The Calcineurin Inhibitor Tacrolimus Reduces Proteinuria in Membranous Nephropathy Accompanied by a Decrease in Angiopoietin-Like-4

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.     

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.     

双向电泳结合质谱初步分析蛇岛蝮蛇毒蛋白质组

采用荧光染料(Cy5)标记中国辽宁蛇岛蝮(Gloydius shedaoensis shedaoensis,GSS)蛇毒(snake venom,SV,GSS-SV)蛋白质,获得了该蛇毒的双向十二烷基硫酸钠聚丙烯酰胺凝胶电泳(2D SDS-PAGE)图谱,经DeCyder软件分析,分辨出1000多个蛋白点,分子量范围在10～150ku间,等电点在4～7的蛋白质点占78.8%。凝胶后染色(post-staining)采用蛋白荧光染料Deep Purple,选取的5个蛋白点经胶内酶解,产生的肽段经高效液相色谱-电喷雾串联质谱(high performance liquid chromatography/HPLC-electro-spray ionization tandem mass spectrometry/ESI-MS/MS,HPLC-ESI-MS/MS)进行序列测定,质谱数据经Sequest Bioworks软件分析,为蛇毒L-氨基酸氧化酶、金属蛋白酶、类凝血酶、纤溶酶原激活物和磷脂酶A2的同源蛋白。本研究采用的荧光标记2DSDS-PAGE结合HPLC-ESI-MS/MS的技术适于高通量研究蛇毒蛋白组成。     

Proximal tubule haptoglobin gene activation is an integral component of the acute kidney injury "stress response"

Haptoglobin (Hp) synthesis occurs almost exclusively in liver, and it is rapidly upregulated in response to stress. Because many of the pathways that initiate hepatic Hp synthesis are also operative during acute kidney injury (AKI), we tested whether AKI activates the renal cortical Hp gene. CD-1 mice were subjected to six diverse AKI models: ischemia-reperfusion, glycerol injection, cisplatin nephrotoxicity, myoglobinuria, endotoxemia, and bilateral ureteral obstruction. Renal cortical Hp gene induction was determined either 4-72 h or 1-3 wk later by measuring Hp mRNA and protein levels. Relative renal vs. hepatic Hp gene induction during endotoxemia was also assessed. Each form of AKI induced striking and sustained Hp mRNA increases, leading to ～10- to 100-fold renal Hp protein elevations (ELISA; Western blot). Immunohistochemistry, and isolated proximal tubule assessments, indicated that the proximal tubule was the dominant (if not only) site of the renal Hp increases. Corresponding urinary and plasma Hp elevations were surrogate markers of this response. Endotoxemia evoked 25-fold greater Hp mRNA increases in kidney vs. liver, indicating marked renal Hp gene reactivity. Clinical relevance of these findings was suggested by observations that urine samples from 16 patients with established AKI had statistically higher (～12×) urinary Hp levels than urine samples from either normal subjects or from 15 patients with chronic kidney disease. These AKI-associated urinary Hp increases mirrored those seen for urinary neutrophil gelatinase-associated lipoprotein, a well accepted AKI biomarker gene. In summary, these studies provide the first evidence that AKI evokes rapid, marked, and sustained induction of the proximal tubule Hp gene. Hp's known antioxidant, as well as its protean pro- and anti-inflammatory, actions imply potentially diverse effects on the evolution of acute tubular injury.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and variability

We performed systematic evaluation of 38 protocols to concentrate normal human urinary proteins prior to 2D-PAGE analysis. Recovery yield and pattern of resolved protein spots were compared among different methods and intra-/inter-individual variabilities were examined. Precipitation with 90% ethanol provided the greatest protein recovery yield (92.99%), whereas precipitation with 10% acetic acid had the least protein recovery (1.91%). In most of precipitation protocols, the higher percentage of applied organic compounds provided the greater recovery yield. With a fixed concentration at 75%, the urine precipitated with acetonitrile had the greatest number of protein spots visualized in 2D gel, whereas the acetic-precipitated sample had the smallest number of spots. For the intra-individual variability, the first morning urine had the greatest amount of total protein but provided the smallest number of protein spots visualized. Excessive water drinking, not caffeine ingestion, caused alterations in the urinary proteome profile with newly presenting spots and also proteins with decreased excretion levels. As expected, there was a considerable degree of inter-individual variability. Coefficients of variation for albumin and transferrin expression were greatest by inter-individual variables. Male urine had greater amount of total protein but provided smaller number of protein spots compared to female urine. These data offer a wealth of useful information for designing a high-quality, large-scale human urine proteome project.     

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.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Study of serum haptoglobin and its glycoforms in the diagnosis of hepatocellular carcinoma: a glycoproteomic approach

Increased serum haptoglobin concentration and changes in its glycosylation have been reported in certain cancer types. Information for hepatocellular carcinoma (HCC) has not yet been available. In this study, we aimed to carry out a systematic analysis of serum concentrations of haptoglobin (Hp) and its glycoforms in the patients with HCC and noncancer patients only with chronic liver diseases (CLD) and to examine their clinical values. This study was divided into two major parts, (1) measurement of serum Hp concentration, and investigation of its value in the diagnosis of HCC, and (2) quantitative analysis of Hp glycoforms with alpha-2,6-sialylation and/or alpha-1,6-fucosylation by using lectin affinity purification and 2D gel electrophoresis and investigation of their relationships with tumor stage. The concentrations of serum Hp in HCC patients were significantly higher than those in noncancer patients with CLD. With the use of serum concentrations of Hp and alpha-fetoprotein, a logistic regression (LR) model was developed from the training data set and used to classify the validation cases. At a specificity of 95%, the sensitivity for HCC detection was 79%. Comparing serum concentrations of alpha-2,6-sialylated Hp (S-Hp) and alpha-1,6-fucosylated Hp (F-Hp) between HCC and CLD patients suggests that purification of S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. 2D gel analysis of S-Hp and F-Hp identified a total of 18 glycoforms. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species was found in the HCC patients. Serum concentrations of these glycoproteins were significantly higher in the patients with advanced tumors, suggesting their tumor-specific nature. We have shown that serum Hp is a potential biomarker in the diagnosis of HCC. The combined use of Hp and AFP could greatly improve the diagnostic accuracy. A unique pattern of Hp glycoforms with altered sialylation and fucosylation is specific to HCC and associated tumor progression.     

Serial changes in urinary proteome profile of membranous nephropathy: implications for pathophysiology and biomarker discovery

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.     

Radiation produces differential changes in cytokine profiles in radiation lung fibrosis sensitive and resistant mice

We studied serum proteomic profiling in patients with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. The expression of a group of proteins, haptoglobin (Hp), alpha-1-antitrypsin, apolipoprotein A-IV, serum paraoxonase and Zn-alpha-glycoprotein were increased and the proteins, clusterin precursor, alpha-2-macroglobulin, serum amyloid protein precursor, sex hormone-binding globulin, serotransferrin and complement C4 were decreased in patients with extensive chronic GVHD (cGVHD). Serum haptoglobin (Hp) levels in patients with cGVHD were demonstrated to be statistically higher than in patients without cGVHD and normal controls (p < 0.01). We used immunoblotting and PCR in combination with 2-DE gel image analysis to determine Hp polymorphisms in 25 allo-HCT patients and 16 normal donors. The results demonstrate that patients with cGVHD had a higher incidence of HP 2-2 phenotype (43.8%), in comparison to the patients without cGVHD (0%) and normal donors (18.7%), suggesting the possibility that specific Hp polymorphism may play a role in the development of cGVHD after allo-HCT. In this study, quantitative serum Hp levels were shown to be related to cGVHD development. Further, the data suggest the possibility that specific Hp polymorphisms may be associated with cGVHD development and warrant further investigation.     

Single molecular observation of the interaction of GroEL with substrate proteins.

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.     

双向凝胶电泳中三种蛋白质检测方法的比较

高通量双向电泳是蛋白质组学的核心 ,双向电泳凝胶上蛋白质点的检测方法应具有灵敏度高、线性范围宽和兼容质谱鉴定等优点 .采用差异凝胶电泳 (differencegelelectrophoresis ,DIGE)技术以Cy3(1 (5 carboxypentyl) 1′ propylindocarbocyaninehalideN hydroxysuccinimidylester)和Cy5 (1 (5 carboxypentyl) 1′ methylindodicarbocyaninehalideN hydroxysuccinimidylester)荧光分别标记正常和TNF α处理细胞的蛋白质 ,用Cy2 (3 (4 carboxymethyl)phenylmethyl) 3′ ethyloxacarbocyaninehalideN hydroxysuccinimidylester)荧光标记正常和TNF α处理细胞蛋白质的等量混合样品作为内标 ,混合 3种荧光标记的蛋白质后 ,在同一等电聚焦胶条进行聚焦 ,然后在聚丙烯酰胺凝胶上进行第二向电泳 ,用 3种波长的激光激发扫描得到凝胶图象 ,DIGE中多个样品在同一条件下电泳 ,因而匹配率高 ,且引入内标使蛋白质点的检测与定量更为准确 .DIGE技术与质谱相结合 ,实现了高通量和相对准确定量 .与硝酸银和考马斯亮蓝染色结果相比较 ,DIGE技术具有灵敏度高、线性范围宽和不影响后续质谱鉴定等优点     

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual.     

Analysis of DIGE data using a linear mixed model allowing for protein-specific dye effects

Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.     

Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis

To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t-test, p-value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer.     

Detection of SNPs in Porcine Haptoglobin and Apolipoprotein Genes

To find differentially expressed protein spots using two-dimensional electrophoresis proteomic analysis, we took blood serum samples from 40 purebred Yorkshire pigs at 12, 18, 24, and 30 weeks. Each growth stage contained 10 male pigs having half-sib pedigrees. With the pooled serum samples, two interesting spots, differentially expressed in the growth stages, were identified using MALDI-TOF-TOF MS/MS analysis as haptoglobin alpha 1S (Hp) and apolipoprotein A-IV (APOA4) gene products. The Hp was down-regulated from 12 to 30 weeks, and APOA4 was not expressed much before 18 weeks but was highly expressed in the late growth stages. There may be an inverse relationship between the Hp and APOA4 genes. Four segments for the Hp and APOA4 genes were successfully amplified with sizes around 500 bp. The porcine Hp and APOA4 genes were screened in the 40 purebred Yorkshire pigs and a random cross population (90 pigs), resulting in the location of 6 single nucleotide polymorphisms (SNPs) in the coding regions. The mutations resulted in amino acid changes in segments of Hp627, Hp742, and APOA41203. Further investigation of the function of the Hp and APOA4 genes with SNPs will be necessary to understand fully the different expression profiles and association studies.     

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.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Activity-dependent neuroprotector homeobox protein: A candidate protein identified in serum as diagnostic biomarker for Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.