Are Protease Inhibitors Required for Gel-Based Proteomics of Kidney and Urine? Phattara-orn Havanapan and Visith Thongboonkerd* Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Received January 9, 2009
Proteolysis is one of the major problems in collection and storage of biological samples for proteome analysis, particularly when the samples undergo freeze-thaw cycles. The use of protease inhibitors for prevention of such proteolysis in some samples is debated because protease inhibitors may interfere with proteome analysis and whether protease inhibitors are useful for renal and urinary proteomics remains unclear. We therefore performed a systematic evaluation of the use of protease inhibitors in gel-based renal and urinary proteomics. Renal proteins were extracted from porcine kidney tissue and stored at -30 or -70 °C without protease inhibitors. After 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles, the 2-D proteome profile was examined. Differential spot analysis and ANOVA with Tukey posthoc multiple comparisons revealed significantly quantitative changes in intensity levels of 12 and 7 renal proteins that were stored at -30 and -70 °C, respectively, after g4 freeze-thaw cycles. Additionally, there were qualitative changes (vertical elongation or streak) in 6 and 1 renal proteins that were stored at -30 and -70 °C, respectively. All these changes could be successfully prevented by the addition of 1% (v/v) protease inhibitors cocktail prior to storage. In contrast, neither quantitative nor qualitative changes were observed in urine samples that were stored without protease inhibitors and processed as for kidney samples. From these data, the addition of protease inhibitors is highly recommended for gel-based renal proteomics, but no longer recommended for gel-based urinary proteomics. Keywords: Kidney • Protease • Protease inhibitor • Protein degradation • Proteolysis • Renal proteome • Urinary proteome • Urine
Introduction During the past decade, proteomics has been widely applied to various fields of medicine to better understand (patho)physiology and to explore the complexity of pathogenic mechanisms of human diseases. There is no exception in the nephrology field, in which renal and urinary proteomics has proven its great potential in the investigation of kidney diseases and in the development of new therapeutic targets.1,2 Moreover, urinary proteomics has become one of the most interesting subdisciplines in clinical proteomics, particularly for biomarker discovery not only in kidney diseases but also in nonkidney diseases.3,4 The rapid growth of renal and urinary proteomics has attracted a number of nephrologists and proteomists worldwide to apply proteomics to address clinical questions in the nephrology field. Consequently, two internationally collaborative networks, namely, the “Human Kidney and Urine Proteome Project” (HKUPP)5 (www.hkupp.org) and the “European Network for Kidney and Urine Proteomics” (EuroKUP)6 (www.eurokup.org) have been established to promote and facilitate clinical applications of renal and urinary * To whom correspondence should be addressed. Visith Thongboonkerd, M.D., FRCPT, Medical Proteomics Unit, Office for Research and Development, 12th Floor Adulyadejvikrom Building, 2 Prannok Road, Siriraj Hospital, Bangkoknoi, Bangkok 10700, Thailand. Phone/Fax: +66-2-4184793. E-mail:
[email protected] or
[email protected]. 10.1021/pr900015q CCC: $40.75
2009 American Chemical Society
proteomics. One of the very first goals of these two networks is to standardize protocols for sample collection, sample preparation and analysis of the kidney and urine proteomes. A major concern during collection and storage of biological samples is proteolysis or protein degradation caused by endogenous proteolytic enzymes, especially when the samples undergo repeated freeze-thaw cycles (due to sample preparation protocols and repetition of experiments). Addition of protease inhibitors had been initially recommended for prevention of such unwanted biological phenomenon in protein chemistry and proteomics studies. Protease inhibitors are molecules that block the function of proteolytic enzymes to degrade proteins. They are classified either by the type of targeted protease enzymes or by their inhibitory mechanisms. There are many protease inhibitors available for various types of protease classes (i.e., serine proteases, cysteine proteases, aminopeptidases, acid proteases, and elastases).7,8 Common protease inhibitors include PMSF (phenylmethylsulfonyl fluoride), AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride hydrochloride), EDTA (ethylenediaminetetraacetic acid) or EGTA (ethyleneglycoltetraacetic acid), peptide protease inhibitors (leupeptin, pepstatin, aprotinin, bestatin), benzamidine, and so forth. Nevertheless, there are some limitations and differences of efficiencies among these protease inhibitors.9 Generally, the protease inhibitors cocktail contains both peptides and Journal of Proteome Research 2009, 8, 3109–3117 3109 Published on Web 04/08/2009
research articles
Havanapan and Thongboonkerd 10
small-molecule inhibitors. Some of these inhibitors are native peptides (e.g., aprotinin) or modified peptides and amino acids (e.g., leupeptin, bestatin and pepstatin A), which serve as competitive, reversible inhibitors that bind to the active sites of proteases. Other inhibitors (e.g., AEBSF and PMSF) are competitive, irreversible inhibitors, which covalently attach to important amino acids in the active sites of the proteases.11,12 Despite their beneficial effects, on the other hand, protease inhibitors may interfere with proteome analysis. For example, some peptide protease inhibitors (e.g., aprotinin), which require microgram per milliliter (µg/mL) range of concentrations to work, can directly compete and interfere with the detection of proteins or polypeptides in the samples by mass spectrometry (MS) because they are also peptides.10 Additionally, AEBSF have been shown to form covalent bonds with proteins and interfere with 2-DE proteome analysis by shifting isoelectric point (pI) of proteins.12 On the basis of the data reported by the Human Plasma Proteome Project,10,12 the universal use of protease inhibitors is no longer recommended for plasma proteomics. The use of protease inhibitors is also one of the most debated issues in the renal and urinary proteomics arena.5,13 Previously, there was no systematic analysis to evaluate the necessity and appropriate concentration of protease inhibitors for analyses of kidney and urine proteomes. Therefore, a systematic evaluation of the use of protease inhibitors in renal and urinary proteomics is urgently needed. In this report, we performed a systematic analysis of the necessity and appropriate dosage of a protease inhibitors cocktail (PIC) for analyses of kidney and urine proteomes. Our data will be useful for the HKUPP and EuroKUP to draft an initial guideline and standardization of sample collection and preparation.
Materials and Methods Preparation and Storage of Kidney Tissue. A porcine kidney was bought from a local fresh poultry market. The tissue was then dissected into thin slices, washed with phosphate buffered saline (PBS), snap-frozen in liquid nitrogen, and ground into powder using prechilled mortar and pestle. Tissue powder was then resuspended in the solubilizing buffer containing 7 M urea, 2 M thiourea, 4% CHAPS (3-[(3 cholamidopropyl)dimethylamino]-1-propanesulfonate), 2% (v/v) ampholytes (pH 3-10), 120 mM dithiothreitol (DTT), and 40 mM Tris-base. The solution was centrifuged at 10 000g at 4 °C for 5 min to collect the supernatant. Protein concentration was measured using Bio-Rad protein assay, based on Bradford’s method.14 The sample was then divided into several 100-µg aliquots without or with various concentrations [0.1%, 0.5%, 1%, or 5% (v/v)] of a protease inhibitors cocktail (PIC) (Sigma-Aldrich; St. Louis, MO) containing 104 mM AEBSF, 1.53 mM pepstatin A, 1.4 mM E-64 (N-[trans-epoxysuccinyl]-L-leucine 4-guanidinobutylamide), 4.22 mM bestatin hydrochloride, 1.9 mM leupeptin hemisulfate salt, and 0.085 mM aprotinin. All these aliquots were frozen at either -30 or -70 °C for 23 h. The samples were then thawed at room temperature (25 °C) for 1 h and then refrozen for 23 h. A total of 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles were performed prior to 2-DE analysis. These experiments were performed in triplicate. Preparation and Storage of Urine. Midstream random urine samples were collected from 5 normal healthy individuals (3 females and 2 males), who had no recent medication. All females had no menstrual cycle at the time of collection. The samples were pooled and immediately subjected to low-speed centrifugation (1500g for 15 min) to remove cell debris and 3110
Journal of Proteome Research • Vol. 8, No. 6, 2009
particulate matters. Proteins in this urine pool were precipitated by 75% ethanol. Briefly, ethanol was added to the urine with a final concentration of 75%. The solution was mixed and incubated at 4 °C for 10 min and the precipitate was isolated by a centrifugation at 12 000g and 4 °C for 5 min. The supernatant was discarded and the pellet was allowed to airdry and then resuspended with the aforementioned solubilizing buffer. The protein solution was dialyzed against 18 MΩ · cm (dI) water at 4 °C overnight. The dialyzed urine was lyophilized and then resuspended with the same solubilizing buffer. Protein concentration was measured by Bradford’s method.14 The sample was then divided into several 100-µg aliquots without or with various concentrations [0.1%, 0.5%, 1%, or 5% (v/v)] of the same PIC as above. All these aliquots were frozen at either -30 or -70 °C for 23 h. The samples were then thawed at room temperature (25 °C) for 1 h and then refrozen for 23 h. A total of 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles were performed prior to 2-DE analysis. These experiments were performed in triplicate. 2-DE and Staining. Immobiline DryStrips (GE Healthcare, Uppsala, Sweden), linear pH 3-10 for urine samples15 and nonlinear pH 3-10 for kidney samples16 (7-cm long) were utilized. Each strip was rehydrated overnight with 100 µg of total protein from each sample aliquot that was premixed with a rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 2% (v/v) ampholytes (pH 3-10), 120 mM DTT, 40 mM Tris-base and bromophenol blue to make the final volume of 150 µL per strip. The first-dimensional separation (IEF) was performed in Ettan IPGphor II IEF System (GE Healthcare) at 20 °C, using a stepwise mode to reach 9083 Vh. After completion of the IEF, proteins on the strip were equilibrated with a buffer containing 6 M urea, 130 mM DTT, 30% glycerol, 112 mM Tris-base, 4% SDS and 0.002% bromophenol blue for 15 min, and then with another buffer containing 6 M urea, 135 mM iodoacetamide, 30% glycerol, 112 mM Tris-base, 4% SDS and 0.002% bromophenol blue for 15 min. The equilibrated IPG strip was then transferred onto 12.5% acrylamide slab gel and the second-dimensional separation was performed in SE260 Mini-Vertical Electrophoresis Unit (GE Healthcare) with a constant voltage of 150 V for 2 h. The separated protein spots were then stained with Deep Purple (GE Healthcare) fluorescence dye. Briefly, the slab gels were fixed in 7.5% acetic acid and 10% ethanol at room temperature for 1 h and then incubated in 200 mM Na2CO3 for 30 min. The solution was removed and 20 mL of Deep Purple was added to each gel and incubated on a continuous rocker at room temperature in the dark for 1 h. Fluorescence-stained proteins were visualized using a Typhoon laser scanner (GE Healthcare). 2-DE experiments were performed in triplicate using 3 independent replicates of the samples as described above. Totally, 168 gels derived from 168 independent (100-µg) aliquots of the samples were run and analyzed. Matching and Quantitative Intensity analysis. Image Master 2D Platinum (version 6.0) software was used for detecting, matching and analysis of protein spots on 2-D gels. Parameters used for spot detection were (i) minimal area ) 10 pixels; (ii) smooth factor ) 2.0; and (iii) saliency ) 2.0. A reference gel was created from an artificial gel combining all of the spots presenting in different gels into one image. The reference gel was then used for matching the corresponding protein spots among 2-D gels. The matched protein spots by an automatic matching were then confirmed and edited manually. Background subtraction was performed, and the intensity volume
research articles
Protease Inhibitors in Renal and Urinary Proteomics
Figure 1. The 2-D proteome map of significantly altered proteins in kidney samples frozen at -30 °C (A) and -70 °C (B) without protease inhibitors. Totally, 100 µg of proteins was resolved by 2-DE using nonlinear pH 3-10 IPG strip and then visualized with Deep Purple fluorescence dye. Significant quantitative and qualitative differences among 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles are indicated in circles and rectangles, respectively, with numbers that correspond to those reported in Tables 1-3. Their intensity levels and statistical data are summarized in Tables 1 and 3, respectively. Two protein spots (nos. 70 and 114) in (A) and one spot (no. 114) in (B) had both quantitative and qualitative changes; the latter were defined as those with altered spot shape (i.e., vertical elongation or streak) (see also Figure 4). All these significantly altered proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses (see Table 2).
of each spot was normalized with total intensity volume (summation of the intensity volumes obtained from all spots within the same 2-D gel). Statistical Analysis. To define significant differences among the different groups of samples (various freeze-thaw cycles), the intensity levels of corresponding (matched) protein spots were compared by ANOVA with Tukey posthoc multiple comparisons using SPSS software (version 10.0). P-values less than 0.05 were considered statistically significant. In-Gel Tryptic Digestion. The protein spots whose intensity levels significantly differed among groups were excised from 2-D gels, washed twice with 200 µL of 50% acetonitrile (ACN)/ 25 mM NH4HCO3 buffer (pH 8.0) at room temperature for 15 min, and then washed once with 200 µL of 100% ACN. After washing, the solvent was removed, and the gel pieces were dried using a SpeedVac concentrator (Savant; Holbrook, NY) and rehydrated with 10 µL of 1% (w/v) trypsin (Promega; Madison, WI) in 25 mM NH4HCO3. After rehydration, the gel pieces were crushed with siliconized blue stick and incubated at 37 °C for at least 16 h. Peptides were subsequently extracted twice with 50 µL of 50% ACN/5% trifluoroacetic acid (TFA); the extracted solutions were then combined and dried using the SpeedVac concentrator. The peptide pellets were resuspended with 10 µL of 0.1% TFA and purified using ZipTipC18 (Millipore; Bedford, MA). The peptide solution was drawn up and down in the ZipTipC18 10 times and then washed with 10 µL of 0.1% formic acid by drawing up and expelling the washing solution three times. The peptides were finally eluted with 5 µL of 75% ACN/0.1% formic acid. Protein Identification by Q-TOF MS and MS/MS Analyses. The trypsinized samples were premixed 1:1 with the matrix solution containing 5 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN, 0.1% (v/v) TFA, and 2% (w/v) ammonium citrate and deposited onto the 96-well MALDI (matrix-assisted laser desorption/ionization) target plate. The samples were analyzed by Q-TOF Ultima MALDI (Micromass, Manchester, U.K.) and instrumental settings were as described previously.16,17 Peptide mass fingerprinting (PMF) (for MS analysis) and MS/MS ions search were performed using the
MASCOT search tool (http://www.matrixscience.com) with assumptions that peptides were monoisotopic, oxidized at methionine residues, and carbamidomethylated at cysteine residues. The search was performed using the mammalian protein database of the NCBI (National Center for Biotechnology Information). Only 1 missed trypsin cleavage was allowed and mass tolerances of 100 and 50 ppm were used for MS and MS/MS analyses, respectively. MS scores >71 or MS/MS ions scores >34 were considered statistically significant (p < 0.05) (significant identification).
Results and Discussion The Kidney Proteome Profiles after 0-12 Freeze-Thaw Cycles without Protease Inhibitors. We examined the effect of freeze-thaw cycles on kidney proteome analysis. The extracted renal proteins were stored at either -30 or -70 °C for 23 h. The frozen samples were then thawed at 25 °C for 1 h and refrozen for 23 h. After 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles, the 2-D proteome profiles were examined for both quantitative (focusing on altered intensity levels, which infer changes in protein amount) and qualitative changes (focusing on new spots and altered spot shape; i.e., vertical elongation or streak, as proteolysis can cause fragmentation products and gradual changes in molecular size of the protein). Three replicated sets of the samples were analyzed by 2-DE. For the samples stored at -30 °C, a total of 500 ( 8 protein spots were detected in each 2-D gel using the predefined criteria of spot detection described in Materials and Methods. There were no significant differences in spot number detected among various conditions. Matching of protein spots across different gels revealed 92.98 ( 3.78% of matched spots among all these gels (coefficient of variation ) 4.06%). There were no significantly new spots (must be consistently found in all triplicated gels) of fragmentation products found or, if any, were under the detection limit of our study. Using a 2-D gel image analysis software and ANOVA with Tukey posthoc multiple comparisons, significant changes (p < 0.05) in abundance levels of 12 protein spots among different freeze-thaw cycles were Journal of Proteome Research • Vol. 8, No. 6, 2009 3111
research articles
Havanapan and Thongboonkerd
Table 1. Renal Proteins with Significant Quantitative Changes in Their Intensity Levels after Storage at -30°C without Protease Inhibitors and Repeated Freeze-Thaw Cycles intensity levels (× 105 pixels) (mean ( SEM) altered spots
0 cycle
2 cycles
4 cycles
6 cycles
8 cycles
10 cycles
12 cycles
p-value
spot 70 spot 114 spot 122 spot 123 spot 125 spot 126 spot 136 spot 176 spot 184 spot 234 spot 301 spot 370
0.282 ( 0.032f,g 0.380 ( 0.008 0.345 ( 0.004c-g 0.292 ( 0.012f 0.260 ( 0.019c,e-g 0.350 ( 0.019c,e 0.246 ( 0.017c 0.329 ( 0.011g 0.281 ( 0.011 0.354 ( 0.004e,g 0.377 ( 0.008e 0.186 ( 0.004f
0.248 ( 0.013 0.396 ( 0.004d 0.324 ( 0.008 0.279 ( 0.013 0.224 ( 0.016 0.288 ( 0.019 0.208 ( 0.007 0.352 ( 0.015c,e-g 0.278 ( 0.019 0.315 ( 0.007 0.364 ( 0.016e 0.123 ( 0.014
0.195 ( 0.039 0.398 ( 0.003d 0.269 ( 0.016a 0.737 ( 0.004 0.186 ( 0.009a 0.753 ( 0.012a 0.176 ( 0.009a 0.302 ( 0.014b 0.249 ( 0.005 0.270 ( 0.024 0.994 ( 0.025 0.393 ( 0.265
0.234 ( 0.020 0.340 ( 0.008b,c 0.285 ( 0.011a 0.250 ( 0.003 0.193 ( 0.009 0.279 ( 0.016 0.202 ( 0.009 0.311 ( 0.016 0.250 ( 0.013 0.284 ( 0.020 0.328 ( 0.020 0.138 ( 0.013
0.175 ( 0.011 0.364 ( 0.008 0.278 ( 0.008a 0.234 ( 0.017 0.166 ( 0.011a 0.265 ( 0.013a 0.226 ( 0.014 0.290 ( 0.016b 0.235 ( 0.009g 0.244 ( 0.023a 0.268 ( 0.016a,b 0.633 ( 0.234
0.094 ( 0.049a 0.369 ( 0.014 0.281 ( 0.012a 0.217 ( 0.008a 0.158 ( 0.008a 0.278 ( 0.003 0.200 ( 0.006 0.291 ( 0.014b 0.274 ( 0.011 0.260 ( 0.011 0.317 ( 0.018 0.576 ( 0.231a
0.103 ( 0.052a 0.354 ( 0.016 0.285 ( 0.016a 0.234 ( 0.022 0.186 ( 0.023a 0.299 ( 0.020 0.207 ( 0.018 0.284 ( 0.010b 0.294 ( 0.004e 0.236 ( 0.042a 0.301 ( 0.014 0.374 ( 0.226
0.0442 0.0119 0.0046 0.0177 0.0037 0.0171 0.0444 0.0465 0.0361 0.0280 0.0164 0.0477
a-g Any cycles to be significantly differed from 0, 2, 4, 6, 8, 10, and 12 cycles, respectively. All spots had p < 0.05 by ANOVA with Tukey posthoc multiple comparisons.
observed (labeled with circles in Figure 1A, whereas intensity data and statistically significant differences are summarized in Table 1). Additionally, there were 6 proteins whose spot shape was gradually changed from cycles 0 to 12; they became vertically elongated (labeled with rectangles in Figure 1A) (serial changes of these spots can be seen in Figure 4). Moreover, there were two proteins (spot nos. 70 and 114), which had both quantitative and qualitative changes (labeled with both circles and rectangles in Figure 1A). All these quantitatively and qualitatively altered proteins were then successfully identified by Q-TOF MS (PMF) and/or MS/MS (peptide sequencing) analyses. Table 2 summarizes identities, identification scores and other detailed information related to protein identification. For the samples stored at -70 °C, there were no significant changes in spot number observed among various conditions (that is, new spots of fragmentation products were not found or under the detection limit of our study). Quantitative intensity analysis and ANOVA with Tukey posthoc multiple comparisons revealed significant changes (p < 0.05) in abundance levels of 7 protein spots among different freeze-thaw cycles (labeled with circles in Figure 1B, whereas intensity data and statistically significant differences are summarized in Table 3). There was only one protein (spot no.114), whose spot shape was gradually changed from cycles 0 to 12 (became vertically elongated) and had both quantitative and qualitative changes (labeled with both rectangle and circle in Figure 1B). All the altered proteins observed in this set of samples were also significantly altered in the samples stored at -30 °C (Table 1). From the list of altered proteins identified by Q-TOF MS and/ or MS/MS analyses (Table 2), all of them have important roles in many biological processes, including tissue integrity, signal transduction, transcription/translation, innate immunity, molecular chaperoning, endoplasmic reticulum (ER)-stress response, and cellular metabolisms. Functional significance and potential mechanisms of proteolysis of some of these proteins by responsible proteases are discussed as follows. Collagen is one of the major extracellular matrix proteins of connective tissues in animals. The matrix metalloproteinases are a family of enzymes, which degrade components of the extracellular matrix, thereby regulating connective tissue turnover.18 The interstitial collagenase, a member of this family, is responsible for degradation of collagens within the extracellular matrix and essential for efficient restructuring and reconstructing the tissues.19 This enzyme is produced by various types of mam3112
Journal of Proteome Research • Vol. 8, No. 6, 2009
malian cells, such as fibroblasts,20,21 macrophages,22 endothelial cells,23 keratinocytes24 and chondrocytes.25 The sequence of collagenolysis in mammalian tissues begins when active collagenase attaches to individual triple-helical molecules of collagen (approximately 3/4 of the N-terminus) and cleaves the protein through the three polypeptide helical chains.26 Transferrin is a major iron-binding protein in vertebrate serum and also found in the kidney.27 Transferrin is suggested to be a natural substrate of the protease and there is evidence demonstrating that papain (a cysteine protease) and chymotrypsin can digest transferrin on the cell surface.28 Papain prevents entering of iron into cultured cells by limiting endocytosis of iron-bound transferrin.29 Protein disulfide isomerase (PDI), a homodimeric enzyme, catalyzes disulfide reactions, thereby facilitating the shuffling of the disulfide bonds in nascent proteins until they achieve their native pairing.30,31 PDI is a member of the thioredoxin superfamily and found mainly in ER of mammalian cells,32 but can be also found in non-ER locales.33 PDI is cleaved by several proteases, that is, serine protease (enterokinase) and cysteine protease (caspase).34 In addition, many proteins participating in cytoskeletal rearrangements, cell membrane blebbing, nuclear condensation and DNA fragmentation have been shown to be other substrates for one or more caspases.32 As the kidney is a tissue enriched with many proteases (including the aforementioned enzymes), it is not unexpected that repeated freeze-thaw cycles would aggravate the proteolytic activity of these proteases in degradation of other kidney proteins. Interestingly, our results showed that the quantitative and qualitative changes were observed obviously after g4 freeze-thaw cycles, whereas the pattern of protein spots at 2 freeze-thaw cycles remain unchanged as compared to the baseline (0 cycle) (Tables 1 and 3). Therefore, the samples that are subjected to no more than 2 freeze-thaw cycles may not require protease inhibitors, whereas those are subjected to >2 freeze-thaw cycles should be considered for the addition of protease inhibitors. It should be noted that we obtained the porcine kidney from the fresh poultry market. One should keep in mind that our sample collection, although fresh, might have a modest degree of difference (e.g., sample treatment, duration from surgical removal to laboratory), comparing to the perfect set up in the hospital where the human kidney tissue is taken from patients. However, there was no evidence of proteolysis observed in the
research articles
Protease Inhibitors in Renal and Urinary Proteomics
Table 2. Q-TOF MS and/or MS/MS Analyses of Renal Proteins with Quantitative and/or Qualitative Changes after Storage at -30 °C without Protease Inhibitors and Repeated Freeze-Thaw Cyclesa
spot no.
protein name
NCBI ID
no. of identification matched scores (MS, %cov (MS, peptides (MS, identified by MS/MS) MS/MS) MS/MS)
48 Type VI collagen alpha-1 chain
gi|92020094 MS, MS/MS
56, 72
13, 13
10, 1
50 Type VI collagen alpha-2 chain
gi|92020103 MS, MS/MS
79, 67
17, 27
12, 2
63 Pyruvate carboxylase
gi|47523756 MS, MS/MS
245, 210
31, 4
27, 4
70 Hexokinase 1 gi|184021 MS/MS 94 Eukaryotic translation elongation gi|109122950 MS factor 2
NA, 43 153, NA
NA, 1 20, NA
NA, 1 25, NA
114 Chain A, steric and conformational features of the aconitase mechanism 122 NADH dehydrogenase (ubiquinone) Fe-S protein 1
gi|157829964 MS, MS/MS
160, 207
35, 7
21, 4
gi|21704020 MS, MS/MS
85, 116
23, 5
15, 3
123 Transferrin
gi|833800
MS, MS/MS
215, 182
40, 8
24, 3
125 Transferrin
gi|833800
MS, MS/MS
138, 64
40, 5
23, 2
126 78 kDa Glucose-regulated protein precursor (GRP78) (Heat shock 70 kDa protein 5) (Immunoglobulin heavy chain-binding protein) (BiP) 136 Heat shock 70 kDa protein 9 (mortalin) 176 ATP synthase, H+ transporting, lysosomal 56/58 kDa, V1 subunit B1 isoform 1 184 Protein disulfide-isomerase precursor (PDI) (Prolyl 4-hydroxylase subunit beta) 234 S-adenosylhomocysteine hydrolase 301 Pyruvate dehydrogenase:SUBUNIT)beta 370 3-Hydroxybutyrate dehydrogenase, type 2 (predicted), isoform CRA_b
gi|121570
MS, MS/MS
256, 233
45, 12
26, 5
gi|12653415 MS/MS
NA, 38
NA, 2
NA, 1
gi|109103302 MS
113, NA
39, NA
16, NA
gi|129729
NA, 163
NA, 6
NA, 2
gi|58801555 MS, MS/MS
97, 36
34, 4
13, 2
gi|448581
77, 77
34, 13
12, 3
NA, 95
NA, 10
NA, 2
MS/MS
MS, MS/MS
gi|149026006 MS/MS
∆ mass (ppm) of individual peptides (MS); (MS/MS)
pl
MW (kDa)
(6, -94, -11, -2, -9, 19, 36, 35, 10, 7); (-2) (16, -31, -72, 0, 37, 55, 28, 34, -3, -4, 13, -49); (-9, -0) (11, 18, -7, -22, 3, 16, -2, 8, -7, -2, -73, -21, -87, 5, -5, -2, 4, 5, -11, 2, 4, 7, 14, -4, 8, 4, 26); (3, 9, 7, 15) (NA); (3) (-6, -7, -58, -51, 25, -8, -26, -4, -12, 16, 26, -19, -26, -11, 10, 28, -1, -4, 5, -6, -10, -4, -5, 0, -9); (NA) (5, 5, -1, -1, -9, 0, -5, -1, 5, 15, 0, 1, 4, 3, 1, 2, -15, -38, 4, 1, 2); (-5, -1, 0, 1) (2, -6, -9, 20, 32, 3, -30, -5, -3, -6, -3, -23, -0, 55, 34); (-6, -4, -7) (-13, -20, 3, -8, 5, 17, -36, -13, 9, 4, -4,-6, 12, 0, -0, -4, 14, 48, 6, 16, 2, 11, -9,-3); (-6, 2, -3) (1, -20, 8, -11, 6, -7, 5, 5, -4, 11, 1, 2, -1, 22, 44, 10, 22, 6, 4, 13, 14, -0, -1); (-4, 5) (-71, -2, 3, 2, 5, -11, -5, -0, -12, 0, -14, -31, -5, 1, 1, -2, 10, -2, -3, -5, -0, -1, -6, 1, 7, -25); (3, 0, -5, -2, -1) (NA); (0)
8.26 127.15
(-56, 6, -4, -6, -7, 1, -9, -16, 1, 4, -8, -1, -6, -3, -38, -16); (NA) (NA); (5, -6)
5.44 55.45
8.90 112.63 6.36 130.27
6.44 103.58 9.40 157.37
7.16 83.29 5.51 80.73 6.73 78.95
6.73 78.95 5.07 72.51
6.03 73.97
4.79 57.51
(-9, 0, 12, 31, 4, 9, -1, -5, -5, 5.88 48.18 -5, 0, 2, 0); (-11, 0) (9, 16, -3, -9, 2, 1, 7, -4, -7, 5.38 36.11 -3, -0, 0); (-3, -11, -3) (NA); (-0, -1) 6.94 26.98
a NCBI ) National Center for Biotechnology Information, %cov ) %Sequence coverage [(number of the matched residues/total number of residues in the entire sequence) × 100%], NA ) not applicable.
Table 3. Renal Proteins with Significant Quantitative Changes in Their Intensity Levels after Storage at -70°C without Protease Inhibitors and Repeated Freeze-Thaw Cycles intensity levels (× 105 pixels) (mean ( SEM) altered spots
0 cycle
2 cycles
4 cycles
6 cycles
8 cycles
10 cycles
12 cycles
p-value
spot 114 spot 122 spot 123 spot 125 spot 184 spot 234 spot 370
0.294 ( 0.027d,e 0.268 ( 0.022 0.273 ( 0.014g 0.240 ( 0.015e-g 0.278 ( 0.005c-g 0.270 ( 0.016f,g 0.219 ( 0.010d,f
0.336 ( 0.006 0.281 ( 0.008e,f 0.264 ( 0.004 0.223 ( 0.033f,g 0.226 ( 0.016 0.202 ( 0.029 0.144 ( 0.026
0.323 ( 0.005 0.209 ( 0.038 0.245 ( 0.001 0.238 ( 0.014e-g 0.149 ( 0.016a 0.215 ( 0.023 0.143 ( 0.020
0.372 ( 0.015a,g 0.284 ( 0.019e,f 0.263 ( 0.025 0.209 ( 0.011 0.162 ( 0.027a 0.222 ( 0.017 0.124 ( 0.004a
0.360 ( 0.014a,g 0.162 ( 0.024b,d 0.238 ( 0.017 0.171 ( 0.014a,c 0.154 ( 0.013a 0.183 ( 0.011 0.133 ( 0.013
0.327 ( 0.018 0.166 ( 0.027b,d 0.217 ( 0.005 0.163 ( 0.020a-c 0.176 ( 0.016a 0.158 ( 0.004a 0.119 ( 0.018a
0.287 ( 0.023e 0.192 ( 0.007a 0.201 ( 0.004a 0.158 ( 0.010a-c 0.175 ( 0.029a 0.175 ( 0.012a 0.131 ( 0.027
0.0360 0.0500 0.0150 0.0220 0.0040 0.0140 0.0330
a-g Any cycles to be significantly differed from 0, 2, 4, 6, 8, 10, and 12 cycles, respectively. All spots had p < 0.05 by ANOVA with Tukey posthoc multiple comparisons.
porcine kidney sample up to 2 freeze-thaw cycles, indicating that there was no proteolysis occurring during the sample collection and transfer. Hence, our data should be also applicable to the human kidney. Successful Prevention of Proteolysis in Kidney Samples by Protease Inhibitors. To examine whether the addition of protease inhibitors could prevent quantitative and qualitative
changes of those altered proteins shown in Figure 1, we performed a parallel experiment and added a protease inhibitors cocktail (PIC) into the samples prior to storage at both -30 and -70 °C. All analyses were performed as for the experiments without protease inhibitors. A PIC from SigmaAldrich contains common protease inhibitors (both small molecular compounds and peptides) and is widely used in Journal of Proteome Research • Vol. 8, No. 6, 2009 3113
research articles
Havanapan and Thongboonkerd
Figure 2. The kidney proteome profiles after 0-12 freeze-thaw cycles of the samples stored at -30 °C without (A) and with (B) 1% (v/v) PIC. Totally, 100 µg of proteins was resolved by 2-DE using nonlinear pH 3-10 IPG strip and then visualized with Deep Purple fluorescence dye. N ) 3 gels per condition derived from 3 independent aliquots; total number of gels in this set of experiment ) 42. Image Master 2D Platinum software (GE Healthcare) and ANOVA with Tukey posthoc multiple comparisons were performed to analyze protein spots across different gels (see Tables 1 and 4).
proteomics studies.35-38 However, the appropriate concentration of this PIC for prevention of proteolysis in kidney tissuederived samples remains unclear. We thus used various concentrations (0.1%, 0.5%, 1%, and 5% (v/v)) of PIC to screen for its minimal concentration that could efficiently suppress the protease activity. 2-DE analysis comparing intensity levels of the altered proteins reported in Tables 1 and 2, without and with various concentrations of PIC, suggested that the concentration at 1% (v/v) of Sigma-Aldrich PIC was an optimum in our setting (data not shown). Thus, this concentration was used throughout our present study. The addition of 1% (v/v) PIC could efficiently prevent both quantitative and qualitative changes of all the altered proteins detected in both sets of samples stored at -30 and -70 °C for at least 12 freeze-thaw cycles (as this was the maximal repeated freeze-thaw cycle performed in our study). Figure 2 illustrates, side-by-side, the 2-D proteome profiles of kidney samples stored at -30 °C without (Figure 2A) and with 1% (v/v) PIC (Figure 2B), whereas Figures 3 and 4 show magnified images of the altered protein spots to demonstrate the inhibitory effects of PIC for prevention of quantitative and qualitative changes, respectively. Also, Table 4 shows the intensity data of 12 protein spots with significant quantitative changes in the samples without PIC (see Table 1), but had no significant differences detected by ANOVA with Tukey posthoc multiple comparisons (p values were not significant) after the addition of 1% PIC, indicating successful prevention by PIC. We found no effects of the PIC on 2-DE analysis of kidney proteome as the small molecules and peptides presented in PIC were too small to be resolved and visualized in 2-D gel (generally, 2-DE has a limitation for proteins with molecular masses
