Unveiling the rat urinary proteome with three complementary proteomics approaches

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS‐PAGE separation, followed by LC‐ESI‐MS/MS; 2DE, followed by MALDI‐TOF‐TOF and 2D‐liquid chromatography‐chromatofocusing, followed by LC‐ESI‐Q‐TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases.     

Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry

The human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin-enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two-dimensional (2-D) maps and subsequent identification of over 100 protein spots by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post-translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold-detergent method yielded a large number of known caveolae residents, including caveolin-1. Thus, a large part of this subproteome was established, including known (trans-)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)-anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP-2, C8ORF2, and XRP-2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.     

Determination of the glycoprotein specificity of lectins on cell membranes through oxidative proteomics

The cell membrane is composed of a network of glycoconjugates including glycoproteins and glycolipids that presents a dense matrix of carbohydrates playing critical roles in many biological processes. Lectin-based technology has been widely used to characterize glycoconjugates in tissues and cell lines. However, their specificity toward their putative glycan ligand and sensitivity in situ have been technologically difficult to study. Additionally, because they recognize primarily glycans, the underlying glycoprotein targets are generally not known. In this study, we employed lectin proximity oxidative labeling (Lectin PROXL) to identify cell surface glycoproteins that contain glycans that are recognized by lectins. Commonly used lectins were modified with a probe to produce hydroxide radicals in the proximity of the labeled lectins. The underlying polypeptides of the glycoproteins recognized by the lectins are oxidized and identified by the standard proteomic workflow. As a result, approximately 70% of identified glycoproteins were oxidized in situ by all the lectin probes, while only 5% of the total proteins were oxidized. The correlation between the glycosites and oxidation sites demonstrated the effectiveness of the lectin probes. The specificity and sensitivity of each lectin were determined using site-specific glycan information obtained through glycomic and glycoproteomic analyses. Notably, the sialic acid-binding lectins and the fucose-binding lectins had higher specificity and sensitivity compared to other lectins, while those that were specific to high mannose glycans have poor sensitivity and specificity. This method offers an unprecedented view of the interactions of lectins with specific glycoproteins as well as protein networks that are mediated by specific glycan types on cell membranes.

A lectin proximity oxidative labeling (Lectin PROXL) tool was developed to identify cell surface glycoproteins that contain glycans that are recognized by lectins.     

差减凝胶渗透色谱在产品浓度和品牌识别中的应用

差减凝胶渗透色谱(D-GPC)以已知的标准样品溶液作淋洗剂,样品溶液作进样试液,通过内部差减,快速准确地给出样品溶液与标准样品溶液各组分之间的差异。以聚乙二醇(PEG200)的浓度识别以及3种啤酒的品牌识别为例,通过比较常规GPC与D-GPC所得到的实验结果,说明D-GPC方法在啤酒质量控制和品牌识别等方面所具有的应用价值。     

Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics

The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.     

Isolation and identification of three urinary metabolites of retinoic acid in the rat.

After the intraperitoneal administration of high doses of ¹⁴C- and ³H-labelled retinoic acid ( 1 ) to rats three major urinary metabolites have been isolated in microgram amounts by use of column, thin-layer and high-pressure liquid chromatography. Their structures were elucidated by mass spectroscopy and Fourier transform ¹H-NMR. spectroscopy as 2 (5-methyl-5-[2-(2,6,6-trimethyl-3-oxo-1-cyclohexen-1-yl)vinyl]-2-tetrahydrofuranone), 3 (5-[2-(6-hydroxymethyl-2,6-dimethyl-3-oxo-1-cyclohexen-1-yl)vinyl]-5-methyl-2-tetrahydrofuranone) and 4 (6-(6-hydroxymethyl-2,6-dimethyl-3-oxo-1-cyclohexen-1-yl)-4-methyl-4-hexenoic acid). In these metabolites the tetraene side chain of 1 is shortened and the cyclohexene ring oxidized. The radioactivity of 2 and 3 accounted for about 10% (0.9% of the dose) each, metabolite 4 for about 6% (0.5% of the dose) of the total urinary radioactivity.     

Identification of alkylpyrocatechols by thin-layer chromatography on silica gel

Conclusions A method was developed for the chromatographic separation of the alkyl derivatives of pyrocatechol employing thin-layer chromatography on plates.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 12, pp. 2816–2817, December, 1971.     

On characterizing proteomics maps by using weighted Voronoi maps

In contrast to the standard construction of Voronoi regions, in which the boundaries between different regions are at equal distance from the given points, we consider the construction of modified Voronoi regions obtained by giving greater weights to spots reported to have higher abundance. Specifically we are interested in applying this approach to 2-D proteomics maps and their numerical characterization. As will be seen, the boundaries of the weighted Voronoi regions are sensitive to the relative abundances of the protein spots and thus the abundances of protein spots, the z component of the (x, y, z) triplet, are automatically incorporated in the numerical analysis of the adjacency matrix, rather than used to augment the adjacency matrix as non-zero diagonal matrix elements. The outlined approach is general and it may be of interest for numerical analyses of other maps that are defined by triplets (x, y, z) as input information.     

Identification of discriminatory variables in proteomics data analysis by clustering of variables

This article presents a data analysis method for biomarker discovery in proteomics data analysis. In factor analysis-based discriminate models, the latent variables (LV's) are calculated from the response data measured at all employed instrument channels. Since some channels are irrelevant and their responses do not possess useful information, the extracted LV's possess mixed information from both useful and irrelevant channels. In this work, clustering of variables (CLoVA) based on unsupervised pattern recognition is suggested as an efficient method to identify the most informative spectral region and then it is used to construct a more predictive multivariate classification model. In the suggested method, the instrument channels (m/z value) are clustered into different clusters via self-organization map. Subsequently, the spectral data of each cluster are separately used as the input variables of classification methods such as partial least square-discriminate analysis (PLS-DA) and extended canonical variate analysis (ECVA). The proposed method is evaluated by the analysis of two experimental data sets (ovarian and prostate cancer data set). It is found that our proposed method is able to detect cancerous from healthy samples with much higher sensitivity and selectivity than conventional PLS-DA and ECVA methods.     

Identification of peanut and hazelnut allergens by native two-dimensional gel electrophoresis

A procedure for the native two-dimensional electrophoresis of peanut and hazelnut proteins is described. Proteins were solubilised after acetone treatment using a combination of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and tetramethylene sulphone. These extracts were analysed by a combination of isoelectric focusing in the presence of lactose in immobilized pH gradients followed by charge shift electrophoresis. Immunoblot analysis, using sera from nut allergic patients, allowed the identification of a peanut and hazelnut allergen with identical isoelectric point and apparent molecular mass. These proteins were recovered from duplicate gels using a mixture of formic acid, acetonitrile (ACN) and isopropanol. The molecular masses for both proteins, determined by matrix assisted laser desorption/ionisation-mass spectrometry (MALDI-MS), were 4826 Da.     

Identification of interactions involved in rennet gel structures using dissociating chemical agents

The dissociation of curds by enzymic coagulation by EDTA, urea, and ionic strength was considered to study the forces involved in the curd matrix structure. Curds were obtained at 25 (C₂₅) and 38 °C (C₃₈) and the effect of temperature on these forces was studied. Curd dissociation was followed by quantifying the release of proteins, calcium, and phosphate by the curds mechanically disrupted in the presence of the dissociating agent. In all the cases, the release of whey proteins and mineral revealed the liberation of entrapped whey. At difference with C₃₈, C₂₅ at low EDTA concentrations or with NaCl liberated α_S- and β-casein in similar proportions and higher amounts of minerals. Then, coagulation at low temperatures could favor the formation of weak electrostatic bonds involving Ca. Their dissociation by urea at a concentration of 4 M or higher was very effective, especially for C₃₈, suggesting that high temperature favored the formation of hydrophobic interactions.     

Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis

Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.     

Mass spectrometric compatibility of Deep Purple and SYPRO Ruby total protein stains for high-throughput proteomics using large-format two-dimensional gel electrophoresis

In order to identify putative biomarkers from two-dimensional (2D) gel electrophoresis it is necessary to use a visualization technique that is sensitive, has a large dynamic range and does not interfere with the identification of the protein. As mass spectrometry increases in sensitivity more pressure is placed on visualization techniques that facilitate proteomic workflows but do not interfere with downstream processing. Two stains reported to meet these requirements are SYPRO Ruby (Invitrogen) and Deep Purple (GE Healthcare). This study examined the compatibility of these stains with protein identification by selecting spots from replicate 2D gels of human plasma and subjecting these to protein identification using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using a test of two populations of proportions it was found that proteins were statistically more likely to be identified from gels stained with Deep Purple. Additionally, the identifications from Deep Purple stained gels are of higher quality because they are based on multiple peptides.     

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.     

纳米纤维在线固相萃取检测尿液中3种儿茶酚胺和5-羟色胺

生物单胺包括儿茶酚胺类以及5-羟色胺等,在中枢神经系统中扮演着非常关键的角色,也是临床上诊断神经内分泌肿瘤疾病的重要生物标志物。由于这类单胺类物质的强化学极性导致传统吸附材料对其吸附效果不佳,从复杂生物样本中同时检测更多的生物单胺存在挑战性。该文建立了一种基于聚冠醚纳米纤维在线固相萃取检测尿液中3种儿茶酚胺(多巴胺、肾上腺素、去甲肾上腺素)和5-羟色胺的方法。采用静电纺丝法制备聚二苯并-18-冠-6醚-聚苯乙烯复合纳米纤维(PCE-PS),制成装填纤维的固相萃取(PFSPE)柱,再将PFSPE柱与HPLC进行在线联用。该在线PFSPE-HPLC方法采用双三元泵进行样品富集净化和分析,左泵连接PFSPE柱,进行样品富集净化;右泵连接分析柱进行样品分离检测。控制切换阀的切换,实现样品富集后洗脱至分析柱中分离检测。结果表明,在线PFSPE-HPLC检测尿液儿茶酚胺(多巴胺、肾上腺素、去甲肾上腺素)和5-羟色胺在1~200 ng/mL范围内有良好的线性关系,线性相关系数达0.996以上。3种儿茶酚胺和5-羟色胺的检出限(S/N=3)分别为1和2.5 ng/mL,定量限(S/N=10)分别为2.5和5 ng/mL。空白尿液和实际尿液加标回收率在83.5%~117.7%之间,日内精密度<10%。PCE-PS复合纳米纤维在多次使用后无明显变化,具有良好的稳定性,可重复使用达95次以上。在线PFSPE-HPLC方法能够集样品在线前处理与分析检测于一体,省时省力,实现分析过程的高度自动化。该方法成功应用于尿液中3种儿茶酚胺和5-羟色胺的检测,可以为临床上相关疾病检测诊断和研究提供有力的技术支持。     

Comparison of three different enrichment strategies for serum low molecular weight protein identification using shotgun proteomics approach

Serum low-molecular weight (LMW) proteins potentially contain useful biological information and their identification can be used to discover novel potential biomarkers. Given the high complexity of serum samples, in the last years several different prefractionation and enrichment strategies have been developed. In this study three different methods, i.e. hydrogel nanoparticles, Proteominer^® peptide ligand affinity beads and Sartorius Vivaspin^® centrifugal ultrafiltration device, were compared and evaluated in order to select the best strategy for the enrichment and prefractionation of LMW proteins. A shotgun proteomics approach was adopted, with in-solution proteolytic digestion of the whole protein mixture and determination of the resulting peptides by nanoHPLC coupled with a high-resolution Orbitrap LTQ-XL mass spectrometer. Data analysis, focusing on the LMW proteome (MW ≤ 40 kDa), has shown that the hydrogel nanoparticles performed better in enriching the LMW protein profiles, with 115 proteins identified against 93 and 95 for Proteominer^® beads and Sartorius Vivaspin^® device, respectively.     

Comparison of L-tryptophan binding capacity of BSA captured by a polymer brush with that of BSA adsorbed onto a gel network

A polymer brush containing a diethylamino group as an anion-exchange group was appended onto a polymer substrate by radiation-induced graft polymerization and subsequent chemical modifications. Bovine serum albumin as a chiral ligand for L-tryptophan was bound to the polymer brush at a density ranging from 17 to 150 g BSA/l. For comparison, BSA was adsorbed onto the gel network containing a diethylaminoethyl group. The molar binding ratio of L-tryptophan to BSA on the polymer brush was 1.7-fold higher than that to BSA on the gel network.