantitation was used to separate and estimate the contribution of each component to the overall allele composition. The Z allele components contained about 15% of the total MZ quantity. The 2-D electrophoresis technique may offer a new approach for molecular structural studies of alpha 1-antitrypsin variants and similar glycoproteins.     

Differential protein phosphorylation in human gastric adenocarcinomas.

A qualitative analysis of endogenous protein phosphorylation in microsomal fractions from surgical specimens of human gastric cancer, benign gastric ulcers and normal gastric tissues is presented. Fractions were incubated in the presence of (gamma-32P)ATP to measure the transfer of (gamma-32P) to natural substrates mediated by endogenous protein kinases. Phosphoproteins were characterized through PAGE-SDS and detected by autoradiography. KOH at high temperature was used to select for tyrosine-phosphorylated polypeptides on dried gels. We report a notorious enhancement in overall protein phosphorylation in gastric cancer samples over benign ulcers and normal controls as well. Moreover, a highly basic-low molecular weight phosphoprotein is found through 2-D protein gel analysis and a 50 kDa protein is detected only in the presence of Mg2+ after KOH treatment. These two proteins might become putative molecular markers to detect this type of neoplasia.     

The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products

An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes. This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs). The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry. The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin. A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis. The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.     

双向电泳分析鸢尾绿白嵌合叶片的蛋白质

利用双向聚丙烯酰胺凝胶电泳对鸢尾（Iris japonica)绿白嵌合叶片的蛋白质进行分离,并初步鉴定了蛋白质的相对分子量和等电点。每个电泳图谱共检测到400余个蛋白点,其中至少13个蛋白的表达变化明显;结果表明,嵌合叶片的绿色与白色叶组织具有明显不同的蛋白质双向电泳图谱。与数据库中拟南芥双向电泳图谱相比较,发现Rubisco大亚基,标记为W和T蛋白的表达变化与产生绿白嵌合叶片的表型密切相关。     

Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract.

A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native/SDS-2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor alpha chain (IL-2Ralpha) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2/IL-2-2Ralpha complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions.     

双向凝胶电泳比较三种常用蛋白质提取方法

组织(或细胞)的蛋白质提取效率直接影响蛋白质双向凝胶电泳(2-DE)的分辨率.为探索建立适用于人乳腺癌细胞株MCF-7蛋白质提取的最佳条件,比较目前在双向凝胶电泳中常用的3种蛋白质提取方法对MCF-7细胞总蛋白的提取效率.MCF-7细胞经培养后,分别采用M-PER试剂、标准裂解液或含硫脲裂解液提取其总蛋白质,然后进行双向凝胶电泳,并根据凝胶上蛋白质斑点的丰度和分布特点判断所得双向电泳图谱的质量,以确定MCF-7细胞蛋白质提取的相对最佳方法.结果显示,M-PER试剂法得到的图谱分辨率较低,蛋白质主要集中分布在分子量15~70kD,pH4.7~6.3的范围内;标准裂解液法得到的图谱分辨率有所提高,蛋白质分布比M-PER试剂法得到的图谱广;硫脲裂解液法得到的图谱是三者中分辨率最高的,尤其是高丰度蛋白和高分子量蛋白分离效果比前两者好.结果表明,在3种常用的蛋白质提取方法中,硫脲裂解液对细胞蛋白质的溶解性最佳,相对更适合于提取MCF-7细胞的蛋白质,并与双向凝胶电泳条件更兼容.