Practical Points in Urinary Proteomics - American Chemical Society

Visith Thongboonkerd*. Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital,. Mahidol University,...
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Practical Points in Urinary Proteomics Visith Thongboonkerd* Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Received May 30, 2007

During the proteomic era, one of the most rapidly growing areas in biomedical research is biomarker discovery, particularly using proteomic technologies. Urinary proteomics has become one of the most attractive subdisciplines in clinical proteomics, as the urine is an ideal source for the discovery of noninvasive biomarkers for human diseases. However, there are several barriers to the success of the field and urinary proteome analysis is not a simple task because the urine has low protein concentration, high levels of salts or other interfering compounds, and more importantly, high degree of variations (both intra-individual and inter-individual variabilities). This article provides step-by-step practical points to perform urinary proteome analysis, covering detailed information for study design, sample collection, sample storage, sample preparation, proteomic analysis, and data interpretation. The discussion herein should stimulate further discussion and refinement to develop guidelines and standardizations for urinary proteome study. Keywords: biomarker discovery • guidelines • kidney • proteome • sample preparation • urine • urinary proteomics • variabilities

Introduction The urine is one of the ideal biological samples for the discovery of noninvasive biomarkers for human diseases, because it is available in almost all patients and its collection is simple and does not require any invasive procedures. Urinary proteomics has thus become one of the most attractive subdisciplines in clinical proteomics, particularly with the aim for biomarker discovery and clinical diagnostics.1,2 However, there are several barriers to the success of the field and urinary proteome analysis can be considered as a sophisticated experiment because the urine has low protein concentration, high levels of salts or other interfering compounds, and more importantly, high degree of variations (both intra-individual and inter-individual variabilities). Some investigators may have been experienced with such barriers already. Therefore, the critical discussion on key issues of concern in urinary proteomics is crucially required. This kind of information can then be used as the reference or guidelines to perform urinary proteome analysis; otherwise, it may end up with no standardizations and several blinded attempts, which most likely lead to difficulties and perhaps failure. This article provides stepby-step practical points and discussion on key issues of concern in urinary proteomics that can be used as the initial guidelines for the urinary proteome study. The practical points to be discussed herein include: A. Study design. B. Selection of the appropriate proteomic technology. C. Sample collection: 24-h, first-morning, second-morning, or random urine? early-stream or midstream urine? D. Should protease inhibitors and/or preservatives be added into the urine sample? E. Removal of cells and debris. * To whom correspondence should be addressed. Visith Thongboonkerd, MD, FRCPT, Medical Molecular Biology Unit, Office for Research and Development, 12th Floor - Adulyadej Vikrom Building, Siriraj Hospital, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand; Tel/Fax, +66-24184793; E-mail, [email protected] or [email protected]. 10.1021/pr070328s CCC: $37.00

 2007 American Chemical Society

F. Sample storage and effects of freeze-thaw cycles. G. Sample preparation methods for concentrating or isolating urinary proteins. H. Removal of interfering compounds. I. Should albumin and/or other high-abundance proteins be removed prior to urinary proteome analysis? J. Intra-individual and inter-individual variabilities. K. Pooled versus individual samples. L. Data interpretation and normalization. A. Study Design. The first important issue of concern that, somehow, might be overlooked in some of recent urinary proteome studies is the study design. For example, selection of the control group(s) is really crucial for defining the “diseasespecific urinary biomarker(s)”. Recently, there has been a consensus by a group of the proteomists (proteomic scientists), clinicians, biochemists, chemists, bioinformaticians, and statisticians from more than 25 institutions worldwide that “it is not justified to define disease-specific biomarkers by comparing the urinary proteome profile of a disease group to that obtained only from normal healthy individuals”.3 Urine samples obtained from patients with other diseases or disorders that may have clinical, biochemical, and metabolic profiles mimicking those of the disease of interest must be included as the other controls. The proteomists should work closely together with clinicians to recruit patients into the study. As is generally known, the spectra of some diseases are too broad to specify the cases. Also, staging of diseases can have significant effects on laboratory findings and the urinary proteome data. The challenge is to carefully characterize patients to be included and to follow these patients for a long time for serial analyses of their urine samples. Another issue of concern is the search for urine markers of nonkidney diseases. There is a fact that “not all the diseases will present marker molecule(s) in the urine”. The investigators have to realize whether it is reasonable that potential marker molecule(s) of nonkidney diseases can be excreted into the urine. If not, the urine is not the right target for biomarker discovery. To search for urinary proteins that may potentially Journal of Proteome Research 2007, 6, 3881-3890

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Figure 1. Current proteomic technologies applied to human urine. Advantages and limitations of each technique are summarized in Table 1.

be biomarkers, the physiology of urinary protein excretion should be reviewed. Normally, urinary proteins can originate from:2,4 (i) Glomerular filtration of plasma proteins. (ii) Secretion of proteins from renal tubular epithelial cells. (iii) Shedding of whole cells along urinary passage, shedding of apical membranes of renal tubular epithelial cells, and exosome secretion. During the normal physiological state, low molecular weight proteins pass freely through the glomerular barriers, whereas almost none of high molecular weight proteins and only a fraction of proteins with middle molecular weight reach renal tubules.5 All proteins that reach tubular lumens are excreted with negligible amounts into the urine because of the high efficacy of the reabsorption process by proximal tubular epithelial cells.2,4 The nonkidney diseases, which have no any renal involvements (or in other words, without disruption of the glomeruli and/or renal tubules) and no abnormal cells in the urine, can present the marker molecules in the urine only when their plasma levels are high enough to overcome the ability of tubular cells to reabsorb.2,4 This mechanism allows only small to middle molecular weight proteins to pass through the glomerular barriers. Therefore, such molecules should have small to medium sizes. Finally, a single ideal biomarker may not exist for each disease. Therefore, evaluating a panel of multiple biomarkers may be required.3 B. Selection of the Appropriate Proteomic Technology. The second important step is the choice of proteomic technology to be used. Analytical approaches for urinary proteomic analysis can be classified into two main categories6 (Figure 1). The “classical approach” is to extensively and systematically examine protein expression and function to better understand 3882

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pathophysiology and pathogenic mechanisms of diseases. Proteomic technologies used in this approach involve expression proteomics (using either gel-based or liquid chromatography-based techniques), bioinformatics, and functional proteomics. The “alternative approach” or “proteome profiling” is to examine the proteome profiles or patterns of protein expression to differentiate types or groups of urine samples (e.g., normal versus diseases, a specific disease versus others), rather than focusing on a specific protein. Proteomic technologies used in the latter approach include surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDITOF MS),7-9 capillary electrophoresis coupled to mass spectrometry (CE-MS),10-12 microarrays,13 and microfluidic technology on a chip14 (Figure 1). The advantage of the alternative approach is that detailed characterizations of a specific protein may not be required. This approach is, therefore, suitable for clinical diagnostics and biomarker discovery, particularly in cases of multi-factorial diseases for which a single marker may not be sufficient for effective detection or diagnosis. To compare them, each technique has its own advantages and limitations (Table 1). Two-dimensional gel electrophoresis (2-DE) is available in most proteomics laboratories and is simple to perform. However, it is time-consuming, not applicable for proteins/polypeptides smaller than 10 kDa, and still limited of use for highly hydrophobic proteins.2,15 Liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) can be automated with a high sensitivity. However, it is time-consuming and too sensitive for interfering compounds.2,15 Moreover, quantitative analysis using LC-MS/MS is not simple. SELDI-TOF MS is an easy-to-use system with an automation and high-throughput manner. Only a small volume, such as 20 kDa.2,15,16 Microarrays technology is one of the ideal methods for urinary proteome profiling.13 This approach allows the high-throughput profiling of multiple proteins in urine samples and offers an opportunity to discover low molecular weight biomarkers that may be missed by other (more commonly used) techniques.17 However, its applications are still limited by the difficulty to make the arrays to cover all the urinary proteins and the availability of antibodies or other ligands.17 Other limitations include the conservation of protein functionality during immobilization, as well as the provision of the required absolute and relative sensitivity.17 Microfluidic technology on a chip is the latest technology applied to urinary proteome profiling.14 However, the identification of the resolved proteins is still problematic, as is the case for SELDI-TOF MS and CE-MS. Regarding the scale or number of proteins or polypeptides in the “normal urine” that can be analyzed by different proteomic technologies, 2-DE can analyze 70-420 protein spots per single gel,18-21 whereas 400-2000 urinary polypeptides can be analyzed by a single run of CE-MS.22-24 It should be noted that 2-DE is more suitable for analyzing intact proteins or their fragments with molecular mass >10 kDa, whereas CE-MS is more suitable for analyzing polypeptides or small proteins with molecular mass 20 kDa Currently does not cover all urinary proteins, conservation of protein functionality during immobilization Subsequent identification of the resolved proteins is still limited

obtained from 24-h or first-morning urine would be best for analysis of urinary proteins. The 24-h urine can provide average information of urinary protein excretion within a day, whereas the first-morning urine can avoid “diurnal variation” (variation by different time-points during the 24-h period). However, several studies have demonstrated that the collection of 24-h and first-morning urine is less practical and may have a problem of contamination of proteins from overgrown bacteria or bladder epithelial cells.35,36 Therefore, the second-morning or random urine is recommended for urinary proteome analysis. Comparing between first-stream (early-stream) and midstream urine, the midstream urine is recommended, particularly for females. There is a recent study by Schaub et al.7 demonstrating that the first-stream (early-stream) urine from females had additional SELDI peptide peaks after 3-day storage, probably due to bacterial contamination. Cleaning at the area surrounding external urethral orifices may be required, particularly in case of physiological or abnormal leukorrhea. D. Should Protease Inhibitors and/or Preservatives Be Added into the Urine Sample? Regarding protease inhibitors, although the addition of protease inhibitors was previously suggested for urine collection,18,37 it is no longer recommended for the nonproteinuric urine (the urine with low amount or concentration of proteins) for the following reasons: First, the normal urine has a much lower amount of proteases as compared to plasma, cells, or tissues. Second, protease inhibitors themselves can interfere with proteomic analysis. Generally, the protease inhibitors cocktail includes both peptide and small molecule inhibitors.38 The inhibitory peptide such as aprotinin requires µg/mL concentrations that may directly compete and interfere with the detection of urinary polypeptides in the mass spectrometer, whereas small molecules such as AEBSF, a sulfonyl fluoride, have been shown to form covalent bonds with proteins and thereby shift the isoelectric point (pI) of the protein.39 For the proteinuric urine, protease inhibitors may still be required. However, this issue needs extensive investigations and further elucidations. For preservatives, unless the urine can be frozen immediately, the addition of a preservative (e.g., sodium azide, boric acid) to prevent bacterial contamination is highly recommended, particularly when the urine is kept at room temperature for >8 h, or at 4 °C for >16 h [manuscript submitted]. For the collection of one-void random urine, 2-20 mM boric acid or 0.1-1 mM sodium azide, which can delay the bacterial overgrowth for 12-20 h at room temperature, should be added. For 24-h urine collection, the addition of 200 mM boric acid or 10 mM sodium azide is highly recommended. Without an appropriate preservation, the overgrown bacteria can dramatically change the urinary proteome profile [manuscript submitted]. Journal of Proteome Research • Vol. 6, No. 10, 2007 3883

perspectives E. Removal of Cells and Debris. This is a crucial step that requires immediate action after the collection. The normal urine generally has 17 years is still analyzable with high-quality data, using gel-based proteomics approach. Freeze-thaw cycles should be avoided. Recently, Schaub et al.7 have reported that urinary proteome profile obtained by SELDI-TOF MS analysis of urine samples before freeze and after 1-4 freeze-thaw cycles remained unchanged. However, an increasing loss of intensity in some peaks was detected and some small peaks were absent (or under the detectability of SELDI-TOF MS) after the fifth freeze-thaw cycle. Similarly, Powell et al.40 have also reported that 4-7 freeze-thaw cycles can cause both an increase and decrease in levels of some urinary proteins. G. Sample Preparation Methods for Concentrating or Isolating Urinary Proteins. Selection of sample preparation methods is also crucial for analyzing the urinary proteome. Although sample preparation to concentrate urinary proteins may not be sophisticated for SELDI-TOF MS25,26 and CEMS,23,41 it is a crucial step in gel-based proteomics study.42 As a model for discussion herein, some data obtained from a recent systematic gel-based urinary proteomics study19 are illustrated. Figure 2A clearly indicates that using different methods to concentrate or isolate urinary proteins provides different results. Ten differential methods used in the mentioned study19 included precipitation with various organic compounds or solvents, centrifugal filtration, lyophilization, and ultracentrifugation. Concerning the “quantitative” data, lyophilization provided the greatest protein recovery yield, whereas precipitation with acetic acid provided the least protein recovery yield when an equal volume of urine was used (Table 2).19 Concerning “qualitative” data, precipitation with acetonitrile provided the greatest number of protein spots visualized, whereas precipitation with acetic acid and lyophilization provided the smallest number of protein spots visualized when an equal amount of total protein was loaded (Table 2).19 The reason why lyophilization resulted in the highest recovery yield could be simply explained by the least protein loss during sample preparation, whereas greater loss was ac3884

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companied with the use of precipitation or filtration. However, the explanation of the lowest number of protein spots offered by lyophilization remained unclear. It should be pointed out that using other concentrating techniques resulted in one more purification step (e.g., precipitation favors hydrophilic proteins, filtration selects proteins by size, ultracentrifugation favors hydrophobic proteins), which can improve resolution of 2-D spots. Perhaps, the better resolution of urinary proteins isolated by other protocols (Figure 2A) was the key factor determining the greater numbers of the resolved protein spots obtained from other protocols, compared to those visualized in the lyophilized sample. Concerning the total number of unique protein spots that could be obtained, any single method provided approximately 100-200 spots, whereas combination of two protocols increased the total number of unique spots to approximately 300 and combinations of all 10 protocols increased the total number of unique spots to approximately 700 (Figure 2B).19 Therefore, selection of sample preparation methods is very important for a high-quality, large-scale urinary proteomics study, and a combination of various sample preparation methods is required to obtain the complete human urinary proteome data. Although the discussion herein focuses on the data obtained from gel-based study, standardizations of sample preparation methods for gel-free techniques are definitely required and a systematic evaluation on this issue using gelfree techniques is urgently needed. H. Removal of Interfering Compounds. Interfering compounds is one of the major obstacles in urinary proteomics. Because there are lots of salts and other charged compounds in the urine that can interfere with proteomic analysis, these charged components should be removed prior to urinary proteome analysis. There are several methods to remove these interfering compounds, e.g., size exclusion using filtrating column, exclusion by precipitation, and removal by dialysis. Figure 3 shows that precipitation method, which most of the time provides satisfactory results on 2-DE, sometimes cannot eliminate all of the interfering compounds. Subsequent washing of the pellet and/or dialysis after resuspension is quite helpful.19 For nongel techniques, removal of these interfering compounds is also recommended, although some techniques do not require a concentrating step. I. Should Albumin and/or Other High-Abundance Proteins Be Removed Prior to Urinary Proteome Analysis? Apparently, major abundant urinary proteins, such as albumin, immunoglobulins, transferrin, and R1-antitrypsin, can obscure the identification of low abundant proteins. Therefore, removal of these major abundant proteins is definitely helpful for enhancing the ability to examine low abundant urinary proteins. However, Candiano et al.43 have shown that repetitive fragmentation products of albumin and R1-antitrypsin are associated with nephrotic syndrome. They have identified a total of 72 fragments of albumin and R1-antitrypsin in the urine.43 Interestingly, several repetitive fragmentation motifs and a few differences among different pathologies were found.43 The same group has also reported that active focal segmental glomerulosclerosis is associated with massive oxidation of plasma albumin.44 Therefore, we definitely miss this kind of information if these major abundant proteins are removed from the urine. Moreover, some of the major abundant proteins, particularly albumin, can bind to several other proteins, which will also be removed together with the major abundant proteins. As there are pros and cons, whether the removal of

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Figure 2. 2-D gel images of urinary proteins isolated by various different protocols (A) and number of unique spots that could be obtained from each single protocol or from combinations (B). Each gel was loaded with equal amounts of 200 µg total protein. Quantitative and qualitative data are summarized in Table 2. (Modified from ref 19 with permission from the American Chemical Society.) Table 2. Protein Recovery Yield and Total Number of Protein Spots Obtained from Various Sample Preparation Methods to Concentrate/Isolate Human Urinary Proteinsa group ID

sample preparation methods

recovered proteinb (mean ( SD) (µg)

recovery yield (average) (%)

total number of spotsc

A B C D E F G H I J

Precipitation with 75% acetic acid Precipitation with 75% acetone Precipitation with 75% acetonitrile Precipitation with 75% ammonium sulfate Precipitation with 75% chloroform Precipitation with 75% ethanol Precipitation with 75% methanol Centrifugal filtration (10 kDa cutoff) Lyophilization Ultracentrifugation

138.40 ( 2.55 460.14 ( 1.78 212.02 ( 27.96 235.83 ( 9.59 174.21 ( 22.59 908.11 ( 54.74 884.56 ( 26.04 254.00 ( 5.25 1,017.10 ( 26.64 292.00 ( 25.88

9.89 32.87 15.14 16.85 12.44 64.87 63.18 18.14 72.64 20.86

72 159 206 142 146 187 189 116 72 76

a Modified from ref 19 with permission from the American Chemical Society. b Equal volume (10 mL) of pooled urine was used (n ) 3 independent experiments). c Equal amount (200 µg) of total protein was loaded.

major abundant proteins is really beneficial for urinary proteomics should be balanced and further elucidated. J. Intra-Individual and Inter-Individual Variabilities. Among the most difficult things to deal with urine samples are intraindividual and inter-individual variabilities. As a model for discussion herein, some data obtained from a recent systematic gel-based urinary proteomics study19 are illustrated. Figure 4A shows the intra-individual variability of urine samples obtained from a healthy subject at different time points within a day. Obviously, there were some differences observed in these urine samples. Also, drinking a cup of coffee or a large volume of water (i.e., a liter within 20 min) caused changes in the urinary proteome profile.19 Figure 4B and C clearly shows the interindividual variability of urine samples obtained from 4 males and 4 females, respectively. Concerning the “quantitative” data, the first-morning urine provided the greatest amount of recovered proteins when equal volume of urine was used, whereas drinking a cup of coffee or a large volume of water resulted in a markedly decreased amount of the recovered proteins (Table 3).19 Comparing between males and females, male urine had the greater amount of the recovered urinary proteins.19 However, only 4 males and 4 females were examined in this study. Extending the study to a larger number of subjects and to other racial and ethnic groups is required. 3886

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Concerning the “qualitative” data, the first-morning urine (though it provided the greatest amount of the recovered proteins) had the smallest number of visualized protein spots in 2-D gels when an equal amount of total protein was loaded (Table 3).19 Physiological changes by caffeine and water loading caused changes in excretion levels of some urinary proteins. Whereas male urine provided the greater amount of the recovered proteins, female urine had more protein spots visualized in 2-D gels, when equal amount of total protein was loaded (Table 3).19 K. Pooled versus Individual Samples. As aforementioned, there is a high degree of variations of urine samples obtained from different individuals (inter-individual variability). Therefore, this factor should be taken into account in urinary proteome analysis. The pooled urine lacks this kind of information and may generate biased or erroneous results. For example, if the level of protein X is extremely high in one of five samples in group A, while remaining normal in the other four samples in group A and in all five samples in group B, the comparison of level of this protein between the two groups, using respective pooled sample of each group, may result in significantly higher level in group A. However, when individual samples are analyzed and compared using statistical analysis, the data may yield no significant difference. Therefore, analysis of individual samples is highly recommended. Analysis of the

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Figure 3. Effects of interfering compounds in the urine on isoelectric focusing of 2-DE and improvement after dialysis to remove such interfering compounds. Each gel was loaded with equal amount of 200 µg total protein. (Modified from ref 19 with permission from the American Chemical Society.)

pooled urine samples can be also done, but with subsequent validation in individual samples to confirm the results obtained from the pooled samples. L. Data Interpretation and Normalization. Whenever possible, the urinary proteome data should be correlated to the well-characterized clinical parameters.45-47 In addition, the discovery of candidate biomarkers from the urine should be correlated to the standard clinical diagnostic tests or wellestablished markers.48 Because urinary concentrating and diluting mechanisms can directly affect urinary protein concentration, normalization of urinary proteome data is required. Most of urinary proteomics studies remain using the relative abundance of a protein compared to the amount of total protein. Other normalized data that are acceptable for urinary proteomics are 24-h excretion, rate of excretion within a certain period of the time, and normalization with urine creatinine. Which one is the best normalization method remains to be elucidated. Perhaps, the way to normalize the urinary proteome data should rely on subsequent applications. For example, if a strip or test kit will be applied to examine a urine biomarker, the rate or absolute quantitation within 24-h period is not the right choice.

Concluding Remarks and Outlook There are several barriers to the success of urinary proteomics. Therefore, the urinary proteome study needs to be carefully designed to overcome such barriers. The practical points discussed herein are based on available references and on

personal view/experience. This kind of information should stimulate further discussion and refinement to develop guidelines and standardizations for urinary proteome analysis. These guidelines and standardizations are most likely achievable via an active international collaborative network, namely, “Human Kidney and Urine Proteome Project” (HKUPP) (http://www. hkupp.org), under an umbrella of the “Disease Biomarkers Initiative” of the Human Proteome Organization (HUPO). All the issues mentioned herein and other additional topics will be further discussed in the coming HKUPP workshop (entitled “Toward Standards for Urine Proteomics”) during the HUPO sixth Annual Congress (October 6-10, 2007; Seoul, Korea) and also in another workshop during the 40th Annual Meeting of the American Society of Nephrology (November 2-5, 2007; San Francisco, CA). Summary of the Key Practical Points. (1) It is not justified to define disease-specific biomarkers by comparing the urinary proteome profile of a disease group to that obtained only from normal healthy individuals. (2) Not all the diseases will present marker molecule(s) in the urine. (3) A single ideal biomarker may not exist for each disease. (4) 2-DE and LC-MS/MS are more suitable for exploratory study of the pathogenic mechanisms and pathophysiology of diseases, whereas SELDI-TOF MS, CE-MS, microarrays, and microfluidic technology on a chip are more suitable for diagnostics and biomarker discovery. Journal of Proteome Research • Vol. 6, No. 10, 2007 3887

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Figure 4. Intra-individual and inter-individual variabilities. (A) Intra-individual variability. There were a few spots present only after water loading (circle area). In contrast, excretion levels (as determined by spot intensity) of the other few spots were decreased to almost absent after water loading (rectangular area). (B) and (C) Inter-individual variability in urine samples of males and females, respectively. Each gel was loaded with equal amount of 200 µg total protein. (Modified from ref 19 with permission from the American Chemical Society.)

(5) The second-morning or random midstream urine is recommended for urinary proteome analysis. (6) The use of protease inhibitors for the nonproteinuric urine is no longer recommended. However, its use for the proteinuric urine needs further investigations and discussion. (7) Unless the urine can be frozen immediately, the addition of a preservative (e.g., sodium azide, boric acid) to prevent bacterial overgrowth is highly recommended, particularly when the urine is kept at room temperature for >8 h, or at 4 °C for >16 h. (8) Removal of cells and debris must be done within 20-30 min after the urine collection to avoid the contamination of proteins originated from RBC, WBC, bladder epithelial cells, or debris. (9) Freeze-thaw cycles should be avoided. 3888

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Table 3. Intra-Individual and Inter-Individual Variabilitiesa sample variables

recovered proteinb (mean ( SD) (µg)

Intra-individual variables first-morning void (n ) 3) 530.37 ( 40.76 afternoon void (n ) 3) 486.47 ( 10.69 effect of caffeine (n ) 3) 319.88 ( 0.86 effect of water loading (n ) 3) 127.89 ( 11.65 Inter-individual variables male (n ) 4) 512.96 ( 65.66 female (n ) 4) 355.01 ( 105.23

total number of spotsc

135 144 166 156 115-153 157-246

a Modified from ref 19 with permission from the American Chemical Society. b 75% ethanol precipitation was applied for all samples and equal volume (10 ml) of pooled urine was used. c Equal amount (200 µg) of total protein was loaded; counted from 1 gel in the set of intra-individual variables and from 4 gels in the set of inter-individual variables.

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(10) Different methods to concentrate or isolate urinary proteins provide different proteome profiles. (11) Removal of interfering compounds, particularly salts, prior to urinary proteome analysis is highly recommended. (12) Whether albumin and/or other high-abundance proteins should be removed prior to urinary proteome analysis needs further investigations and discussion. (13) The high degree of intra-individual and inter-individual variabilities of the urine proteome must be concerned. (14) Analysis of individual samples is highly recommended, whereas analysis of the pooled urine can be also done, but with subsequent validation in individual samples to confirm the results obtained from the pooled samples. (15) Whenever possible, the urinary proteome data should be correlated to the well-characterized clinical parameters. (16) The best way to normalize urinary proteome data needs further discussion. Abbreviations: 2-DE, two-dimensional gel electrophoresis; CE-MS, capillary electrophoresis coupled to mass spectrometry; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; RBC, red blood cells; SELDI-TOF MS, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry; UTI, urinary tract infection; WBC, white blood cells.

Acknowledgment. This work was supported by Siriraj Grant for Research and Development, The Thailand Research Fund, Commission on Higher Education, Mahidol University, the National Research Council of Thailand, and the National Center for Genetic Engineering and Biotechnology. References (1) Thongboonkerd, V.; Malasit, P. Renal and urinary proteomics: Current applications and challenges. Proteomics 2005, 5, 10331042. (2) Thongboonkerd, V. Recent progress in urinary proteomics. Proteomics Clin. Appl. 2007, 1, 780-791. (3) Mischak, H.; Apweiler, R.; Banks, R. E.; Conaway, M.; Coon, J.; Dominicsak, A.; Ehrich, J. H. H.; Fliser, D.; Girolami, M.; Goodsaid, F.; Hermjakob, H.; Hochstrasser, D.; Jankowski, J.; Julian, B. A.; Kolch, W.; Massy, Z. A.; Neusuess, C.; Novak, J.; Peter, K.; Rossing, K.; Schanstra, J.; Semmes, J.; Theodorescue, D.; Thongboonkerd, V.; Weissinger, E. M.; Van Eyk, J. E.; Yamamoto, T. Clinical proteomics: a need to define the field and to begin to set adequate standards. Proteomics Clin. Appl. 2007, 1, 148-156. (4) Pisitkun, T.; Johnstone, R.; Knepper, M. A. Discovery of urinary biomarkers. Mol. Cell Proteomics 2006, 5, 1760-1771. (5) D’Amico, G.; Bazzi, C. Pathophysiology of proteinuria. Kidney Int. 2003, 63, 809-825. (6) Thongboonkerd, V. Proteomic analysis of renal diseases: Unraveling the pathophysiology and biomarker discovery. Expert Rev. Proteomics 2005, 2, 349-366. (7) Schaub, S.; Wilkins, J.; Weiler, T.; Sangster, K.; Rush, D.; Nickerson, P. Urine protein profiling with surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry. Kidney Int. 2004, 65, 323-332. (8) Nguyen, M. T.; Ross, G. F.; Dent, C. L.; Devarajan, P. Early prediction of acute renal injury using urinary proteomics. Am. J. Nephrol. 2005, 25, 318-326. (9) Woroniecki, R. P.; Orlova, T. N.; Mendelev, N.; Shatat, I. F.; Hailpern, S. M.; Kaskel, F. J.; Goligorsky, M. S.; O’Riordan, E. Urinary proteome of steroid-sensitive and steroid-resistant idiopathic nephrotic syndrome of childhood. Am. J. Nephrol. 2006, 26, 258-267. (10) Fliser, D.; Wittke, S.; Mischak, H. Capillary electrophoresis coupled to mass spectrometry for clinical diagnostic purposes. Electrophoresis 2005, 26, 2708-2716. (11) Kolch, W.; Neususs, C.; Pelzing, M.; Mischak, H. Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery. Mass Spectrom. Rev. 2005, 24, 959-977.

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