Simple Urinary Sample Preparation for Proteomic Analysis - American

Simple Urinary Sample Preparation for Proteomic Analysis Alamgir Khan*,† and Nicolle H. Packer‡ Australian Proteome Analysis Facility (APAF), Level 4, Building F7B, Research Park Drive, Macquarie University, Sydney NSW 2109, Australia, and Proteome Systems Ltd., 1/35-41 Waterloo Road, North Ryde, NSW 2113, Australia Received June 23, 2006

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. Keywords: urine • precipitation • 2-D gel electrophoresis • urinary proteome • ovarian cancer • MALDI-TOF

Introduction In recent years, there has been increased interest in exploring the human urinary proteome and particularly in the establishment of reference maps to assist in biomarker discovery. Urine is an easily accessible, noninvasive body fluid that carries proteins, peptides, and amino acids related to renal systems that have not been found in plasma.1 There are several reports on the use of urine analysis for diagnosis and/or prognosis in the following diseases: bladder cancer,2 acute inflammation due to a pilonidal abcess,3 urogenital diseases such as prostate cancer4 and benign hypertrophy of prostate,5 adenolysuccinase deficiency, 5-oxoprolinuria, propionic acidemia and disorders having orotic acid excreted,6 glomerular nephrotoxicity,7 nephrotic syndrome,8 diabetic nephropathy,9 in the study of gentamicin toxicity, and in exposure to xenobiotics.10 In sports, the World Anti Doping Agency prescribes urine to be analyzed for the detection of the performance-enhancing drug erythropoietin.11 Because urine collection is noninvasive, it is of interest as a diagnostic medium for the identification of protein and peptide biomarkers.2,3,12-14 Identification of urinary proteins and peptides may lead to an enhanced understanding of renal physiology as well as being a tool for diagnosis of renal systemic * To whom correspondence should be addressed at Australian Proteome Analysis Facility, Level 4, Building F7B, Research Park Drive, Macquarie University, Sydney NSW 2109, Australia. Tel, +61-2-9850 6204; fax, +61-29850 6200; e-mail, [email protected]. † Australian Proteome Analysis Facility (APAF). ‡ Proteome Systems Ltd.

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diseases. An efficient method for the treatment and analysis of urine is essential for these applications. Two-dimensional (2-D) gel electrophoresis is a powerful protein array technique and is widely used in biomarker discovery for diagnosis and clinical monitoring of diseases (reviewed in ref 15). Hence we have focused on the development of a rapid, robust, and efficient urine preparation method for 2-D gel electrophoresis and describe the application of this method to the analysis of the urinary proteome. Although there is the potential to discover novel biomarkers of renal and urogenital diseases by arraying human urinary proteins on 2-D gels as the first separation step for proteomic analysis, there are problems associated with the electrophoretic separation of urinary proteins. The protein concentration of healthy urine is far less than in human plasma (approximately 60 mg/mL) with healthy adults excreting approximately 5070 g of solids per day in the urine of which approximately 50120 mg is protein.1 Twenty milliliters of healthy urine precipitated with acetonitrile yielded only approximately 200 µg of protein. Additionally, urine contains anions and cations (salts)16 that interfere with the isoelectric focusing (IEF) of proteins when conducting 2-D gel electrophoresis. Therefore, preconcentration of urine and removal of nonprotein solid materials and ions from urine prior to the eletrophoretic separation of urinary proteins is essential to obtain high resolution. Several urine preconcentration methods have been reported, among the most common methods being dialysis and lyophilization,1,17 filtration,18 ultracentrifugation, and precipitation.19 The pre10.1021/pr060305y CCC: $33.50

 2006 American Chemical Society

Urinary Sample Preparation for Proteomic Analysis

cipitation approach is less time-consuming and is more compatible with larger numbers and volumes of urine compared with other methods. Ultracentrifugation uses expensive equipment, and acidic proteins are lost during protein concentration.19 Reported protein losses were much higher in ultrafiltration20,21 and in the dialysis-lyophilization method22 compared to a precipitation method.23,24 However, previous work separating urinary proteins precipitated by acetone resulted in only about 150 protein spots being detected in healthy urine.19 There have been many approaches to the separation of urinary proteins such as gas chromatography,6 high-performance liquid chromatography (HPLC),6 immunoassay,17,25-27 capillary electrophoresis (CE),6 surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS),2 mass spectrometric immunoassay (MSIA),12 and liquid chromatographytandem mass spectrometry (LC-MS/MS).28 The utility of these techniques for the identification of urinary proteins is limited to specific protein targets and has not been optimized for global proteome analysis and differential display. Recently, proteomic techniques have been applied to establish reference 2-D gel maps of the urinary proteome.3,13,14,17-19,29-32 In these reports, a relatively low number of protein spots were shown on the 2-D gels or were identified with poor overall resolution of the proteins. This indicates nonoptimal urine preparation, particularly in the case of desalting by dialysis and lyophilization. Since the sequence of the human genome is now known, and with the advent of sensitive analytical tools (e.g., mass spectrometers),33 there is the potential to identify more urinary proteins for potential biomarker discovery using 2-D gel electrophoresis coupled with mass spectrometry. The aim of this study was to develop and use a simple, robust, and efficient urine preparation method for 2-D gel electrophoresis and establish a protein reference map of healthy urine using mass spectrometry for protein identification.

Materials and Methods Collection of Urine. Urine samples were collected at various times over a 6 week period from a healthy male volunteer. The volunteer had no history of taking any prescription medicine in the past 10 years. Urine was collected once in the 1st and 2nd weeks in the morning (5 a.m.); three times in the 3rd week in the morning (5 a.m.), afternoon (3 p.m.), and evening (10 p.m.); and once in the 6th week in the afternoon (3 p.m.). A complete protease inhibitor cocktail (AEBSF, Aprotinin, Leupeptin, Bestatin, Pepstatin A, and E-64) for mammalian tissues (Sigma) was added to the urine immediately after collection at 1 µL/mL from a stock prepared according to the instructions provided by the manufacturer. Another urine sample was collected from an ovarian cancer (OVCA) patient (OVCA stage IIIc, epithelial serous adenocarcinoma) to observe the suitability of the urine preparation method described in this manuscript for the analysis of the urinary proteome of a diseased subject. Aliquots (20 mL) of urine samples were stored at -80 °C and thawed prior to use. Preparation of Urine. A thawed 20 mL urine sample was centrifuged at 1500g for 10 min at room temperature, and the sediment of broken cells or tissues and other solid materials in the pellet were discarded. The proteins were precipitated from the clarified urine by single or double precipitation methods. For the single precipitation method, urinary proteins were precipitated at 4 °C overnight using a urine-to-solvent ratio of 1:5 (1) acetone, (2) ethanol, (3) methanol, (4) acetoni-

research articles trile, (5) a mixture of an equal volume of acetone and ethanol, and (6) ultrafiltration followed by acetone precipitation. The ultrafiltration of urine(s) was carried out according to the procedure provided with the device (Amicon ultrafiltration cell, series 800, 3 kDa cutoff Membrane, Millipore) in order to reduce the initial volume of urine. This urine was initially concentrated 10-fold by ultrafiltration and further concentrated to 26.6-fold by the acetone precipitation. After overnight precipitation, proteins from all the treatments were pelleted by centrifugation at 4000g for 40 min at 10 °C. For the double precipitation method, the urinary proteins were first precipitated with acetonitrile at the pH of the urine (pH 6.2). After centrifugation, the pellet was collected (first pellet), and the supernatant was reprecipitated at -20 °C for 2.5 h after using 100 mM Tris base to increase the pH to 9.4 before the proteins were pelleted as for single precipitation with acetonitrile (second pellet). The precipitated pellets of all the methods were resuspended, and proteins were reduced with 5 mM TBP, alkylated with 10 mM acrylamide, and desalted by buffer exchange using centrifugal filtration with a 5kDa cutoff membrane according to our previous report.11 Variation in the conductivity of the urine was observed on different days from the same person, and the conductivity was generally too high for electrophoresis. For example, the conductivity of a collected sample of urine was 6 mS/cm, whereas the conductivity of the solubilized acetoneprecipitated urinary proteins only decreased to 4 mS/cm which was still too high for electrophoretic separation. The desalting step reduced the conductivity to less than 300 µS/cm, while simultaneously removing excess TBP, acrylamide, and Tris. The precipitated urinary protein solution was then rehydrated into the immobiline pH gradient (IPG) strips or stored at -80 °C until used. Unless indicated, urine was concentrated 100-fold (20 mL of urine to 200 µL of urinary proteins) by precipitation in all cases. Protein concentration was measured using Bradford reagent (Sigma) using human serum albumin as a standard. 2-D Gel Electrophoresis. Protein solution (200 µL of each sample equal to approximately 200 µg of protein obtained from 20 mL of urine) was loaded onto 11 cm long IPG strips (pH 3-5, 4-7, and 6-11) by in-gel rehydration for IEF, and 13 × 8 cm 6-15% (w/v) acrylamide gradient gels (both IPG strips and gels were from Proteome Systems Ltd., Australia) were used for the second dimension separation of proteins. IPG strip rehydration, IEF, equilibration of the strips, and the second dimension gel run were performed as previously reported.34 The gels were stained with ProteomIQ blue35 (Proteome Systems) according to the instructions provided with the kit. Spot Detection. Gel images were captured in tagged image file format (TIFF) with a UMAX PowerLook III flatbed scanner (UMAX Technologies, Inc.) at 300 dpi. The images were uploaded into Progenesis Discovery 2005 image analysis software (Nonlinear Dynamics Ltd., U.K.) using the analysis wizard. Protein spots were detected from triplicate gels except OVCA urine (detected from a single gel due to insufficient urine available). Statistical Analysis. Single factor analysis of variance (ANOVA) was carried out at 1% level of significance (F-test) with urine samples collected at three different times in 1 day or on four different days over a 6 week period. Sample Preparation for Mass Spectrometric Analysis. Relatively high-intensity Coomassie-stained protein spots from urine gels were selected for identification purposes. Spots were cut, washed, dried, and digested, and the peptide solution was Journal of Proteome Research • Vol. 5, No. 10, 2006 2825

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Figure 1. Comparison of urinary protein precipitation methods. Organic solvents used for precipitation are (a) acetone, (b) ethanol, (c) methanol, (d) acetonitrile, (e) ultrafiltration followed by acetone precipitation, and (f) a mixture of equal volume of acetone and ethanol (50:50) precipitation. Urinary proteins (urine collected at 1st week) were concentrated 100-fold (20 mL of urine) in all cases except for e, which was concentrated 266-fold (53.2 mL of urine), and the entire concentrated urinary proteins were loaded on each IPG strip by in-gel rehydration method. The pH ranges of gels are 4-7 (left to right) and stained with ProteomIQ Blue. The numbers of spots detected in each method are shown in Table 1.

desalted and concentrated using the Xcise robotic protein processing system (Shimadzu Biotech, Japan). Gel plugs were washed and digested, and the peptide solution was processed using the sample preparation kit for MALDI-MS analysis (Proteome Systems). Thirty microliters of 5 µg/mL trypsin was added to each spot, and the proteins were digested in the gel plugs overnight at 30 °C. After digestion, the peptide solution was desalted and concentrated using the C18 ZipTip micro column (Millipore) prior to placing on the MALDI target. MALDI-TOF MS and Database Searching. The MALDI-MS was performed on an Axima CFR instrument (Shimadzu Kratos, Manchester, U.K.), equipped with an N2 laser (337 nm, 10 Hz repetition rate) as described earlier.36 Internal mass calibration was performed using the trypsin autolysis peak (peak m/z 842.51) and adrenocorticotrophic hormone (ACTH, peak m/z 2466.2) spiked into matrix solution. Two blank gel plugs were cut and digested from an area outside of the IPG strip, and the mass signals generated from these plugs were subtracted from the peptide mass list of each protein spot. This removed autolyzed trypsin, matrix, and Coomassie peaks. The database search engines MS Fit in ProteinProspector (http://prospector.ucsf.edu/) and IonIQ peptide mass fingerprinting program (Proteome Systems) were used to identify the proteins (primary level search) using the Swiss-Prot, NCBI, or Owl databases, allowing an error margin of 50 ppm and one missed cleavage. For modification of peptides, cysteine alkylation (by acrylamide), methionine oxidation, and lysine methylation were considered. When peptide masses were matched to protein sequences in the databases (human only), a number of parameters was considered for the identification of proteins (secondary level search), such as (i) matched number of peptides (a minimum of four peptides matches were considered; however, if any peptide mass was matched to both modified and unmodified forms, then three peptide matches were also accepted; modifications considered were either methionine oxidation or lysine methylation); (ii) number of missed cleavage peptides within the matched peptides (if a 2826

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protein matched with more than 50% missed cleavage peptides, it was not considered to be an identification. If amino acid P was C-terminal to K and R, it was considered to be a true missed cleavage); (iii) intensities of matched peptides (if less than 50% of highest intensity peptides matched to a protein, it was not considered to be an identification); (iv) number of modified peptides matched (if more than 33% matched peptides reported as modified peptides, it was not considered to be an identification); and (v) sequence coverage (for >60 kDa proteins, >10% sequence coverage accepted as an identification. However, if any peptide mass was matched to both modified and unmodified forms (methionine oxidation or lysine methylation), then 30 kDa but 20% sequence coverage was accepted as an identification; however, if any peptide mass was matched in both modified and unmodified forms as mentioned above, then pH 7). However, resolution of the protein spots is higher in the narrow rather than the broader pH range (pH 3-5, 4-7, and 6-11). There were some variation in the number of spots and protein separation pattern on the gels between the urine samples collected at three time points in a day (Figure 3). Table 2 shows the number of spots detected (from triplicate gels) from the urine collected in the morning 798 (SD ( 12), afternoon 703 (SD ( 10), and evening 729 (SD ( 11), and the difference in the spot numbers between sample collection times is significant (P ) 0.01, F ) 59). On the other hand, a greater variation in the proteome pattern was observed between urine of the same subject collected on different days. The average number of spots (averaged from triplicate gels) detected on the 1st week was 567 (SD ( 12), 2nd week 672 (SD ( 12), 3rd week 798 (SD ( 12), and 6th week 640 (SD ( 13) spots in the pH range 4-7 (Table 2), and the difference in the spot numbers between different days urine collection is significant (P ) 0.01, F ) 196). Urine was collected in the morning in all the weeks except the 6th week which was collected in the afternoon. However, spot numbers also differed in urine collected in the afternoon in both the 3rd (703, SD ( 10) and 6th week (640, SD ( 13) (Table 2). There were unique proteins present, and spot intensity varied over the day of urine sampling (Figure 4).

Urinary Proteome of an Ovarian Cancer Patient. To test the applicability of this method on urine of a diseased subject, urine from an ovarian cancer patient was analyzed. Spatial location of spots on the gels and spot numbers of an OVCA subject were different from the healthy male subject. The number of proteins was higher in the OVCA urine (1098) than the healthy male subject (highest 798) in the pH range 4-7 (Tables 2 and 3), but the separation method was applicable. Identification of Urinary Proteins. Proteins were separated from the 3rd week morning urine of the male subject and were identified by MALDI-MS peptide mass fingerprinting. Spots were cut, and MS analyses were carried out from the gels in the pH ranges 3-5, 4-7, and 6-11. Three hundred and ninetyfive proteins from 178 genes were identified on these gels (Table 3, Figures 5 and 6). However, some identified proteins overlapped on the gels of different pH ranges. Accounting for this, 339 proteins (45, 272, 22 in the pH ranges 3-5, 4-7, 6-11, respectively) from 159 genes (16, 125, 18 in the pH ranges 3-5, 4-7, 6-11, respectively) were identified in the pH range 3-11. Thirty-seven protein spots (29 genes) contained two or more proteins. Classification of Identified Proteins. The identified urinary proteins were classified according to their subcellular and tissue origins based on the information available in the Swiss-Prot entry and/or NCBI databases. Out of the 339 identified proteins in the urine, 19% (63) were classified as membrane-associated, 53% (181) cytoplasmic or secreted, 3% (11) both membraneassociated and cytoplasmic or secreted, and 25% (83) had unknown subcellular origin (Table 4). Within the abovementioned sucellular origin, proteins were further grouped according to their tissue specificity. Of the identified proteins, 12% (39) were specific to renal systems (urine and kidney related), 25% (86) specific to plasma, 12% (42) specific to both renal systems and plasma, 9% (30) specific to both renal systems and other tissues, 6% (19) specific to both plasma and other tissues, 4% (13) ubiquitous, 24% (80) other tissues or organs, and 9% (30) from unknown tissues (Table 4). Other tissue origins include liver, bone marrow, lung, heart, brain, skeletal muscle, eye, fibroblast, skin, salivary gland, tonsil, thyroid, and trachea.

Discussion Our data confirms that urine contains far less soluble protein by weight compared to nonsoluble solid materials.1 Therefore, it was important to remove these solid materials which are primarily cells, broken tissue of the urinary tract, and crystalstructured material (observed under microscope) prior to electrophoretic separation of the soluble proteins for improved high resolution and sensitivity. A high content of solid contaminants limits the amount of protein able to be loaded onto the IPG strips because of their limited loading capacity. In addition to nonsoluble solid materials, urine also contains a high content of ions,16 particularly salts, which are incompatible with the IEF process. The conductivity of the urinary protein solutions varied between samples, which were desalted to maintain conductivity at 300 µS/cm or less, and to remove excess TBP, acrylamide, and Tris for better IEF separation.37 It should be noted that we buffer-exchanged with 5 kDa cutoff membranes for desalting so any urinary small proteins, peptides, and amino acids1 would not be retained. In this work, proteins were concentrated by precipitation using various organic solvents to determine the best method for maximal protein recovery from urine. Variable amounts of Journal of Proteome Research • Vol. 5, No. 10, 2006 2829

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Table 3. Summary of the Identified Urinary Proteins of a Healthy Male Subject

acc. no.

name of the protein

peptide matched

coverage (%)

mass (Da)

pI

4 8 8 12 10 17 15 16 5 23 31 27 12 11 16 7 4 6 9 16 19 16 28 9 20 17 16 6 12 8 11 15 8 14 10 13 20 11 18 11 13 11 15 5 5 5 7 6 4 4 5 5 5 6 5 5 8 7 5 9 6 5 5 7 6 9 5 3 7 5 4

8 11 12 19 16 23 21 21 5 24 37 28 14 12 30 13 8 11 28 24 27 26 36 15 31 42 27 11 23 13 20 25 19 34 32 40 31 21 31 19 22 18 23 18 19 14 17 15 18 19 21 15 16 12 12 13 34 29 18 17 33 37 37 11 33 63 8 16 22 13 37

69715 69715 69715 69715 69715 69715 69715 69715 69715 69715 69715 69715 108548 108548 83263 83263 83263 83263 42469 69322 69322 69322 69322 69715 69322 61988 69322 69715 69322 69715 69322 61988 53675 47651 47651 47651 71946 71946 71946 71946 71946 69715 69322 28375 28375 28375 49124 49124 31434 31434 31434 39325 39301 41793 41793 53822 26672 39000 39000 54467 26599 15595 15595 69762 21534 21534 69762 31434 47155 47155 10438

5.05 5.05 5.05 5.05 5.05 5.05 5.05 5.05 5.05 5.05 5.05 5.05 5.3 5.3 5.6 5.6 5.6 5.6 5.3 5.92 5.92 5.92 5.92 5.05 5.92 5.1 5.92 5.05 5.92 5.05 5.92 5.1 5.5 5.3 5.3 5.3 6.3 6.3 6.3 6.3 6.3 5.05 5.92 4.7 4.7 4.7 5.4 5.4 4.7 4.7 4.7 5.4 5.43 5.3 5.3 5.1 6.7 5.9 5.9 6.2 4.6 4.6 4.6 5.1 4.9 4.9 5.1 4.7 4.9 4.9 4.8

spot on map

3-5a

P07911 P07911 P07911 P07911 P07911 P07911 P07911 P07911 P07911 P07911 P07911 P07911 P12109 P12109 P01833 P01833 P01833 P01833 P48667 P02768 P02768 P02768 P02768 P07911 P02768 P35527 P02768 P07911 P02768 P07911 P02768 P13645 P05787 P01011 P01011 P01011 P01042 P01042 P01042 P01042 P01042 P07911 P02768 P61981 P61981 P61981 P55010 P55010 P24855 P24855 P24855 P02765 P02765 P63261 P63261 Q02818 Q9UNN8 P02760 P02760 P10619 P56537 P01591 P01591 P07911 27500341 27500341 P07911 P24855 P09104 P09104 Q9H299 2830

pH Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Tamm-Horsfall urinary glycoprotein Collagen alpha 1(VI) chain precursor Collagen alpha 1(VI) chain precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Keratin, type II cytoskeletal 6D Serum albumin precursor Serum albumin precursor Serum albumin precursor Serum albumin precursor Tamm-Horsfall urinary glycoprotein Serum albumin precursor Keratin, type I cytoskeletal 9 Serum albumin precursor Tamm-Horsfall urinary glycoprotein Serum albumin precursor Tamm-Horsfall urinary glycoprotein Serum albumin precursor Keratin, type I cytoskeletal 10 Keratin, type II cytoskeletal 8 Alpha-1-antichymotrypsin precursor Alpha-1-antichymotrypsin precursor Alpha-1-antichymotrypsin precursor Alpha-2-Thiol Proteinase Inhibitor Alpha-2-Thiol Proteinase Inhibitor Alpha-2-Thiol Proteinase Inhibitor Alpha-2-Thiol Proteinase Inhibitor Alpha-2-Thiol Proteinase Inhibitor Tamm-Horsfall urinary glycoprotein Serum albumin precursor 14-3-3 gamma protein 14-3-3 gamma protein 14-3-3 gamma protein Eukaryotic translation initiation factor 5 Eukaryotic translation initiation factor 5 Deoxyribonuclease I precursor Deoxyribonuclease I precursor Deoxyribonuclease I precursor Alpha-2-HS-glycoprotein precursor Alpha-2-HS-glycoprotein precursor Actin, cytoplasmic 2 (Gamma) Actin, cytoplasmic 2 (Gamma) Nucleobindin 1 precursor Endothelial protein C receptor precursor AMBP protein precursor AMBP protein precursor Lysosomal protective protein precursor Eukaryotic translation initiation factor 6 Immunoglobulin J Immunoglobulin J Tamm-Horsfall urinary glycoprotein Similar to vitelline membrane outer layer protein Similar to vitelline membrane outer layer protein Tamm-Horsfall urinary glycoprotein Deoxyribonuclease I precursor Gamma Enolase Gamma Enolase SH3 domain binding glutamic acid-rich protein like 3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 23 24 24 25 25 26 26 27 27 28 29 30 31 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Urinary Sample Preparation for Proteomic Analysis Table 3 (Continued)

acc. no.

P07911 P07911 P01133 P12109 P12109 P12109 P00450 P00450 P00450 P07911 P27487 P07911 P12830 P01833 P01833 P01833 P01833 P13645 P01833 P01833 P01833 P01833 P01833 P00450 P04217 Q86T22 P00450 P11142 P11142 P01833 P02787 P49221 P49221 P02768 P07911 P02768 P02768 P02768 P02768 P01042 P02768 P01042 P02768 P01042 P02768 P01042 P02768 P01042 P02768 P01042 P02768 P55287 P04004 P04217 P04217 P04217 P04217 P02768 P08133 P02768 P02768 P02790 P02768 P02768 P02790 P02768 P02768 P02768 P02768 P02768

name of the protein

pH Tamm-horsfall urinary glycoprotein Tamm-horsfall urinary glycoprotein Pro-epidermal growth factor precursor Collagen alpha 1(VI) chain precursor Collagen alpha 1(VI) chain precursor Collagen alpha 1(VI) chain precursor Ceruloplasmin precursor Ceruloplasmin precursor Ceruloplasmin precursor Tamm-horsfall urinary glycoprotein Dipeptidyl peptidase IV Tamm-horsfall urinary glycoprotein Epithelial-cadherin precur. Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Keratin, type I cytoskeletal 10 Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Polymeric-immunoglobulin receptor precursor Ceruloplasmin Precursor Alpha-1B-glycoprotein precursor OLFM4 protein [Frag.] Ceruloplasmin Precursor Heat shock cognate 71 kDa protein Heat shock cognate 71 kDa protein Polymeric-immunoglobulin receptor precursor Serotransferrin precursor Protein-glutamine glutamyltransferase 4 Protein-glutamine glutamyltransferase 4 Serum albumin precursor Tamm-horsfall urinary glycoprotein Serum albumin precursor Serum albumin precursor Serum albumin precursor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Cadherin-11 precursor Vitronectin precursor Alpha-1B-glycoprotein precursor Alpha-1B-glycoprotein precursor Alpha-1B-glycoprotein precursor Alpha-1B-glycoprotein precursor Serum albumin precursor Annexin VI Serum albumin precursor Serum albumin precursor Hemopexin precursor Serum albumin precursor Serum albumin precursor Hemopexin precursor Serum albumin precursor Serum albumin precursor Serum albumin precursor Serum albumin precursor Serum albumin precursor

peptide matched

coverage (%)

mass (Da)

pI

spot on map

22 4 14 12 15 11 4 12 12 27 19 30 14 10 21 23 20 19 19 27 29 21 29 7 23 22 7 27 36 18 21 27 13 41 16 36 28 25 7 12 24 18 31 24 46 23 34 23 26 15 27 14 20 18 21 21 21 26 15 17 33 28 30 34 31 42 41 49 54 62

69762 69762 133948 108548 108548 108548 122206 122206 122206 69762 88279 69762 97457 83314 83314 83314 83314 59519 83314 83314 83314 83314 83314 122206 54273 48197 122206 70855 70855 83314 77051 77146 77146 69367 69762 69367 69367 69367 69367 71946 69367 71946 69367 71946 69367 71946 69367 71946 69367 71946 69367 88050 54306 54210 54210 54210 54210 69367 75874 69227 69367 51677 69367 69367 51677 69367 69367 69367 69367 69367

5.1 5.1 5.6 5.3 5.3 5.3 5.4 5.4 5.4 5.1 5.7 5.1 4.6 5.6 5.6 5.6 5.6 5.1 5.6 5.6 5.6 5.6 5.6 5.4 5.3 5.7 5.4 5.37 5.37 5.6 6.8 6.3 6.3 5.9 5.1 5.9 5.9 5.9 5.9 6.3 5.9 6.3 5.9 6.3 5.9 6.3 5.9 6.3 5.9 6.3 5.9 4.7 5.6 5.5 5.5 5.5 5.5 5.9 5.4 5.9 5.9 6.5 5.9 5.9 6.5 5.9 5.9 5.9 5.9 5.9

1a 2a 3a 4a 5a 6a 7a 8b 9a 10a 11a 12a 13a 14a 15b 16a 17b 18a 19b 20a 21b 22a 23b 24b 25b 25b 26a 27a 28b 29b 30b 31a 32b 33a 33a 34b 35b 36b 37b 38a 38a 39b 39b 40a 40a 41b 41b 42a 42a 43b 43b 44a 45b 46b 47a 48b 49a 50a 51b 52a 53b 53b 54a 55b 55b 56a 57b 58a 59b 60a

4-7b 16 5 12 9 13 10 3 9 10 24 14 25 8 7 13 14 13 11 10 19 17 13 19 5 9 8 5 15 18 11 13 14 7 21 10 18 13 10 4 7 11 10 16 14 24 12 22 14 13 8 12 6 9 7 9 9 9 12 8 8 15 12 16 18 15 27 24 32 32 35

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Table 3 (Continued)

acc. no.

name of the protein

peptide matched

coverage (%)

mass (Da)

28 14 6 8 7 7 10 7 9 5 5 5 28 14 14 13 11 8 17 18 16 17 18 14 12 13 9 9 6 8 10 6 10 12 12 11 6 6 4 5 6 6 9 5 6 5 13 7 9 5 4 7 6 12 24 25 10 19 6 8 7 8 8 8 6 7 16 17 12 6

53 36 14 15 15 14 22 13 18 14 13 13 53 27 29 26 23 17 31 33 29 35 35 25 24 30 30 30 24 21 34 12 28 38 28 26 18 17 13 19 20 15 30 11 18 14 33 13 15 29 24 31 20 45 64 64 37 62 27 36 32 26 33 30 19 14 43 51 41 27

69367 57707 57707 71946 69367 71946 69367 71946 71946 53158 44787 55186 61988 71946 71946 71946 69367 71946 71946 71946 71946 71946 71946 71946 71946 71946 39325 39325 39325 44567 40077 52964 52964 40077 44567 44567 37655 36509 37655 65719 44567 69367 45885 69367 54514 67719 48286 69762 69715 23603 23603 34347 34347 33872 33872 33872 30996 33872 33780 33872 33872 30996 30996 30996 30996 69367 41737 41793 33872 42618

pI

spot on map

4-7b

P02768 P04746 P04746 P01042 P02768 P01042 P02768 P01042 P01042 34533060 9367869 34527351 P35527 P01042 P01042 P01042 P02768 P01042 P01042 P01042 P01042 P01042 P01042 P01042 P01042 P01042 P02765 P02765 P02765 P15309 P08571 P02774 P02774 P08571 P15309 P15309 P01876 P01877 P01876 7662260 P15309 P02768 Q03154 P02768 P12955 Q05513 P11117 P07911 P07911 P19652 P19652 P02750 P02750 P25311 P25311 P25311 Q8WVV7 P25311 Q06520 P25311 P25311 Q8WVV7 Q8WVV7 Q8WVV7 Q8WVV7 P02768 P60709 P63261 P25311 P12277 2832

pH Serum albumin precursor Alpha-amylase, pancreatic precursor Alpha-amylase, pancreatic precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Unnamed protein product Immunoglobulin heavy chain variant Unnamed protein product Keratin, type I cytoskeletal 9 Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Serum albumin precursor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-thiol proteinase inhibitor Alpha-2-HS-glycoprotein precursor Alpha-2-HS-glycoprotein precursor Alpha-2-HS-glycoprotein precursor Prostatic acid phosphatase precursor Monocyte differentiation antigen CD14 precursor Vitamin D-binding protein precursor Vitamin D-binding protein precursor Monocyte differentiation antigen CD14 precursor Prostatic acid phosphatase precursor Prostatic acid phosphatase precursor Ig alpha-1 chain C region Ig alpha-2 chain C region Ig alpha-1 chain C region hypothetical protein LOC9920 Prostatic acid phosphatase precursor Serum albumin precursor Aminoacylase-1 Serum albumin precursor Xaa-Pro dipeptidase Protein kinase C, zeta type Acid phosphatase 2, lysosomal Tamm-horsfall urinary glycoprotein Tamm-horsfall urinary glycoprotein Alpha-1-Acid Glycoprotein 2 Precursor Alpha-1-Acid Glycoprotein 2 Precursor Leucine-rich alpha-2-glycoprotein precursor Leucine-rich alpha-2-glycoprotein precursor Zinc-alpha-2-glycoprotein precursor Zinc-alpha-2-glycoprotein precursor Zinc-alpha-2-glycoprotein precursor Gelsolin Zinc-alpha-2-glycoprotein precursor Alcohol sulfotransferase Zinc-alpha-2-glycoprotein precursor Zinc-alpha-2-glycoprotein precursor Gelsolin Gelsolin Gelsolin Gelsolin Serum albumin precursor Actin, cytoplasmic 1 Actin, cytoplasmic 2 Zinc-alpha-2-glycoprotein precursor Creatine kinase, B chain


5.9 6.6 6.6 6.3 5.9 6.3 5.9 6.3 6.3 5.7 5.7 6.4 5.1 6.3 6.3 6.3 5.9 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 5.4 5.4 5.4 5.8 5.8 5.4 5.4 5.8 5.8 5.8 6.1 5.7 6.1 5.78 5.8 5.8 5.64 5.4 6.15 5.1 5.05 5 5 5.7 5.7 5.6 5.6 5.6 4.58 5.6 5.7 5.6 5.6 4.58 4.58 4.58 4.58 5.3 5.3 5.6 5.34

61b 62b 63a 64a 64a 65a 65a 66b 67a 68b 69b 70a 71a 72a 73b 74a 74a 75b 76a 77a 78b 79a 80b 81a 82b 83a 84b 85a 86b 87a 88a 89b 90b 91a 92a 93b 94a 94a 95b 96a 97b 98a 99a 100b 101b 102b 103b 104b 105a 106a 107b 108a 109a 110a 111b 112a 113a 114b 115a 116b 117a 118a 119a 120b 121a 122b 123a 124b 125b 126a

research articles


acc. no.

name of the protein

peptide matched

coverage (%)

mass (Da)

pI

spot on map

10 8 11 8 9 7 3 7 14 10 9 9 10 10 10 12 8 12 10 13 12 14 7 9 7 9 14 8 9 6 8 7 9 5 5 7 12 6 5 8 5 5 6 6 9 11 8 6 9 8 8 8 10 7 7 7 8 4 4 5 9 7 9 5 6 4 6 4 4 7 6

32 26 38 31 34 18 14 23 33 24 23 23 19 19 21 23 21 37 27 40 32 36 20 30 25 25 37 39 54 28 47 36 44 12 31 43 47 35 25 38 20 39 38 38 42 53 46 28 46 54 40 38 47 35 26 31 27 34 24 32 41 54 41 30 30 27 31 23 25 23 15

40877 40877 36639 39000 39000 47651 39000 39000 39000 59373 53822 53822 53822 53822 53822 68178 53822 39000 39000 39000 38975 38975 34956 40229 35420 47066 39000 41426 23518 26025 23518 26441 27547 44553 23365 22333 30454 25521 26441 26025 38910 19516 33297 33179 28459 28482 23419 29089 25834 25521 26441 25521 23044 27709 37808 18731 30454 17473 17473 17473 20171 15878 20172 31649 27709 22876 18731 18731 14177 42469 36154

6.1 6.1 5.7 5.9 5.9 5.3 5.9 5.9 5.9 4.9 5.1 5.1 5.1 5.1 5.1 5.2 5.1 5.9 5.9 5.9 5.96 5.96 5.51 6.5 5.8 5.8 5.9 5.6 5.5 5.9 5.5 5.7 5.7 6.1 6.2 5.95 6.1 6.1 5.7 5.9 5.5 5.5 6.83 6.61 6.7 6.7 6.9 6.2 6.1 6.1 5.7 6.1 5.8 5.7 5.9 5.6 6.1 5.2 5.2 5.2 5.77 5.5 5.8 5.6 5.7 5.4 5.6 5.6 6 5.3 5.6

127b 128b 129a 130b 131a 132a 133a 134b 135b 136a 136a 137a 138b 139b 140a 141a 141a 142a 143b 144a 145b 146a 147a 148b 149a 150b 151b 152b 153a 154b 155a 156b 157a 158a 159a 160b 161b 162a 163b 164a 165b 166a 167b 167b 168a 169b 170a 171a 172b 173b 174a 175b 176b 177a 178b 179a 180a 181b 182a 183b 184a 185b 186a 187b 188a 189a 190b 191a 192b 192b 193a

4-7b

Q16769 Q16769 P07195 P02760 P02760 P01011 P02760 P02760 P02760 32425553 Q02818 Q02818 Q02818 Q02818 Q02818 P38607 Q02818 P02760 P02760 P02760 P02760 P02760 Q9NP67 Q12907 Q7Z4Z9 P49407 P02760 P50148 JE0242 21410817 JE0242 17511825 O95336 P07339 5360675 Q96IU4 32967272 16741061 17511825 21410817 22671682 21706410 Q16762 P25325 21669485 21669451 4176418 21669453 21619606 16741061 17511825 16741061 P02753 21669311 P07858 P05451 32967272 O75780 O75780 O75780 Q9ULC7 P02766 Q9ULC7 33590472 21669311 9506653 P05451 P05451 P13987 P48667 P02649

pH Glutaminyl-peptide cyclotransferase precursor Glutaminyl-peptide cyclotransferase precursor L-lactate dehydrogenase B chain AMBP protein precursor AMBP protein precursor Alpha-1-antichymotrypsin precursor AMBP protein precursor AMBP protein precursor AMBP protein precursor Unknown (protein for IMAGE:3343931) Nucleobindin 1 precursor Nucleobindin 1 precursor Nucleobindin 1 precursor Nucleobindin 1 precursor Nucleobindin 1 precursor Vacuolar ATP synthase A, osteoclast isoform Nucleobindin 1 precursor AMBP protein precursor AMBP protein precursor AMBP protein precursor AMBP protein precursor AMBP protein precursor Intelectin Vesicular integral-membrane protein VIP36 precursor Lambda-Crystallin Beta-arrestin 1 AMBP protein precur. Guanine nucleotide-binding protein G(q), alpha Ig kappa chain NIG26 precursor IGKC protein Ig kappa chain NIG26 precursor Unknown (protein for MGC:31980) 6-phosphogluconolactonase Cathepsin D precursor Anti-Entamoeba histolytica Ig kappa light chain CCG1-interacting factor B Neurofibromin 2 isoform 10 Unknown (protein for MGC:27376) Unknown (protein for MGC:31980) IGKC protein Thyroid peroxidase isoform 2/4 AGRN protein Thiosulfate sulfurtransferase 3-mercaptopyruvate sulfurtransferase Immunoglobulin kappa light chain VLJ region Immunoglobulin kappa light chain VLJ region IgG kappa chain Immunoglobulin kappa light chain VLJ region Unknown (protein for MGC:40426) Unknown (protein for MGC:27376) Unknown (protein for MGC:31980) Unknown (protein for MGC:27376) Plasma retinol-binding protein precursor Immunoglobulin kappa light chain VLJ region Cathepsin B precursor Lithostathine 1 alpha precursor Neurofibromin 2 isoform 10 Peroxisome proliferator-activated receptor alpha (Frag.) Peroxisome proliferator-activated receptor alpha (Frag.) Peroxisome proliferator-activated receptor alpha (Frag.) MBL-associated serine protease(MASP)-2 Transthyretin precursor MBL-associated serine protease (MASP)-2 MHC class I antigen Immunoglobulin kappa light chain VLJ region ADP-ribosylation factor related protein 2 Lithostathine 1 alpha precursor Lithostathine 1 alpha precursor CD59 glycoprotein precursor Keratin, type II cytoskeletal 6D Apolipoprotein E precursor


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Khan and Packer

Table 3 (Continued)

acc. no.

name of the protein

peptide matched

coverage (%)

mass (Da)

pI

spot on map

6 4 4 3 3 5 4 4 3 5 5 4 5 5 3 3 3 4 3 4 7 5 5 14 17 27 29 29 27 24 15 9 7 11 12 6 13 10 13 10 8 9 9 6 10 4 7 9 7 13 7 20 7 8 6 19 8 8 8 9 6 11 8 11 7 20 13 7 6 15

21 33 37 21 28 27 37 32 18 44 27 27 44 27 31 30 35 32 24 23 48 24 37 37 43 62 65 63 66 63 41 24 21 26 38 13 24 22 37 21 26 21 19 21 41 21 13 17 47 22 38 63 22 47 36 77 39 57 44 43 56 72 39 40 23 36 37 23 14 24

38224 20128 13997 22456 20363 14177 14177 20363 14177 15627 14177 14177 15627 14177 11535 15348 13097 15595 15595 28866 13715 21276 26599 47884 47884 46737 46737 46737 46737 46737 52603 47884 50152 44488 52879 61229 70954 68554 40229 70954 40229 73835 52221 33338 29644 25231 58158 54353 21534 59519 23400 30778 34144 23051 28594 26560 22567 23211 22567 19493 13815 15945 19493 40229 45665 59519 46737 41793 70899 76092

5.5 5.32 5.3 4.98 5.2 6 6 5.2 6 4.8 6 6 4.8 6 5.6 5.1 4.3 4.6 4.6 5.3 6.06 5.1 4.6 6.3 6.3 5.4 5.4 5.4 5.4 5.4 6.3 6.3 4.9 5.93 5.7 6 5.4 5.3 6.5 5.4 6.5 6 5.7 5.68 5.7 6 6.3 6.2 4.9 5.1 5.7 5.6 5.6 5.8 6.1 5.4 5.5 5.44 5.5 5.45 5.7 5.5 5.45 6.5 5.6 5.1 5.4 5.3 5.4 6.1

193a 194b 195a 196a 197a 197a 198b 199a 199a 200a 200a 201b 202a 202a 203a 203a 204b 205b 206a 207a 208a 209a 210a 211b 212a 213b 214a 215b 216a 217a 218b 219b 220b 221a 222a 223a 224a 224a 225a 226a 226a 227a 228b 229b 230a 231a 232a 233a 234b 235a 236a 237a 238b 239a 240b 241b 242b 243a 244b 245a 246a 247a 248a 249b 250b 251b 252a 253b 254a 255a

4-7b

P17693 1694789 9789610 Q99653 673416 P13987 P13987 673416 P13987 3288499 P13987 P13987 3288499 P13987 P13987 E57233 23954348 P01591 P01591 21669555 P01884 P05090 P56537 4504893 4504893 P01009 P01009 P01009 P01009 P01009 P01008 4504893 P68363 Q96QK9 Q96KP4 O14773 7770149 O15287 Q12907 7770149 Q12907 O43572 4503521 Q9P0G7 O95865 558216 27502930 12653639 27500341 547749 P62070 P02647 O75225 A23746 21669345 P21266 P05452 P09211 P05452 Q9UMV3 Q9NRX4 Q14019 Q9UMV3 Q12907 P63092 P13645 P01009 P63261 P11142 P16278 2834

pH HLA class I histocompatibility antigen, alpha chain GRS protein BC282485_1 Calcium-binding protein p22 GM2 activator protein CD59 glycoprotein precursor CD59 glycoprotein precursor GM2 activator protein CD59 glycoprotein precursor COL1A1 and PDGFB fusion transcript CD59 glycoprotein precursor CD59 glycoprotein precursor COL1A1 and PDGFB fusion transcript CD59 glycoprotein precursor CD59 glycoprotein precursor Complexin II Breast carcinoma amplified sequence 4 protein Immunoglobulin J chain Immunoglobulin J chain Immunoglobulin lambda light chain VLJ region Beta-2-microglobulin precursor Apolipoprotein D precursor Eukaryotic translation initiation factor 6 Alpha-2-Thiol Proteinase Inhibitor Alpha-2-Thiol Proteinase Inhibitor Alpha-1-antitrypsin precursor Alpha-1-antitrypsin precursor Alpha-1-antitrypsin precursor Alpha-1-antitrypsin precursor Alpha-1-antitrypsin precursor Antithrombin-III precursor Alpha-2-Thiol Proteinase Inhibitor Tubulin alpha-1 chain Acid phosphatase, prostate Glutamate carboxypeptidase-like protein 1 Tripeptidyl-peptidase I precursor PRO1851 Fanconi anemia group G protein Vesicular integral-membrane protein VIP36 precursor PRO1851 Vesicular integral-membrane protein VIP36 precursor A kinase anchor protein 10, mitochondrial precursor Murine mammary tumor integration site 6 Lambda-Crystallin NG,NG-dimethylarginine dimethylaminohydrolase 2 ketohexokinase GK protein Similar to protective protein for beta-galactosidase Similar to vitelline membrane outer layer protein Keratin, type I cytoskeletal 10 Ras-Related Protein R-Ras2 Apolipoprotein A-I precursor Hypothetical protein CPVL [Frag.] Ig kappa chain V-III (KAU cold agglutinin) Immunoglobulin kappa light chain VLJ region Glutathione S-transferase Mu 3 Tetranectin precursor Glutathione S-transferase P Tetranectin precursor MBL-associated serine protease-2 related protein 14 kDa phosphohistidine phosphatase CLP protein (Coactosin-like protein) MBL-associated serine protease-2 related protein Vesicular integral-membrane protein VIP36 precursor Guanine nucleotide-binding protein G(S), alpha Keratin, type I cytoskeletal 10 Alpha-1-antitrypsin precursor Actin, cytoplasmic 2 Heat shock cognate 71 kDa protein Beta-galactosidase precursor


research articles


acc. no.

name of the protein

pH

peptide matched

coverage (%)

mass (Da)

pI

spot on map

4-7b

P16279 P16278 P16279 P04745 1515427 P02647 Q9NP86 Q9BRV8 21670289

Beta-galactosidase-related protein precursor Beta-galactosidase precursor Beta-galactosidase-related protein precursor Alpha-amylase, salivary precursor Nel homolog Apolipoprotein A-I precursor Calcium-binding protein CaBP5 Hypothetical protein FLJ21168 Immunoglobulin heavy chain VHDJ region

14 15 13 10 5 6 3 4 3

26 23 25 23 12 21 27 23 40

60552 76092 60552 57768 48860 30778 19826 23720 13165

6.5 6.1 6.5 6.5 5.1 5.6 4.5 5.09 5.7

255a 256b 256b 257b 258b 259b 260b 261b 262b

P07911 P02768 P08571 Q96MI8 Q9NUZ1 21758117 Q8TC36 P04424 Q9NWL0 O00560 P04220 A23746 A23746 11968003 21669343 106586 7438710 P07911 O75916 P07911 O75916 P07911 O75916 O75916 P02768 P02768 P02768 P02768 P02768 20135645 O96020 3420277 P07911 Q7Z7F6

pH 6-11c Tamm-Horsfall urinary glycoprotein Serum Albumin Precursor Monocyte differentiation antigen CD14 precursor Hypothetical protein FLJ32312 Hypothetical protein FLJ11042 Unnamed protein product Sperm-associated antigen 4-like protein Argininosuccinate lyase Hypothetical protein FLJ20758 Syntenin 1 (Melanoma differentiation associated protein-9) Ig MU heavy chain disease protein Ig kappa chain V-III (KAU cold agglutinin) Ig kappa chain V-III (KAU cold agglutinin) 5-azacytidine induced gene 2 Immunoglobulin kappa light chain VLJ region Ig kappa chain V-III (KAU cold agglutinin) Ig kappa chain NIG2 precursor Tamm-Horsfall urinary glycoprotein Regulator of G-protein signaling 9 Tamm-Horsfall urinary glycoprotein Regulator of G-protein signaling 9 Tamm-Horsfall urinary glycoprotein Regulator of G-protein signaling 9 Regulator of G-protein signaling 9 Serum Albumin Precursor Serum Albumin Precursor Serum Albumin Precursor Serum Albumin Precursor Serum Albumin Precursor SVAP1 protein G1/S-Specific cyclin E2 Glypican 4 Tamm-Horsfall urinary glycoprotein OFD1 protein

26 45 9 6 5 6 5 8 7 9 7 7 6 10 6 4 4 17 12 16 14 19 19 14 15 14 17 10 14 8 15 8 10 6

29 70 31 11 30 12 18 18 23 34 23 32 32 33 22 21 24 23 21 21 22 24 26 21 30 25 31 18 24 21 41 20 15 30

69762 69367 40077 60211 42336 42632 43082 51744 31863 32445 43058 23051 23051 44935 28886 23051 23463 69762 76967 69762 76967 69762 76967 76967 69367 69367 69367 69367 69367 52595 46758 62399 69762 27732

5.1 5.9 5.8 6.1 9.2 9.11 8.6 6.2 5.13 7.1 5.1 5.8 5.8 6.15 6.7 5.75 8.3 5.1 9.4 5.1 9.4 5.1 9.4 9.4 5.9 5.9 5.9 5.9 5.9 8.9 8 6.3 5.1 8.26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18 19 19 20 20 21 22 23 24 25 26 27 28 29 30 31

a The map is shown for pH 3-5 in Figure 5a. b The map is shown for pH 4-7 in Figure 6. The map pH 4-7 is divided into two, the spot numbers indicated as ‘a’ and ‘b’ at the end of spot numbers are shown in panels a and b, respectively. c The map is shown for pH 6-11 in Figure 5b.

proteins were precipitated by different solvents, and acetonitrile proved to be the most efficient. It has been reported that acetone precipitated more acidic and hydrophilic proteins compared to ultracentrifugation from urine.19 In our hands, acetonitrile precipitated even more proteins than acetone from urine. Acetone precipitation of proteins has also been found to be less effective compared to perchloric acid precipitation of proteins from rat liver homogenate.38 We attempted concentration of urine by ultrafiltration prior to precipitation by acetone to reduce the amount of organic solvent required for the precipitation process, but this resulted in less protein spots than acetonitrile precipitation even when 2.5 times more urine was precipitated. We also attempted to improve protein precipitation by acidifying the urine prior to precipitation (data not shown), but this also did not improve upon acetonitrile precipitation. As another alternative, we carried out a double precipitation of urinary proteins using acetonitrile, by first precipitating with acetonitrile at the native pH of the urine and reprecipitating the supernatant at alkaline

pH. Most of the protein species were precipitated in the first precipitation of normal urine, but some new proteins were detected on the gel when the supernatant from the precipitation of urine of a cancer patient was reprecipitated. The protein concentration in OVCA urine was higher than in the healthy subject, which is consistent with the report that excessive protein in urine is an indicator of the diseased state of a subject.15 Although the double acetonitrile precipitation of protein from the ovarian cancer sample was incomplete, this discriminatory protein precipitation may be useful for the enrichment of particular proteins of interest. We found that acetonitrile precipitation was the best method (including precipitation, pellet solubilization, and buffer exchange) for the preparation of urine for the analysis of its protein composition. A number of reports3,13,14,17-19,29-31,39 have been published describing different urinary protein preparation methods for 2-D gel analysis. Most of the reported methods are either complicated and/or do not result in a comparable number of resolved gel-separated proteins. The urine sample Journal of Proteome Research • Vol. 5, No. 10, 2006 2835

research articles

Khan and Packer

Figure 6. Maps of human urinary proteome of a healthy male subject pH range 4-7 (left to right). Labeled spots are identified by MALDI-MS, and the summary of identified proteins is shown in Table 3. Spots in panels a and b are shown in Table 3 as ‘a’ and ‘b’ at the end of the spot numbers, respectively. The map is divided into two for clear visualization of the spot numbers. A total of 334 spots was analyzed, and 290 spots were identified.

preparation in our method involves three steps only: concentration of urinary proteins by acetonitrile precipitation, reduction and alkylation of the protein, and buffer exchange. Nearly all (90-97%) of plasma proteins can be precipitated with acetonitrile.23,24 Maximal protein recovery from urine is essential for detecting trace quantities of proteins present in urine 2836


for potential biomarker discovery. Our urine preparation method is simple but resulted in the separation of a substantial number of well-resolved urinary proteins. To apply our method, we have analyzed urine from a healthy male subject collected at three times in a day and four times over a 6 week period to observe how the urinary proteome

research articles

Urinary Sample Preparation for Proteomic Analysis Table 4. Classification of Identified Proteins Based on Their Subcellular and Tissue Origin in the pH Range 3-11

classification of proteinsa

pH 3-11 without overlap between pH ranges (3-5, 4-7, 6-11)

Subcellular Origin Membrane-associated 63 (4, 56, 3) Cytoplasmic/secreted 181 (39, 140, 2) Both membrane and 11 (2, 9, 0) cytoplasmic/secreted Other tissues/organs 1 (0, 0, 1) Not reported (unknown) 83 (0, 67, 16) Tissue Origin Specific to renal systems 39 (22, 15, 2) (urine/kidney) Specific to plasma 86 (3, 83, 0) Both renal systems and 42 (2, 40, 0) plasma Both renal systems and 30 (0, 28, 2) other tissues Both plasma and other 19 (0, 19, 0) tissues Ubiquitous 13 (1, 12, 0) Other tissues/organs 80 (13, 59, 8) Not reported (unknown) 30 (4, 16, 10)

% of total identified proteins

19 53 3 0 25 12 25 12 9 6 4 24 9

a Proteins were classified according to the information available in the respective protein entry databases.

changes over time. Over a single day, the number of protein spots and the proteome pattern differed significantly with the highest number of proteins found in the urine collected in the morning. Similar results have been obtained by Lafitte et al.13 with more protein present in morning urine when compared with pooled 24-h urine. A greater difference in the proteome pattern was observed in the urine collected on different days. This is consistent with the reported findings30 that significant differences in the urinary profiles are observed across individuals and between different days of sample collection. This is to be expected due to changes in the proteome caused by diet, stress, physiological conditions, psychological status, and sleeping pattern between days. To determine if the acetonitrile precipitation method is suitable for the analysis of urine from a diseased subject, we precipitated the protein from an ovarian cancer patient and a large number of proteins were well-resolved on 2-D gels. The amount and number of protein from the same volume of urine was greater in the cancer urine (1098 spots) than healthy male urine (highest 798 spots). Furthermore, there were protein spots which were not present in the healthy male urine. It is not possible to say whether any changes in the cancer proteome are related to the disease or specific to female urinary proteins in such a small sample number and without adequate controls, but as urine is a specific filtrate of blood, the variation of urinary proteins across and within individuals seems to be inevitable.30 In total, 491 spots (in the pH ranges 3-6, 4-7, and 6-11) were cut and analyzed for protein identification, and 395 spots (80%) were identified by MALDI-TOF MS peptide mass fingerprinting. This is a significantly higher proportion of the urinary proteome identified, using a single analytical technique, compared to other recently published reports.3,13,14,17-19,29-31,39 There have been reports in the literature of up to 1400 protein spots29 or 383 unique gene products39 identified from human urine. However, these numbers depend on unsubstantiated image analysis programs and less rigorous criteria of MS data for protein identification. Image analysis programs result in sig-

nificantly different number of spots by auto detection methods compared with manually edited methods such as those we used, which defined spot boundaries and manually omitted false spots. For example, in our case, auto spot detection resulted in 1876 spots in healthy and 2983 spots in ovarian cancer which was reduced to 798 and 1098, respectively, by manual spot editing. The peptide mass fingerprinting from the MS data used the currently accepted criteria (see Materials and Methods) for proteomics40 to ensure accurate protein identification. We conclude that precipitation of proteins with acetonitrile is superior to other preparation methods tested for the 2-D gel analysis of the human urinary proteome of both healthy and diseased subjects, and this method may facilitate biomarker discovery in urine.

Acknowledgment. We thank Julie Soon, Australian Proteome Analysis Facility (APAF) Ltd. for skilful technical assistance in image analysis, AP Clinical for collecting OVCA urine, and Mark P. Molloy (APAF) for preliminary review of the manuscript. All the laboratory work was carried out during A. Khan’s employment at Proteome Systems; image analysis and database searching for classification of proteins was conducted at APAF (established under the Australian Government’s Major National Research Facilities program). References (1) Anderson, N. G.; Anderson, N. L.; Tollaksen, S. L.; Hahn, H.; Giere, F.; Edwards, J. Concentration and two-dimensional electrophoretic analysis of human urinary proteins. Anal. Biochem. 1979, 95, 48-61. (2) Vlahou, A.; Schellhammer, P. F.; Mendrinos, S.; Patel, K.; Kondylis, F. I.; Gong, L.; Nasim, S.; Wright, G. L., Jr. Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine. Am. J. Pathol. 2001, 158, 14911502. (3) Pang, J. X.; Ginanni, N.; Dongre, A. R.; Hefta, S. A.; Opitek, G. J. Biomarker discovery in urine by proteomics. J. Proteome Res. 2002, 1, 161-169. (4) Bratt, O. Hereditary prostate cancer: clinical aspects. J. Urol. 2002, 168, 906-913. (5) Breul, J.; Pickl, U.; Hartung, R. Prostate-specific antigen in urine. Eur. Urol. 1994, 26, 18-21. (6) Jellum, E.; Dollekamp, H.; Blessum, C. Capillary electrophoresis for clinical problem solving: Analysis of urinary diagnostic metabolites and serum proteins. J. Chromatogr., B. 1996, 683, 55-65. (7) Cutler, P.; Bell, D. J.; Birrell, H. C.; Connelly, J. C.; Connor, S. C.; Holmes, E.; Mitchell, B. C.; Monte, S. Y.; Neville, B. A.; Pickford, R.; Polley, S.; Schneider, K.; Skehel, J. M. An integrated proteomic approach to studying glomerular nephrotoxicity. Electrophoresis 1999, 20, 3647-3658. (8) Aviles-Santa, L.; Alpern, R.; Raskin, P. Reversible acute renal failure and nephrotic syndrome in a Type 1 diabetic patient. J. Diabetes Complications 2002, 16, 249-254. (9) Svensson, M.; Sundkvist, G.; Arnqvist, H. J.; Bjork, E.; Blohme, G.; Bolinder, J.; Henricsson, M.; Nystrom, L.; Torffvit, O.; Waernbaum, I.; Ostman, J.; Eriksson, J. W. Signs of nephropathy may occur early in young adults with diabetes despite modern diabetes management. Diabetes Care 2003, 26, 2903-2909. (10) Kennedy, S. Proteomic profiling from human samples: the body fluid alternative. Toxicol. Lett. 2001, 120, 379-384. (11) Khan, A.; Grinyer, J.; Truong, S. T.; Breen, E. J.; Packer, N. H. New urinary EPO drug testing method using two-dimensional gel electrophoresis. Clin. Chim. Acta 2005, 358, 119-130. (12) Kiernan, U. A.; Tubbs, K. A.; Nedelkov, D.; Niederkofler, E. E.; McConnell, E.; Nelson, R. W. Comparative urine protein phenotyping using mass spectrometric immunoassay. J. Proteome Res. 2003, 2, 191-197. (13) Lafitte, D.; Dussol, B.; Andersen, S.; Vazi, A.; Dupuy, P.; Jensen, O. N.; Berland, Y.; Verdier, J. M. Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples. Clin. Biochem. 2002, 35, 581-589.


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PR060305Y

Simple Urinary Sample Preparation for Proteomic Analysis - American

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