Simple urinary sample preparation for proteomic analysis

Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.     

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.     

Urine protein profile of IgA nephropathy patients may predict the response to ACE-inhibitor therapy

This study was aimed at the search of urinary biomarkers which might help to predict the clinical response of IgA nephropathy (IgAN) patients to angiotensin converting enzyme inhibitors (ACEi). First, we studied the urinary proteome of 18 IgAN patients (toward 20 healthy controls) who had been chronically treated with ACEi by using 2-D PAGE coupled to nano-HPLC-ESI-MS/MS analysis. We identified 3 proteins, kininogen (p = 0.02), inter-alpha-trypsin-inhibitor heavy chain 4 (35 kDa fragment) (p = 0.02) and transthyretin (p<0.0001), whose urinary excretion was different in IgAN patients' responders when compared to those who had not responded to ACEi. A reduction of daily proteinuria >50% and a stable renal function over time were used to classify patients as responders. Then, we adopted immunoblotting to confirm the predictive power of one of the above proteins, kininogen, in 20 patients with biopsy-proven IgAN, before starting any therapy. Thus, we confirmed that very low levels of kininogen urine excretion were indeed predictive of an inadequate or absent clinical response to ACEi therapy of IgAN patients, after 6-month follow-up. Concluding, the analysis of urine proteome of IgAN patients generated a set of proteins which distinguished subjects responsive to ACEi from those unresponsive to the inhibition of renin-angiotensin system (RAS).     

Analysis of complex protein-polypeptide systems for proteomic studies

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by protein extraction and characterization with chemical sequencing or mass spectrometry (MS), is the most commonly used method to analyze complex protein systems such as cells and organelles. However, it is claimed that 2-D PAGE is a slow and labor-intensive technique and also needs subsequent efforts for one-by-one identification of proteins. Recently, the combined methods of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, with preceding separation techniques such as capillary isoelectric focusing (CIEF) or liquid chromatography, have been demonstrated as high-throughput techniques suitable for proteomic analysis of protein systems. The studies which employ FTICR MS, aimed at the analysis of complex protein systems, have been reviewed, comparing their performance with that of 2-D PAGE. Also, the possibilities of combining 2-D PAGE and the FTICR MS method to analyze and reconstruct the structures and functions of complex systems are discussed.     

Establishment of a near-standard two-dimensional human urine proteomic map

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.     

Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool

The combination of laser microdissection and two-dimensional gel electrophoresis (2-D PAGE) has been developed to perform proteomic analysis on specific populations of cells in cancer tissues. However, as conventional low sensitivity silver staining was used for spot detection, the microdissection required to obtain an adequate amount of protein for 2-D PAGE is laborious and only a restricted number of protein spots could be visualized. As a consequence, this technology was impractical for direct clinical applications and had a limited impact on cancer studies. To solve these problems, we developed an application in which fluorescent dyes label the proteins extracted from microdissected tissues prior to 2-D PAGE separation. In this application, a small amount of protein, less than 6.6 microg, was enough to generate a 2-D profile with approximately 1500 protein spots. This technique was applied to compare the proteome of normal intestinal epithelium with that of adenoma in Min mice. Thirty-seven protein spots reproducibly showed significant differences in intensities. Mass spectrometric analysis and Western blotting identified eight of them, including prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84. These results indicate that fluorescence labeling of proteins from microdissected tissues prior to 2-D PAGE is a powerful cancer proteomic study tool.     

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.     

Heterozygosity and ethnic variation in Japanese platelet proteins

Summary Sixty-two polypeptides visualized on silver-stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) preparations of platelets from 85 Japanese subjects, in total 5,252 spots, have been scored for genetic variation. Inherited variants of 11 of the polypeptides were recognized; the index of heterozygosity was 2.4%±0.2%. Thus far, 10 genetic polymorphisms identified by 2-D PAGE of plasma, erythrocytes, or platelets have been identified in both Japanese and Caucasian subjects. A comparison of allele frequencies reveals four significant ethnic differences. We also observed four polypeptides exhibiting a low frequency polymorphism in one group but not in the other, as well as three polymorphisms in Caucasians for which no counterpart polypeptide has thus far been recognized in the Japanese group and, vice versa, 11 such polymorphisms in Japanese. Although a similar comparison of 7 enzyme polymorphisms studied with one-dimensional electrophoresis (1-D E) in the same populations revealed a relatively higher number of significant ethnic differences, evidence is presented that this is due primarily to the greater number of 1-D E observations entering into the calculation. It is argued that this similarity in the frequency of ethnic differences among the polypeptides studied by 2-D PAGE and by 1-D E is further evidence that the proteins revealed by 2-D PAGE do not differ greatly in their response to the interplay of mutation, selection, and drift from those revealed by 1-D E studies of plasma proteins and erythrocyte enzymes.     

Human urine proteome analysis by three separation approaches

The urinary proteome is known to be a valuable field of study related to organ functions. There have been several extensive urine proteome studies. However, the overlapping rate among different studies is relatively low. Whether the low overlapping rate was caused by different sample sources, preparation, separation and identification methods is unknown. Moreover, low molecular mass (<10 kDa) proteins have not been studied extensively. In this report, male and female pooled urine samples were collected from healthy volunteers. The urinary proteins were acetone precipitated, separated and identified by three approaches, 1-DE plus 1-D LC/MS/MS, direct 1-D LC/MS/MS and 2-D LC/MS/MS. 1-D tricine gels were used to separate low molecular mass proteins. The tandem mass spectra of positive identifications were quality controlled both by manual validation and using advanced mass spectrum scanner software. A total of 226 urinary proteins were identified; 171 proteins were identified by proteomics approach for the first time, including 4 male-specific proteins. Twelve low molecular mass proteins were identified. Most urinary proteins had a molecular mass between 30 and 60 kDa and a pI between 4 and 10. The apparent molecular masses of many proteins were different from theoretical ones, which indicated their post-translational modification and degradation. The effects of sample preparation, separation and identification methods on the overlapping rate of different experiments are discussed.     

Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.     

The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis.

SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), created and maintained at the University Hospital of Geneva in collaboration with the Department of Medical Biochemistry of Geneva University. The proteins have been identified on various 2-D PAGE reference maps by microsequencing, immunoblotting, gel comparison and amino acid composition.     

Two-dimensional gel electrophoresis as a taxonomic tool: Evidence from the centrospermae

Two-dimensional polyacrylamide gel (2-D PAGE) electrophoresis was appraised as an experimental technique for assessing systematic relationships among higher plants and to determine at which level in the taxonomic hierarchy this technique is most generally applicable. 2-D PAGE was performed on denatured extracts of mature leaves from 25 species representing five families of the order Centrospermae (Caryophyllales, Chenopodiales) in the Angiospermae as well as Welwitschia mirabilis (Gymnospermae). Cluster analysis of a 256 spot binary-coded data set derived from the computer-encoded spot patterns of the 25 species successfully separated taxa from the individual to the familial levels of the taxonomic hierarchy in accordance with traditional taxonomic delineations of the taxa.     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

A specific method for the determination of millimicrogram amounts of urinary oestrone

1. A new combined radioactivity-fluorescence method is described for urinary oestrone, which involves acid hydrolysis, extraction and purification of the phenolic fraction, saponification, a Girard T separation and alumina chromatography of acetylated oestrone. 2. Sulphuric acid fluorescence is used for quantitation and specificity is achieved by the addition of tritiated oestrone to the urine hydrolysate. This radioactive tracer functions both as an internal corrector for purification losses and enables the demonstration of constant specific activity through the oestrone peaks to act as an index of specificity in each determination. 3. By using one-fifth of a 24hr. urine sample, 1mug. of urinary oestrone/24hr. can be determined with an accuracy of +/-4%. Fluorescence emission spectra from processed urine samples are identical with that of authentic oestrone acetate. 4. The advantages of the method are its high sensitivity and specificity, which is achieved with relative convenience.     

An alternative strategy for the membrane proteome analysis of the green sulfur bacterium Chlorobium tepidum using blue native PAGE and 2-D PAGE on purified membranes

To avoid the specific problems concerning intrinsic membrane proteins in proteome analysis, an alternative strategy is described that is complementary to previous investigations using 2-D polyacrylamide gel electrophoresis (PAGE) techniques. The strategy involves (a) obtaining purified preparations of the membranes from Chlorobium tepidum by washing with 2 M NaBr, which removed membrane-associated soluble proteins and membrane-associated organelles; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-beta-d-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE; (d) high-throughput mass spectrometric analysis after gel band excision, in-gel digestion, and MALDI target spotting; and (e) protein identification from mixtures of tryptic peptides by peptide mass fingerprinting. Using this approach, we identified 143 different proteins, 70 of which have not been previously reported using 2-D PAGE techniques. Membrane proteins with up to 14 transmembrane helices were found, and this procedure proved to be efficient with proteins within a wide pI range (4.4-11.6). About 54% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, and protein translocation, while for the others, a function is not yet known, indicating the potential of the method for the elucidation of the membrane proteomes in general.     

Novel finding of caveolin-2 in apical membranes of proximal tubule and first detection of caveolin-2 in urine: A promising biomarker of renal disease

Caveolin-2 (Cav-2) is expressed in a variety of cell tissue, and it has also been found in renal tissue. The expression of Cav-2 in proximal tubules is still unclear. The aim of this study was to carry out a complete evaluation of the expression pattern of Cav-2 in rat renal cortex to clarify and deepen the knowledge about the localization of Cav-2 in the proximal tubules and also to evaluate its presence in urine. Male Wistar rats were used to assess Cav-2 expression by Western blot analysis in homogenates, apical, and basolateral membranes from kidney cortex, in lysates and total plasma membranes from renal cortical cell suspensions, in urine, and in urinary exosomes. Cav-2 was clearly expressed in renal cortex homogenates and in both apical and basolateral membranes isolated from kidney cortex, with a greater expression on the former membranes. It was also observed in lysates and in plasma membranes from cortical cell suspensions. Moreover, Cav-2 was found in urine and in its exosomal fraction. These results confirmed the presence of Cav-2 in proximal tubule cells in the kidney of healthy rats, and showed for the first time its expression at the apical membrane of these cells and in urine. Besides, urinary exosomal pathway could be involved in Cav-2 urinary excretion under normal conditions. We observed an increase in the urinary abundance of Cav-2 in two models of acute kidney injury, and thus we proposed the urinary excretion of Cav-2 as a potential biomarker of kidney injury.     

尿液蛋白质组在膀胱癌临床诊治中的应用

膀胱癌是一种常见的泌尿系统疾病,尿细胞学检查与膀胱镜检查是膀胱癌的主要临床诊断手段,但尿细胞学检查敏感性较差,膀胱镜检查为侵入性检查,易给病人带来强烈的不适感;且膀胱癌具有易复发的特点,大部分患者必须面临频繁的检查,临床亟需发展舒适、准确的检查手段.尿液存储是膀胱的主要生理作用,尿液可以直接接触肿瘤实体,肿瘤分泌的一些蛋白质分子极可能进入尿液中,并且患者尿液样本便于足量多次收集.同时,蛋白质组技术以及尿液蛋白质组研究的快速发展,为我们利用尿液研究膀胱癌提供了便利的途径.本文系统总结了尿液蛋白质组研究的主要技术手段,重点关注膀胱癌尿液蛋白质组研究趋势和应用方向,以期为利用尿液蛋白质组研究膀胱癌提供助力.     

Protein expression analysis of Halobacillus dabanensis D-8T subjected to salt shock

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and Imagemaster 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.     

Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure

Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We found protein expression in the double deletion strain to be highly similar to wild-type. In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa). Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein. Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues. We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases. Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points. However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared. In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini. Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini.     

Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.