Differential Proteomic Shotgun Analysis Elucidates Involvement of

Differential Proteomic Shotgun Analysis Elucidates Involvement of Water Channel Aquaporin 8 in Presence of r-Amylase in the Colon Sameh Magdeldin,*,†,‡ Huiping Li,† Yutaka Yoshida,† Ichiro Satokata,† Yoshitaka Maeda,§ Munesuke Yokoyama,§ Shymaa Enany,†,| Ying Zhang,† Bo Xu,† Hidehiko Fujinaka,† Eishin Yaoita,† and Tadashi Yamamoto† Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Japan, Department of Physiology, Faculty of Veterinary Medicine, Suez Canal University, Egypt, Animal Resource Branch, Center of Bio-Based Researches, Brain Research Institute, Niigata University, Japan, and Department of Microbiology and Immunology, Faculty of Pharmacy, Suez Canal University, Egypt Received July 29, 2010

Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC-MS alignment based approach, we compared wildtype and AQP8 knockout mice colon proteomes. Shared result between both experiments revealed down-regulation of R-amylase 2 in AQP8-deleted mice model. Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in R-amylase 2 regulation. Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating R-amylase 2 synthesis by colonic epithelial cells as well. Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts. Keywords: Aquaporin 8 • knockout • colon • proteomics • amylase

1. Introduction Aquaporins (AQPs) are ubiquitous membrane water channel proteins expressed in various tissues.1,2 At present, 13 AQPs are known and classified into 2 subgroups: water transporters (aquaporins) or also glycerol and other small solutes transporters (aquaglyceroporins).3 Among this family, at least 6 aquaporin isoforms are known to be expressed in the digestive tract and its associated glands.4,5 Since cloning of aquaporin 8 (AQP8), it was reported to be widely distributed in the absorptive epithelial cells of duodenum, jejunum, colon,6-9 as well as salivary gland, pancreatic acini, and hepatocytes.10,11 Recently, AQP8 was implicated in several physiological processes rather than modulating water and solutes movements across cell membrane, for instance, spermatogenesis,12 bile content rearrangement,13 and apoptosis.14,15 These observations refute the only confinement of AQP8 in transmembrane water and solutes movement. Moreover, despite all these functional evidence, it is still unclear how AQP8 achieves its functionality and what proteins are regulated. To disclose this question, we * Correspondence to: Sameh Magdeldin, Department of Structural Pathology Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Japan. E-mails: samehmagd@ med.niigata-u.ac.jp; [email protected]. † Department of Structural Pathology, Niigata University. ‡ Department of Physiology, Suez Canal University. § Animal Resource Branch, Niigata University. | Department of Microbiology and Immunology, Suez Canal University. 10.1021/pr100789v

 2010 American Chemical Society

comprehensively compared wild-type and AQP8 knockout mouse (AQP8 KO) colons using different analytical and comparative platforms. The impetus of this study is to focus on the identification of possible regulatory proteins as a result of knocking out AQP8.

2. Experimental Section 2.1. Animals. Twelve C57BL/6J male mice (8 weeks old) bred at the animal Care Research Center (Niigata University, Japan) were used in the current study (n ) 6/group). All animals were treated following the statement of animal use according to the animal center committee at school of medical and dental sciences, Niigata University. 2.2. Generation of Aquaporin 8 (AQP8) Knockout Mice. The targeting vector was constructed using a 14-kb genomic fragment containing the full-length AQP8 gene. In targeted allele of AQP8, as shown in Figure 1, the 3.8 kb fragment containing a partial sequence of exon II, and full sequence of exons III and IV, was substituted with 4.8 kb sequence encoding galactosidase gene (LacZ) and neomycin phophotransferase (Neo) expression cassettes. The coding sequence of diphtheria toxin A (DT-A) was inserted upstream for the negative selection. Targeting vector was linearized at the downstream Sac II site and electroporated into J1 Embryonic Stem (ES) cells (generous gift from Dr. Satokata, Niigata University). Targeted ES cells were microinjected into PC 2.5 day 8-cell morula stage zygotes. Journal of Proteome Research 2010, 9, 6635–6646 6635 Published on Web 10/12/2010

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Figure 1. Schematic diagram of AQP8 gene interruption. (A) Restriction map of mouse AQP8 gene. AQP8 exons tagged (I to VI). The upstream and downstream untranslated sequences were shown as blank box. SC, Starting codon. (B) Construct of the targeting vector. A 3.8 kb fragment was replaced with galactosidase gene (LacZ) derived from pSV-LacZ vector and neomycin cassette (Neo) derived from pMC1-neo vector. A diphtheria toxin A sequence (DT-A) was inserted upstream of the targeting gene for negative selection. (C) After homologous recombination, partial exon II, and full sequence of exon III and IV of AQP8 gene were disrupted. (Lower panel) Verification of successful deletion of AQP8 gene and corresponding protein: (a) on genomic level (tail DNA extract), (b) successful interruption of AQP8 gene with LacZ insert, (c) on transcriptional (mRNA) level, and (d) protein level. Further details can be found in ref 16. Table 1. Comparative Table of Experimental Procedures Used in the Current Study

Number of experimental animals Method of protein digestion Design of analysis Total lc-Ms runs Solvent Gradient Acquisition time Mode of analysis File type Merging process Processing and comparison Principle of comparison

experiment A (in-gel digestion)

experiment B (in-solution digestion)

3/group In-gel digested samples Single analysis/sample [14 slice] 84 [14 × 6] acetonitrile 7.5-70% B gradienta 140 min conventional modeb mgf files mascot daemon (V 2.0) spreadsheet with integrated visual basic macro all and none strategy

3/group In-solution digested samples 4 replicate analysis/sample 24 [4 × 6] acetonitrile 7.5-70% B gradient 140 min IBAc mode/4 replicates m/z XML files aggregated by progenesis LC-MS progenesis LC-MS abundance based on sum of peak area within isotope boundaries

a Gradient B [98% acetonitrile/2% H2O in 0.1% formic acid]. b Conventional mode is a dynamic exclusion and repeat tandem setting allowing each ion to be selected once and excluded in the subsequent parent ion selection within run. c Information-based acquisition mode, intelligent education and program setting allows selecting of parent ion only once within replicate runs of the same sample.

After overnight culture, the microinjected blastocytes were transplanted into pseudopregnant C57BL/6J female mice. Chimeric mice were bred with C57BL/6J mice. Finally, the presence of truncated AQP8 was confirmed on genomic, transcriptional, and translational levels. Further details for constructing the targeting vector of AQP8 knockout model were precisely discussed elsewhere.16 2.3. Extraction and Preparation of Colon Proteins. Mice were sacrificed and decapitated. Colon was quickly removed, 6636

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longitudinally cut, and rinsed in ice-cold PBS buffer. Tissues were sliced into small pieces, immediately frozen in liquid nitrogen, and stored at -80 °C until usage. For protein extraction, approximately 100 mg of wet tissue was homogenized in Destreak rehydration solution (9.8 M urea, 2% nonidet, 0.2% Pharmalyte; pH 3-10, 12 µL/mL Destreak buffer, 0.5 µg/mL E-64, 0.5 mM PMSF, 40 µg/mL TLCK, 1.0 µg/µL chymostatin, 0.5 mM EDTA, 0.01% bromophenol blue, and 2 µg/µL aprotonin). Samples were homogenized using Polytron

Comparative Study of AQP8 Knockout Mouse Colon

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Figure 2. Illustration of ‘All and None’ comparative strategy. (A) Principle depends mainly on selecting protein candidates appearing in all replicate runs of one group assigned for comparison [all] but not in any replicate of the counterpart group [none] followed by refining based on its peptide confidence. Presence or absence of protein candidate is controlled by many factors, for most, abundance of given protein and corresponding peptides in the sample (other factors such as peptide ionization, protein extraction efficiency, complexity of the sample, and sensitivity of mass spectrometry are also considerable). As shown, protein A with high abundance will yield an ample amount of peptides, increasing the chance to be captured for MS/MS fragmentation and finally appear in the final search report and vice versa in case of protein B. (B) For refining most significantly regulated protein candidates, replicates of same group were compared with each other to generate shared protein list appearing in all replicates (zone A and B) as shown in step 1. In step 2, protein candidates appearing in all replicates of experimental groups were compared with each other producing (zone S), protein candidates present in all replicates of wild-type but not in counterpart, and (zone U), protein candidates present in all replicates of AQP 8 knockout but not in counterpart. In step 3, these lists (S and U) will be compared with other lists of proteins appearing either once or as shared between 2 samples of the same group. This will generate a final candidate list of proteins appearing in all individual of one experimental group [all] but never shown elsewhere in counterpart individuals [none].

PT1200 homogenizer (Kinematica AG, Switzerland), 5-10 bursts with 45 s interval in ice. Homogenized samples were kept in 37 °C for 1 h with occasional vortexing and centrifuged at 12 000 rpm for 20 min. Protein assay for the extracts was carried out using Ramagli’s modified method of Bradford (BioRad, Japan) with bovine serum albumin as a standard.17 2.4. In-Gel Trypsin Digestion [Experiment A]. Ten micrograms of colon protein extract from each sample was run on 12.5% SDS-PAGE. Gel was stained with Coomassie Brilliant Blue stain (CBB R-250, Wako, Japan). Each lane was sliced into 14 slices (2 cm/slice). Samples were reduced with 10 mM dithiothreitol (DTT), alkylated with 55 mM iodoacetamide (IAA), and digested with 6 ng/µL of trypsin overnight.18 Peptide was extracted with 0.3% formic acid and 5 µL (0.25 µg digested protein) from each sample was loaded onto nano-LC-ESI-ITTOF-MS/MS (Hitachi NanoFrontier LD, Tokyo, Japan).

2.5. In-Solution Trypsin Digestion [Experiment B]. In parallel, a gel- free based digestion approach was conducted following Mawuenyega et al.19 with modification. In brief, protein extract was precipitated with cold acetone and dissolved in 7 M guanidine-HCl buffer with 500 mM Tris-HCl (pH 8.0) containing 10 mM EDTA. The preparations were reduced by addition of 1 mM DTT and alkylated with 10 mM IAA. The S-carbamoylmethylated proteins were further precipitated by methanol/chloroform method to remove excess reagents. Pellets were then dissolved in 6 M urea in 100 mM Tris HCl (pH 8.8), sonicated, and diluted to final concentration 1 M urea with 100 mM Tris-HCl, (pH 8.8). Samples were digested overnight at 37 °C with proteomic grade trypsin (Sigma-Aldrich, St. Louis, MO) at an enzyme-substrate ratio of 1:50 (w/w). The digests were acidified to pH 2 by the addition of an aliquot of trifluoruacetic acid (TFA) and used directly for LC-MS/Ms Journal of Proteome Research • Vol. 9, No. 12, 2010 6637

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Figure 3. Workflow of experiment A (in-gel digestion). (A) CBBR-250-stained gel image of wild-type (lanes 2, 3, and 4) and AQP8 KO (lanes 5, 6, and 7) samples were run on 12.5% SDS-PAGE. Each lane was loaded with 10 µg of protein extract of colon and subsequently sliced into 14 slices (2 cm/slice). Lanes 1 and 7 are high molecular weight marker (BioRad, Hercules, CA). (B) Overlapped Venn diagram showing unique and shared proteins (numbers and percentage) between replicates of same group. Percentage of each sector is shown in relation to the whole sample in color [step 1]. (C) Represents shared and unique proteins between wild-type and AQP8 knockout common hits [step 2]. Table 2. List of Significantly Differential Protein Candidates in Experiment (A) ‘In-Digestion’ before Peptide Refining accession number

c

protein name

gene symbol

protein massa

pI

58065 113457 57422 16183 40284

7.18 5.39 4.77 7.9 8.09

38.8 17.2 42.4 18.4 41

35.6 17 52.8 21.1 40.5

15.6 12.9 18.9 12.9 32.9

26 13 32 19 56

13 13 30 7 40

4 9 12 6 21

Up-Regulatedc 112 55 93 71 63 41 595 382 184 57 45 56 42 43 56

29633 12514 22594 29159 34920

6.89 4.89 8.97 6.82 9.04

46.5 10.1 85.8 15.7 31.4

39.6 14.7 69 25.2 14.3

25.8 14.7 70.5 12 31.4

20 17 55 5 12

13 20 33 5 4

10 12 30 3 11

protein score

IPI00133544* IPI00420569 IPI00122815* IPI00555131 IPI00849879

Amylase 2 Na-K ATPase 1R2 Prolyl 4 hydrolase Hemoglobin subunit epsilon y2 Similar to GAPDH

Down-Regulatedb Amy2 206 239 40 Atp1a2 52 62 48 P4hb 285 346 133 Hbb-y 84 41 101 822 778 156

IPI00221890* IPI00551282* IPI00754071 IPI00153234 IPI00807902*

Carbonic anhydrase 3 Similar to Lacremal binding protein delta Perodoxin 6 Pyrroline 5 carboxilate reductase 3 Mitochondrial carrier homologue

Car3 Scgb2b1 Prdx6 Pycrl Mtch2

protein coverage

peptide match

a Shown in daltons (Da). b Down-regulated protein candidates were detected in wild-type samples but not shown anywhere in counterpart group. Up-regulated protein candidates were detected only in AQP8 knockout samples but not shown any where in counterpart group.

analysis. Efficiency of protein digestion was confirmed by separating assigned samples before and after trypsinization on SDS-PAGE (Supplement 1 in Supporting Information). For both experiments, digested peptides were purified and concentrated on a trap column, monolith trap C18-50-150 (Merck, Darmstadt, Germany). Peptides were separated using the separation column, monocap for Fast-Flow, 0.05 × 150 mm. The injected peptides were eluted as shown in Table 1 at 200 nL/min. Nano-LC-ESI-IT-TOF-MS/MS was performed on the top of two ions in each MS scan. Dynamic exclusion and repeat settings ensured each ion was selected only once and excluded in the subsequent parent ion selection. Precursor ions were selected using the following MS to MS/MS switch criteria: ion range m/z 100-1800, charge state 2-5, and former target ions were excluded for 20 ms. Colloidal ion dissociation (CID) was performed using nitrogen as collision gas. For experiment B, LC-MS analysis was allowed for intelligent exclusion program6638


ming using information-based acquisition mode (IBA).20 Each sample was analyzed in 4 replicates. Data were merged using Mascot daemon (V 2.0) or aggregated using progenesis LC-MS for experiment A and B, respectively. 2.6. Protein Identification by MS/MS Ion Search. For both experiments, peak lists were generated using NanoFrontier LD data processing software (V 1.0). Product ion data were searched against Mouse International protein index (IPI_ mouse; version 3.71, 169 347 entries) using a locally stored copy of the Mascot search engine (version 2.2.1, Matrix Science, London, U.K.).21 The following parameters were used for database search: MudPIT scoring, precursor mass tolerance 0.3 Da, product ion mass tolerance 0.3 Da, 2 missed cleavages allowed, fully tryptic peptides only, fixed modification of Carbamoidomethyl (C), variable modifications of glutamine (Gln) to pyroglutamate (pyro-Glu) (N-term Q); glutamate (Glu) to pyroglutamate (pyro-Glu) (N-term E), Oxidation of histidine


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Figure 4. Differentially expressed protein hits between wild-type and AQP8 knockout groups in “in-gel” experiment. (A) Number of peptide hits for up-regulated (5) and down-regulated (5) candidates in replicate runs before refining. LBP, lacrimal binding protein. MCH, mitochondrial carrier homologue. Down-regulated protein hits appeared only in wild-type replicates but not at any run of counter parts. Up-regulated proteins appeared in AQP8 knockout mice replicates but not at any run of counter parts. (B) Number of unique top ranked peptides above identity and homology threshold of mascot algorithm (MOWSE) for down-regulated (appeared only in wildtype) and up-regulated (appeared only in AQP8 knockout) protein candidates.

and tryptophan (HW); Oxidation of methionine (M), mass values of monoisotopic and peptide charge state of 2+ and 3+. Protein was accepted if at least two peptides passed the identity and homology threshold of Mascot (MOWSE) algorithm. The false discovery rate (FDR) against decoy database was below 2%. 2.7. Comparative Strategy. 2.7.1. All and None Strategy [Experiment A]. We have created a simple, macro visual basic “VBA” code embedded in Microsoft spreadsheet that enables high-throughput assessment and comparison of large-scale proteomic outputs (available in Supplement 2). The principle of comparison is shown in Figure 2 and we termed “all and none strategy” which relies mainly on selecting protein candidates appearing in all replicate runs of one group assigned for comparison [all] but not in any replicate of the counterpart

group [none]. Following this step, selected protein candidates were further refined based on its peptide profiling. This inclusion should fulfill the presence of at least 2 unique top ranked peptides (marked bold red in mascot search) with a score above the identity and homology threshold of (MOWSE) mascot algorithm (high confidence). Therefore, the all and none strategy provides a high stringent approach and paradigm for selecting most significantly differential protein candidates between 2 groups assigned for comparison based on its abundance and further refined based on peptide scoring. The main advantages of all and none approach are that it finally selects candidates which are expected to be unambiguously significant between both experimental groups with multiple folds [high expression in one group that is shown in all replicates and low or absence in counterpart that is not shown Journal of Proteome Research • Vol. 9, No. 12, 2010 6639

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Figure 5. (A) Simulated two-dimensional (2D) view of mass spectra using Progenesis LC-MS software. Horizontal axis represents retention time (min) and vertical axis represents mass to charge ratio (m/z). (B) Venn diagram showing data refinement strategy of in-solution experiment. Raw data (red) was filtered to remove variant mass spectra (outliers) within same group with P < 0.0001. Outputs (orange) was then refined again based on peptide charge [+2 to +6]. Output data (yellow) were finally subjected for comparison between both experimental groups and segregated into 4 categories of significance as shown in the inner pie (white). Significantly differential mass spectra are shown in numbers and percentage.

in all replicates] as a feature of a biomarker and thus limits final output result from unimportant hits that usually mask the appropriate significant candidate. In contrast, it ignores differentially expressed candidates which are detected in both experimental groups. 2.7.2. Quantitative Label- Free Comparison of Mass Spectra [Experiment B]. Mass spectra of IBA mode replicates runs were converted into a simulated 2D gel view based on retention time and mass to charge (m/z) using progenesis LC-MS software (Nonliner Dynamics, TX). All images/group were aligned automatically, then manually. A set of inclusion criteria was further assigned to eliminate false positive outputs resulting from noise or low reliable mass peaks. These criteria 6640


include (1) initial selection of mass peaks with p < 0.0001 between replicates of the same group to eliminate significantly different biased outputs resulted from experimental error (type I error); (2) mass peaks charged between +2 and +6; (3) statistical significance (ANOVA) of assigned peaks between experimental groups with probability of 95% (p < 0.05); (4) Peptide MS/MS ion search set to a significant threshold with p < 0.03 (MOWSE algorithm) and false discovery rate (FDR) against decoy database with less than 2%. Peptide abundance was calculated as the sum of peak areas within the isotope boundaries. Finally, peptides which passed all inclusion criteria were then normalized based on calculating their quantitative abundance ratio compared to reference run to determine their


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Figure 6. Significance of R-amylase 2 between wild-type and AQP8 knockout groups in experiment B “in solution digestion”. (A) Normalized abundance volume of 19 peptides related to R-amylase 2. Horizontal axis represents IBA mode replicates for wild-type and AQP8 knockout groups. Vertical axis represents normalized abundance volume (log). (B) Average normalized abundances of R-amylase 2 protein in wild-type and AQP8 knockout groups. ***P < 0.001. (C) Representative three-dimensional (3D) views of differentially significant R-amylase peptides between wild-type (red) and AQP 8 knockout groups (blue). Number on the top left panels represents peptide IDs. (D) Heat map analysis of differentially significant peptides between experimental groups. Horizontal axis represents IBA mode replicate runs, vertical axis represents peptide IDs (tagged by software). Arrows represent 3 down-regulated R-amylase peptides between wild-type (left) and AQP8 knockout (right) groups. Color panel represents peptide intensity. Heat map analysis was generated using VBA code (available in Supplement 2).

global scaling factor and expressed as antilog of the average of the log (ratios). 2.8. Immunoblot and Immunocytochemical Analysis. Twenty micrograms of protein extract for R-amylase 2 and 10 µg for GAPDH diluted in 2× SDS sample buffer [0.125 M TrisHCl (pH 6.8), 4% SDS, 20% (w/v) glycerol, 0.01% bromophenol blue, and 10% β mercaptoethanol] were electrophoresed on 12.5% SDS-polyacrylamide gel, and the bands were transferred to a polyvinylidene difluoride (PVDF) membrane (ImmobilonP, Millipore Corp., Bedford, MA). PVDF membranes were then immersed in blocking buffer (10% nonfat milk, 0.05% Tween

20, 0.5% NaN3 in PBS) for 1 h. For primary antibody incubation, 0.25 µg/mL mouse monoclonal antibody targeting amino acid sequence 212-492 of pancreatic amylase 2 (Santa Cruz Biotechnology, CA) or 0.25 µg/mL mouse monoclonal antibody to GAPDH (Ambion, Applied Biosystem, Japan) was used. After several washes in 0.05% Tween 20 PBS, membranes were incubated for 1 h with goat anti-mouse immunoglobulins conjugated to peroxidase-labeled dextran polymer (EnVision, DAKO, Japan). Reaction was visualized by ECL chemiluminescence kit (Amersham Pharmacia Biotech, Tokyo). For immunohistochemistry (IHC), tissue sections (4 µm) were deparafJournal of Proteome Research • Vol. 9, No. 12, 2010 6641

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Table 3. List of Differentially Significant Peptides of R-Amylase 2 in Experiment B

a

ID

score

mass

RT (mins)

charge

ANOVA

abundance

peptide sequence

1114 4931 296 357 1050 693 252 698 1561 1776 2860 2100 2463 2526 3103 1232 5626 9239 5647

41.3 29.73 30.2 51.2 27.72 38.15 34 37.9 38.1 26.9 33.3 29.2 30.4 26 27.4 28.8 12.8 19.2 26.4

1475.689 2135.132 1270.713 1889.979 730.421 1364.796 1426.737 1630.839 1696.861 1471.718 1426.727 1717.986 1898.886 1898.881 3409.611 1184.728 2272.192 2272.187 1829.818

0.386 1.24 1.07 1.35 0.493 1.08 0.511 1.08 1.13 1.05 0.511 1.24 1.22 1.22 1.09 1.17 1.14 1.11 0.668

3 3 2 3 2 3 3 3 3 2 2 3 3 2 3 2 3 3 2

P < 0.01 P < 0.05 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.05 P < 0.01 P < 0.0001

473 110 2.81 × 103 3.18 × 103 902 900 2.63 × 103 1.86 × 103 1.36 × 103 860 713 735 583 958 837 782 142 121 312

WRQIRNMVAFR MYKMAVGFMLAHPYGFTR TAIVHLFEWR VADYMNHLIDIGVAGFR WVDIAK AVLDKLHNLNTK ALVFVDNHDNQR GHGAGGSSILTFWDAR MAVGFMLAHPYGFTR NWGEGWGLVPSDR ALVFVDNHDNQR LTGLLDLALEKDYVR EFPAVPYSAWDFNDNK EFPAVPYSAWDFNDNK IYVDAVINHMCGAGNPAGTSSTCGSYLNPNNR LTGLLDLALEK AHFSISNSAEDPFIAIHADSK AHFSISNSAEDPFIAIHADSK CNGEIDNYNDAYQVR

a

modifications Oxidation (M) Oxidation (HW)

Oxidation (HW)

Oxidation (HW)

Oxidation (HW)

ID, peptide identification number; RT, retention time. (M), Oxidation of methionine. (HW) Oxidation of histidine and tryptophan.

Figure 7. Annotated tandem mass spectrometry (Ms/Ms) of the [M+2H]2+ ion of TAIVHLFEWR tryptic peptide digest of alpha amylase 2 showing b- and y- ions. Shaded boxes represent peaks detected by Ms/Ms fragmentation.

finized, rehydrated, and incubated with previously described primary (1:500) and secondary antibodies. Staining was visualized using horseradish peroxidase activity in the presence of 3,3′diaminobenzidine (DAB) and nuclei were counterstained with hematoxylin prior to imaging.

3. Results and Discussion 3.1. In-Gel Trypsin Digestion [Experiment A]. So far, this is the first comprehensive proteomic attempt to investigate differentially regulated proteins in AQP8 KO mice. In this experiment, we sought to speculate the most significant protein candidates by comparing wild-type and AQP8 KO strains. However, due to the large data outputs produced by comprehensive proteomic studies that usually mask the appropriate candidate of interest, we created a simple and robust refining methodology for selecting most reliable and significant candidates. As shown in Figure 3A, samples were prefractionated on SDS-PAGE, processed as previously described, and analyzed by LC-MS/MS. Replicate runs of both experimental groups 6642


yielded an average of 16 000 MS/MS spectra/merged single sample (MS/MS spectra can be accessed via proteomic data repository, PRIDE, http://ebi.ac.uk/pride, under accession numbers 13321-13326) and matched to 730 and 720 proteins (nonredundant within single run based on IPI accession number) on average for wild-type and AQP8 KO groups, respectively. Shared proteins between replicates of each group averaged 38% for wild-type and 40% for AQP8 KO group (Figure 3B) which reflects possible individual sample variations or differences in peptides mass ionization. We further assessed the precision of reproducibility of LC-MS/MS to exclude any possible experimental error variation (Supplement 1). To obtain the most significantly reliable protein candidates, “all and none” strategy with 3 refinement steps was applied as illustrated in Figure 2. Initial selection of common candidates appearing in replicates of each group [step 1] revealed 277 and 285 shared proteins for wild-type and AQP8 KO model, respectively (Figure 3B). In the second step, a comparison between outputs of previous step was achieved in order to


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Figure 8. Distribution and appearance of R-amylase 2 peptides in the current study. (A) Amino acid sequence of R-amylase 2. Underlined amino acid sequences tagged by circled number are peptides identified within whole study. (B) Distribution of R-amylase peptides within both experiments. For in-gel digestion experiment (experiment A), peptides appeared in wild-type runs only but never shown in counterpart group [all and none], while in experiment B, in-solution digestion, only sample no. 2 of AQP8 knockout group showed some R-amylase peptides.

Figure 9. Immuno-validation of R-amylase 2. (A) Western blot analysis of R-amylase 2 in wild-type and AQP8 knockout colon samples. Expected 55 kDa bands were detected with higher intensity in wild-type (left triplicates) compared with AQP8 knockout group (right triplicates). Lower bands are GAPDH of the same sample (38 kDa). (B) Corresponding band intensity drawn by image J software showing 2-fold differences in the averaged intensities between experimental groups. **P < 0.01.

identify unique candidates present in all runs of one group but not in the shared counterpart [Step 2]. As shown in Figure 3C, 106 protein hits (38.2%) and 114 proteins (40%) were unique to wild-type and AQP8 KO groups, respectively. In the third step, unique protein candidates of wild-type were searched against all other sections of AQP8 KO replicates rather than the shared area and vice versa. This step filtered the most significantly differential protein candidates that appeared in all replicate runs of one group and never shown elsewhere in the counterpart. As a result, only 10 annotated proteins (5 down-regulated and 5 up-regulated) expressed possible regulation as a result of AQP8 KO (Table 2 and Figure 4). Manual reviewing of those proteins has suggested that some of them were unique to one experimental group but with a low peptide match scoring (below mascot identity and homology threshold) and/or with top ranked peptides (marked bold red in mascot) less than 2. After manual filtration to select protein candidates

with at least 2 peptides above the identity and homology threshold and least 2 top ranked peptides (shown bold red in mascot report), only 2 down-regulated (amylase 2 and prolyl 4 hydrolase) and 3 up-regulated candidates (carbonic anhydrase 3, similar to lacremal binding protein and mitochondrial carrier homologue 2) were considered unambiguous and with highly confidence. The later candidates illustrated in Table 2 were assigned to final selection. For the identified proteins, details of the corresponding peptides can be found in Supplement 3. 3.2. In-Solution Trypsin Digestion [Experiment B]. Replicate runs generated from IBA mode analysis were converted into a simulated 2D gel view as exemplarily shown in Figure 5A. Selection criteria were applied to all spots assigned for comparison (as previously described in Experimental Section). Summarized illustration is shown in Figure 5B. As initially, 9932 features (mass peaks) with low experimental errors (P < 0.0001) were selected. Alignment was driven across all runs with Journal of Proteome Research • Vol. 9, No. 12, 2010 6643

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Figure 10. Immuno-localization of R-amylase 2 in wild-type colon (A) and AQP8 knockout colon (B). R-Amylase 2 immunostaining was confined to colonic epithelial cytoplasm of both groups with low intensity in AQP8 knockout. (C and D) Immunostaining of R-amylase 2 in wild-type and AQP8 knockout mice pancreatic tissues, respectively. Scale bar is 0.5 µm. (E) Normalized quantitative real-time PCR (bars) of R-amylase 2 transcripts in wild-type and AQP8 knockout colon and pancreas with conventional PCR view (bands). mRNA measurements confirmed our proteomic data. For both PCR tests, a primer pair (5′-GCACATGTGGCCTAGAGACATAAAG-3′), (5′-TAATTGCCTCACCACCCAGA-3′) was used. Real-time PCR was performed using syber green method. For conventional PCR, a product size of 118 bp was detected. CW, wild-type colon. CK, AQP8 KO colon. PW, wild-type pancreas. PK, AQP8 KO pancreas.

correction of possible drift in retention time. Mass peaks charged +2 to +6 were further refined (9074 mass peaks) prior to statistical comparison between experimental groups. With selection of mass peaks that showed a significant statistical difference (P < 0.05), 3823 peaks (42%) were assigned to identification using mascot search engine (P < 0.03, FDR< 2). Final search result revealed identification of 146 proteins (95 up-regulated and 50 down-regulated). Peptide and protein abundances can be found in Supplements 3 and 4, respectively. 3.3. Experimental Comparison and Validation. Comparison between experimental groups (wild-type versus AQP8 KO model) is of practical importance to ascertain whether there is any biological relevance of AQP8 rather than its water trafficking capabilities or not.23 Surprisingly, only R-amylase 2 was found to be shared between both experiments. In experiment A, a typical (all and none) finding was observed for R-amylase 2. We reported 43 peptides of R-amylase in wild-type runs that never show elsewhere in AQP8 replicates, which can be interpreted by the significant differences in peptide abundance between both experimental groups, and in turn has led to increase the chance of corresponding parent ion to be selected for further fragmentation (MS/MS). On the other hand, simu6644


lated 2D gel view comparison (experiment B) revealed obvious down-regulation of 19 peptides corresponding to R-amylase 2 in AQP8 KO group with 2-fold differences in the average normalized protein abundance (Figure 6A,B). Most notably, the recognition of R-amylase 2 simulated 2D peptide spots in one sample of AQP8 KO group and its absence in the other 2 samples reflects its low abundance. In contrast, around 19 peptides could be recognized in all wild-type individuals with prominent significance (Table 3). In all runs of AQP8 KO replicates, a significant down-regulating pattern of mass peaks related to R-amylase 2 was reported as shown in the 3D pattern and heat map analysis (Figure 6C,D). In addition, we noticed a characteristic regulatory pattern in the R-amylase 2 proteotypic peptide (TAIVHLFEWR) (Figure 7). For further clarification, an overall distribution and appearance of R-amylase 2 peptides in both experiments is illustrated in Figure 8. To validate our findings, Western blotting using a mouse monoclonal anti-amylase antibody was used. Immunoblotting was coincidental with our mass spectrometric analysis by 2-fold differences between both experimental groups (Figure 9).To further clarify whether AQP8 modulates pancreatic R-amylase production, colonic mucosal amylase synthesis, or colonic

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Comparative Study of AQP8 Knockout Mouse Colon mucosal amylase uptake from the lumen, we first immunovalidated amylase in the pancreatic tissue of both groups. Unexpectedly, our investigation did not show any significance in amylase visibility neither by IHC nor Western blotting between pancreatic cells of both groups (Figure 10). In contrast, IHC staining revealed a remarkable reactivity in the colonic epithelia of wild-type compared to AQP8 KO group (Figure 10). Taken together, these findings possibly refute the involvement of AQP8 in pancreatic amylase synthesis and likely augments the proposition of either its role in controlling amylase uptake by the colonic epithelial cells or its synthesis in colonic epithelial cytoplasm. To disclose this arguements, quantitative transcriptional expression (q RT-PCR) of R-amylase 2 was examined. Interestingly, our finding showed similar downregulating pattern in AQP8 KO group supporting the possible control of AQP8 on colonic R-amylase 2 synthesis (Figure 10E). Whether AQP8 regulates R-amylase 2 directly or through secondary mediator, additional experimentation would be required to elucidate this mechanism. Recently, there has been mounting evidence that production of amylase might be originated from nonclassical (parotid and pancreatic) sources such as liver, intestine, and fallopian tube.24,25 In a previous study by Hokari et al.,24 they reported a significant level of R-amylase mRNA in different intestinal segments (mainly duodenum), denoting probable in situ amylase synthesis from intestinal mucosal cells. Interestingly, when they examined the crude intestinal amylase on cellulose acetate membrane (zymography), amylase isozyme was found to be mainly of pancreatic nature which further supports our proteomics finding.

4. Conclusion In conclusion, this study describes a simple and robust methodology for refining large-scale proteomic data by implementing different analytical and comparative approaches. Shared candidates of final outputs augment the highly confidence protein candidate existence in the experimental samples and its possible regulation between experimental groups. As exemplarily shown in this study, ID, peptide identification number, RT, retention time. (M), Oxidation of methionine. (HW) Oxidation of histidine and tryptophan-amylase 2, which has been confirmed to be regulated in the two experimental platforms and validated immunologically later on, discloses a novel involvement of AQP8 in colonic R-amylase 2 synthesis and supports our experimental approach. In generalized term, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process that can be used for initial screening of biological biomarkers in complex specimens and tissue extracts. Abbreviations: AQP8, aquaporin 8; AQP8 KO, aquaporin knockout; LacZ; galactosidase gene; Neo, neomycin phophotransferase gene; DT-A, diphtheria toxin A; ES, Embryonic Stem cells; TLCK, tosyl-L-lysine chloromethyl ketone; PMSF, phenylmethanesulfonylfluoride; DTT, dithiothreitol; IAA, iodoacetamide; LC-ESI-IT-TOF, liquid chromatographyelectospray ionization-time-of-flight; EDTA, ethylenediaminetetraacetic acid; TFA, trifluoruacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IBA, informatiom based acquisition; CID, colloidal ion dissociation; VBA, visual basic code; FDR, false discovary rate; PVDF, polyvinylidene difluoride, GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ECL, enhanced chemiluminescence; IHC, immunohistochemistry; DAB, 3,3′diaminobenzidine.

Acknowledgment. We thank all members of animal care center, Niigata University, Japan for maintaining experimental mice. This work was supported by Grant-in-Aid for scientific research, B (21390262) from Ministry of Education, Culture, Sports, Science and Technology of Japan. Supporting Information Available: Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Agre, P.; King, L. S.; Yasui, M.; Guggino, W. B.; Ottersen, O. P.; Fujiyoshi, Y.; Engel, A.; Nielsen, S. Aquaporin water channels-from atomic structure to clinical medicine. J. Physiol. 2002, 542 (1), 3– 16. (2) Agre, P.; Bonhivers, M.; Borgnia, M. The aquaporins, blueprints for cellular plumping system. J. Biol. Chem. 1998, 12 (24), 14659– 14662. (3) Magni, F.; Sarto, C.; Ticozzi, D.; Soldi, M.; Bosso, N.; Mocarelli, P.; Kienle, M. G. Proteomic knowledge of human aquaporins. Proteomics 2006, 20 (6), 5637–5649. (4) Matuzaki, T.; Tajika, Y.; Ablimit, A.; Aoki, T.; Hagiwara, H.; Takata, K. Aquaporins in the digestive system. Med. Electron. Microsc. 2004, 2 (37), 71–80. (5) Tani, T.; Koyama, Y.; Nihei, K.; Hatakeyama, S.; Oshiro, K.; Yoshida, Y.; Yaoita, E.; Hatakeyama, Y.; Yamamoto, T. Immunolocalization of aquaporin 8 in rat digestive organs and testis. Arch. Histol. Cytol. 2001, 2 (64), 159–168. (6) Koyama, Y.; Yamamoto, T.; Kondo, D.; Funaki, H.; Yaoita, E.; Kawasaki, K.; Sato, N.; Hatakeyama, K.; Kihara, I. Molecular cloning of a new Aquaporin from rat pancreas and liver. J. Biol. Chem. 1997, 272 (28), 30329–30333. (7) Koyama, Y.; Yamamoto, T.; Tani, T.; Nihei, K.; Kondo, D.; Funaki, H.; Yaoita, E.; Sato, N.; Hatakeyama, K.; Kihara, I. Expression and localization of aquaporins in rat gastrointestinal tract. Am. J. Physiol. 1999, 1 (276), C621–C627. (8) Calamita, G.; Mazzone, A.; Bizzoca, A.; Cavalier, A.; Cassano, D.; Thomas, M.; Svelto, A. Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract. Eur. J. Cell Biol. 2001, 11 (80), 711–719. (9) Yang, B.; Zhao, D.; Solenov, E.; Verkman, A. S. Evidence from knockout mice against physiologically significant aquaporin 8facilitated ammonia transport. Am. J. Physiol. 2006, 3 (291), C417– C423. (10) Ma, T.; Yang, B.; Verkman, A. S. Cloning of a novel water and ureapermeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem. Biophys. Res. Commun. 1997, 240 (17), 423–428. (11) Huebert, R.; Splinter, P.; Garcia, F.; Marinelli, R.; LaRusso, N. Expression and localization of aquaporin water channels in rat hepatocytes. Evidance for a role in canalicular bile secretion. J. Biol. Chem. 2002, 25 (277), 22710–22717. (12) Calamita, G.; Mazzone, A.; Bizzoca, A.; Svelto, M. Possible involvement of aquaporin 7 and 8 in rat testis development and spermatogenesis. Biochem. Biophys. Res. Commun. 2001, 3 (288), 619–625. (13) Larocca, M. C.; Soria, L. R.; Espelt, M. V.; Lehmann, G. L.; Marinelli, R. A. Knockout of hepatocyte aquaporin 8 by RNA interference induces defective bile canalicular water transport. Am. J. Physiol.: Gastrointest. Liver Physiol. 2009, 3 (12), G93–G100. (14) Jablonski, E. M.; Mattocks, M. A.; Sokolov, E.; Koniaris, L. G.; Hughes, F. M.; Fausto, N.; Pierce, R. H.; McKillop, I. H. Decreased aquaporin expression leads to increased resistance to apoptosis in hepatocellular carcinoma. Cancer Lett. 2007, 1 (250), 36–46. (15) Elizabeth, J.; Webb, W. A.; McConnell, N.; Riley, M.; Hughes, F. Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease. Am. J. Cell Physiol. 2003, 4 (286), C975–C985. (16) Magdeldin, S.; Li, H.; Yoshida, Y.; Enany, S.; Zhang, Y.; Xu, B.; Fujinaka, H.; Yaoita, E.; Yamamoto, T. Comparison of two dimensional electrophoresis mouse colon proteomes before and after knocking out aquaporin 8. J. Proteomics 2010, 10 (73), 2031–2040. (17) Ramagli, L. S.; Rodriguez, L. V. Quantification of microgram amounts of protein in two dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 1985, 23 (3), 559–563.

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