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Physiol. Genomics 25: 263-276, 2006. First published January 31, 2006; doi:10.1152/physiolgenomics.00214.2005
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Received 22 August 2005; accepted in final form 25 January 2006.
Physiological Genomics 25:263-276 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

High-throughput identification of IMCD proteins using LC-MS/MS

Trairak Pisitkun 1, Jared Bieniek 1, Dmitry Tchapyjnikov 1, Guanghui Wang 2, Wells W. Wu 2, Rong-Fong Shen 2 and Mark A. Knepper 1

1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
2 Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and {gamma}-epithelial Na channel ({gamma}-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

inner medullary collecting duct; systems biology; mass spectrometry; liquid chromatography; aquaporin-2; epithelial Na channel; vasopressin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VASOPRESSIN CONTROLS RENAL WATER excretion in part by regulating the permeability of collecting duct cells to water. The main protein target for this process is the water channel aquaporin-2 (AQP2). Vasopressin regulates AQP2 in two ways to increase collecting duct water permeability (28). 1) Over a period of minutes, vasopressin stimulates trafficking of AQP2-containing vesicles to the apical region of the collecting duct cells where they fuse with the plasma membrane to increase water permeability. 2) Over a period of hours to days, vasopressin increases AQP2 protein abundance in the collecting duct cells, in part due to increased transcription of the AQP2 gene. The signaling pathways involved in these responses remain incompletely understood.

In recent years, large-scale identification of proteins by mass spectrometry has become practical, and such techniques are finding increasing use in the discovery of signaling networks involved in a variety of physiological processes. An initial goal in identification of regulatory processes in a given cell type is to identify its proteome as completely as possible. To describe the proteome of the inner medullary collecting duct (IMCD) cell, we have previously carried out studies using two-dimensional (2-D) electrophoresis with protein identification by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (15, 34). A general drawback of 2-D electrophoresis is that certain classes of proteins are excluded from the analysis including hydrophobic proteins, proteins with very high or low molecular mass, and proteins with very high or low isoelectric points. Thus complementary methods are needed to fully describe the set of proteins expressed in the IMCD. One viable approach combines SDS solubilization of proteins, 1-D SDS-PAGE, in-gel trypsinization, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) (1, 30), which in principle provides a way to overcome the limitations of 2-D electrophoresis. Here we use such an approach to expand the known proteome of the native rat IMCD cell. Using the new data, we present a new WWW-based "IMCD Proteome Database" that lists all proteins heretofore identified in native IMCD cells by protein MS.

Another goal in identification of regulatory processes in a given cell type is to identify proteins whose abundances, phosphorylation states, or cellular localizations change in response to a stimulus. In a previous study using differential in-gel electrophoresis (DIGE) applied in a 2-D electrophoresis format (34), we identified several proteins whose abundances in IMCD cells are altered by vasopressin. In the present study, to expand the list of IMCD proteins whose abundances are regulated by vasopressin, we use isotope-coded affinity tagging (ICAT) (11), which allows quantification in the setting of LC-MS/MS analysis. In ICAT analysis, cysteine moieties of two protein samples are derivatized via a thiol reaction using chemically identical reagents except for the substitution of some of its natural H, C, or O atoms with different stable (nonradioactive) isotopes. The resulting difference in molecular mass allows tryptic peptides from the two original samples to be distinguished and quantified by the mass spectrometer. In the current study, we use an ICAT reagent that labels cysteine side chains with a tag that contains either nine 13C carbons or nine 12C carbons, giving a mass difference of 9 amu for individual derivatized peptides with single cysteine. For quantification, the relative peak height for paired heavy and light peptides can be integrated over time to estimate the relative abundance of the corresponding proteins in the two original samples. Here we employ the ICAT method for the investigation of proteins regulated in response to long-term 1-desamino-8-D-arginine vasopressin (vasopressin analog; dDAVP) infusion. The animal protocol was the same as that used for our previous study, which reported DIGE-based identification of vasopressin-regulated proteins (34). Finally, we generated a protein network for vasopressin signaling in the IMCD based on previously demonstrated responses to vasopressin in native IMCD cells, combined with newly hypothesized pathways based on proteomic findings of this study.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of IMCD Samples vs. Inner Medullary "Non-IMCD" Samples
IMCD and non-IMCD sample preparation.
Inner medullary collecting ducts were purified from rat renal inner medullas as described by Chou et al. (4). Four male Sprague Dawley rats were euthanized [National Heart, Lung, and Blood Institute (NHLBI) Animal Care and Use Committee (ACUC)-approved Protocol 2-KE-3]. The renal inner medullas from each animal were dissected out, minced to obtain ~1-mm3 pieces of tissue, and separately transferred to four glass tubes containing digestion solution (3 mg/ml collagenase B, 2,000 U/ml hyaluronidase, 250 mM sucrose, 10 mM triethanolamine, pH 7.6). The suspensions were incubated at 37°C with 95% air-5% CO2 bubbling and continuous stirring for 60 min. Low-speed centrifugation (70 g for 10 s) was carried out to sediment the heavier IMCD cells, separating them from the lighter non-IMCD cells. The supernatants were removed and centrifuged at 1,500 g for 10 min to pellet the non-IMCD cells. The quality of separation was examined under a dissection microscope (Wild M8, Heerbrugg, Switzerland). The IMCD and non-IMCD pellets were resuspended in 50 and 100 µl of denaturing buffer, respectively, followed by homogenization with a sonicator probe (XL-2020 Sonicator; Misonix, Farmingdale, NY). Lysates were centrifuged at 14,000 g for 15 min to remove any insoluble material. Protein concentrations of the resulting supernatants were determined using Bradford reagent (USB, Cleveland, OH).

Quantitative LC-MS/MS analysis of IMCD vs. non-IMCD using ICAT.
Samples were pooled from four rats; 250 µg each of pooled IMCD and pooled non-IMCD samples were employed for ICAT analysis as described below.

Quantitative LC-MS/MS Analysis of Response to Long-Term dDAVP Administration in IMCD from Brattleboro Rats
Animal procedures.
Eight male Brattleboro rats (360–430 g body wt; Harlan-Sprague Dawley, Indianapolis, IN) were used to investigate the response to long-term dDAVP administration in IMCD (NHLBI ACUC-approved Protocol 2-KE-3). Four rats were infused with the V2 vasopressin receptor (V2R)-selective vasopressin analog dDAVP (Rhone-Poulenc Rorer, Collegeville, PA) at 5 ng/h for 3 days by subcutaneous osmotic minipumps (model no. 2001; Alzet, Palo Alto, CA). Another four rats were used as controls by receiving osmotic minipumps delivering isotonic saline solution. Rats were maintained in metabolic cages in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. They had free access to water and regular pelleted rat chow. Urine collections were made for quantitative analysis and osmolality measurement using a vapor pressure osmometer (Vapro 5520; Wescor, Logan, UT). After 3 days, the rats were killed by rapid decapitation, and inner medullas were rapidly isolated for IMCD sample preparation as described above.

Quantitative LC-MS/MS analysis of response to long-term dDAVP administration in IMCD using ICAT.
Four hundred micrograms of IMCD cell homogenate from pooled dDAVP samples (100 µg per rat)and pooled control samples (100 µg per rat) were employed for ICAT analysis as described below. The flow-through samples from biotin-avidin affinity purification step containing nonlabeled peptides were also analyzed by nanospray LC-MS/MS to further expand the IMCD Proteome Database (see below).

ICAT Analysis
ICAT analysis used reagents purchased from Applied Biosystems (part no. 4339035 and 4339036; Foster City, CA) and followed the manufacturer’s protocol. The two samples to be compared were denatured by addition of a prescribed "denaturing buffer" (50 mM Tris, 0.1% SDS, pH 8.5) and reduced with 1.2 mM tris-(2-carboxyethyl)-phosphine (TCEP) then boiled for 10 min. The two samples were then labeled with either 12C (light) or 13C (heavy) cleavable ICAT reagents (100 µg of protein per vial of ICAT reagent), respectively, for 2 h at 37°C. Subsequently, the light and heavy ICAT reagent-labeled samples were mixed. The mixed sample was concentrated using a Speed Vac, and then 5x SDS-Laemmli sample buffer was added (1:2 vol/vol Laemmli buffer-sample) before boiling for 10 min.

One-dimensional SDS-PAGE was performed using a 10% polyacrylamide Ready Gel (BioRad, Hercules, CA) to simplify the complexity of proteins in the sample. The gel was stained with colloidal Coomassie blue stain (GelCode Blue Stain Reagent, G-250; Pierce Biotechnology, Rockford, IL) for 5 min and then destained in deionized H2O for 1 h. The gel was then sliced into small blocks from the top of the stacking gel down to the dye front for a total of 16–20 blocks. Each block was minced into small pieces (1–1.5 mm3) and placed into a 1.7-ml prelubricated centrifuge tube (PGC Scientifics, Frederick, MD). The gel pieces were further destained and dehydrated by incubating with 25 mM NH4HCO3-50% acetonitrile (ACN) solution for 10 min three times, and then the gel pieces were dried using a Speed Vac.

In-gel trypsin digestion was performed by rehydrating the gel pieces with 2.5 µg of Sequencing Grade Modified Trypsin (Promega, Madison, WI) diluted in 25 mM NH4HCO3 solution (final concentration, 12.5 ng/µl) for 30 min on ice. The remaining trypsin solution was then removed, and the gel pieces were briefly washed with 25 mM NH4HCO3 to remove excess trypsin. The gel pieces were covered with 25 mM NH4HCO3 solution and incubated at 37°C overnight. After trypsin digestion, the peptides were extracted by incubating the gel pieces with 50% ACN-0.1% formic acid (FA) and then sonicating the gel pieces in water bath for 20 min. This extraction step was repeated two more times. The extracted samples were dried by a Speed Vac and then reconstituted with 500 µl of 2x PBS (20 mM NaH2PO4, 300 mM NaCl, pH 7.2) before purification of the ICAT reagent-labeled peptides using the biotin-avidin affinity purification step as recommended by the manufacturer (ICAT Cartridge, Avidin; Applied Biosystems). The affinity tag portion of ICAT reagent was then cleaved off using cleaving reagent containing concentrated trifluoroacetic acid (TFA) for 2 h at 37°C. The ICAT reagent-labeled peptides were concentrated and cleaned up using a ZipTip C18 pipette tip and then dried and reconstituted with 0.1% FA before analysis by nanospray LC-MS/MS.

Validation of ICAT method.
Two 25-µg bovine serum albumin (BSA) samples labeled with heavy or light ICAT reagents were prepared as described above. The two samples were denatured, reduced, derivatized with heavy or light reagents, and mixed in specified ratios (either 1:1 or 1:2). The mixed samples were digested with trypsin (62.5 ng/µl) in solution at 37°C overnight (SDS gel separation was not performed on BSA samples in contrast to other studies in this paper). The tryptic peptides were separated from the TCEP, SDS, and excess ICAT reagents by cation exchange chromatography (ICAT Cartridge, Cation Exchange; Applied Biosystems). The biotin-containing derivatized peptides were affinity purified and cleaved as described above. MALDI-TOF/TOF analysis (4700 Proteomics Analyzer, Applied Biosystems) was performed on the 1:1 and 1:2 mixed samples. Mascot (Matrix Science, Boston, MA) software was used to search raw data files. GPS Explorer (Applied Biosystems) software was used to quantify the ICAT results. Results were confirmed with nanospray LC-MS/MS analysis (LCQ Deca XP Plus; Thermo Finnigan, San Jose, CA) performed on the 1:1 mixed sample.

Nanospray LC-MS/MS.
One-dimensional LC-MS/MS using a modified configuration of the ProteomeX 2-D LC/MS workstation was employed for ICAT analysis (LCQ Deca XP Plus, Thermo Finnigan). Chromatographic separation of peptides was accomplished using two Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilminton, DE), working in alternating fashion (replacing the standard strong cation exchange and reverse-phase columns), while the standard electrospray ionization (ESI) source was replaced by a nanospray ionization source and a reverse-phase PicoFrit column (BioBasic C18, 75 µm x 10 cm, tip = 15 µm; New Objective, Woburn, MA). The peptides were loaded onto the traps in alternating fashion using an autosampler. After a washing with 0.1% formic acid, the peptides were eluted by 0–60% solvent B in solvent A (solvent A, 0.1% formic acid; solvent B, acetonitrile) for 30 min at a flow rate of ~200 nl/min. The flow-through samples from the avidin affinity column were analyzed using an LTQ linear trap tandem mass spectrometer (Thermo Finnigan).

Inclusion criteria for identified peptides.
The mass-charge (m/z) ratios of peptides and their fragmented ions were recorded by a method that allows the acquisition of three (LCQ mass spectrometer) or five (LTQ mass spectrometer) MS2 scans following each full MS scan. The raw data files were searched against the rat protein database from the National Center for Biotechnology Information (NCBI) using BioWorks 3.1 software (Thermo Finnigan) based on the Sequest algorithm. The search parameters included the following: precursor-ion mass accuracy = 3.0 amu (LCQ) or 1.5 amu (LTQ), fragment-ion mass accuracy = 1.0 amu (LCQ) or 0.0 amu (LTQ), modification allowed for addition of light or heavy ICAT reagents on cysteine, and two missed cleavages allowed. After the peptide sequence and protein identification from BioWorks software was carried out, the identified peptide sequences were initially filtered using the cross-correlation score (Xcorr) at the following threshold: Xcorr >1.5 for 1+ ion, 2.0 for 2+ ion, and 2.5 for 3+ ion.

For each identified ICAT reagent-labeled peptide that passed the filter threshold, proteins identified were selected if they achieved the following criteria: 1) peptide sequence had the highest Xcorr score for a particular collision-induced dissociation (CID) spectrum, 2) peptide sequence had a delta-normalized correlation score ≥0.08, and 3) peptide sequence had good-quality CID spectra by visual inspection. All identified peptide sequences were searched using basic local alignment search tool (BLAST) to obtain the best possible unique protein identification, thus eliminating redundant annotations.

For each identified peptide from the flow-through samples that passed the initial filter threshold, proteins identified from two or more different peptides were selected if they achieved the following criteria: 1) peptide sequence had the highest Xcorr score for a particular CID spectrum, 2) peptide sequence had a delta-normalized correlation score ≥0.08, and 3) peptide sequence had the ranking of the preliminary raw score ≤10.

Quantification of ICAT results.
The XPRESS algorithm implemented in BioWorks 3.1 software was used to calculate the ICAT ratio of each identified ICAT reagent-labeled peptide. The parameters used for this calculation were 1) light/heavy ICAT reagent-labeled cysteine mass difference = 9 amu, 2) mass tolerance = 1.0–1.5 amu, and 3) scan window = 60 full MS scans. Manual inspection of reconstructed ion chromatogram was performed to validate the quantification results.

Immunoblotting
Immunoblotting was performed as described (7). Briefly, proteins were resolved by SDS-PAGE on 7.5, 10, or 12% polyacrylamide gels and transferred electrophoretically onto nitrocellulose membranes. The membranes were then blocked with 5% nonfat dry milk in immunoblot wash buffer (42 mM Na2HPO4, 8 mM NaH2PO4, 150 mM NaCl, and 0.05% Tween 20, pH 7.5), rinsed, and probed with primary antibody overnight at room temperature. After a washing, blots were incubated with species-specific secondary antibodies conjugated to horseradish peroxidase. After the final wash, antibody binding was visualized by chemiluminescence (LumiGLO; KPL, Gaithersburg, MD) using light-sensitive film developed on the Kodak M35A X-OMAT Processor.

Antibodies.
The rabbit polyclonal antibodies to AQP1, AQP2, ß-epithelial Na channel (ß-ENaC), and {gamma}-ENaC were previously generated in our laboratory (21), and a rabbit polyclonal antibody to the {alpha}-1 subunit of Na/K-ATPase was newly prepared using a synthetic peptide (sequence: CDEVRKLIIRRRPGGWVEKETYY) conjugated to keyhole limpit hemocyanin. The anti-myosin IIA rabbit polyclonal was a gift of Dr. Robert Adelstein (NHLBI, Bethesda, MD). The commercial antibodies used are listed as follows: ß-actin (rabbit polyclonal, 4967; Cell Signaling Technology, Beverly, MA); aldose reductase (goat polyclonal, sc-17735), annexin II (goat polyclonal, sc-1924), annexin IV (goat polyclonal, sc-1930), cathepsin D (goat polyclonal, sc-6486), heat shock protein (HSP)70 (goat polyclonal, sc-1060), RhoA (mouse monoclonal, sc-418), RhoGDI (rabbit polyclonal, sc-360), receptor of activated protein kinase C 1 (RACK1; mouse monoclonal, sc-17754), Rap1 (rabbit polyclonal, sc-65), and Cdc42 (rabbit polyclonal, sc-87) from Santa Cruz Biotechnology (Santa Cruz, CA); transglutaminase 2 (goat polyclonal, 06–471; Upstate, Waltham, MA); GAPDH (mouse monoclonal, NB 300–221; Novus Biologicals, Littleton, CO); ß-spectrin II (mouse monoclonal, 612562; BD Biosciences Pharmingen, San Jose, CA); Grp58 (rabbit polyclonal, P7496; Sigma-Aldrich, St. Louis, MO); and syntaxin-7 (rabbit polyclonal, 110 072; Synaptic Systems, Goettingen, Germany).

Bioinformatic Network Analysis
Proteins regulated in response to long-term dDAVP administration that were validated by immunoblotting were analyzed further by bioinformatic network analysis. This analysis used the core signaling pathway downstream from V2R occupation in IMCD demonstrated by previous studies (2, 3, 5, 8, 13, 14, 18, 23, 29, 36) as the core network. The connections between the newly identified proteins and the core network were created through manual and computer-aided literature searching [Ingenuity Pathway Analysis (IPA), Ingenuity Systems, Mountain View, CA, http://www.ingenuity.com; and MetaCore, GeneGo, St. Joseph, MI, http://www.genego.com]. The network was displayed graphically as nodes (individual proteins or molecules) and edges (the biological interactions between the nodes).

IMCD Proteome Database
A database of all proteins identified by protein MS in IMCD in this study and prior studies (1, 15, 16, 34) was constructed as an Excel spreadsheet. The spreadsheet was used to generate HTML files that are posted on a central server at the following URL: http://dir.nhlbi.nih.gov/papers/lkem/imcd/index.htm. The database is limited to protein MS data from freshly isolated IMCDs of rats prepared as above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Validation of ICAT Method
As a preliminary test of the validity of the ICAT method in our setting, we have carried out labeling of samples containing differing amounts of BSA. Figure 1A shows examples of MALDI-TOF spectra with 1:1 and 1:2 ratios of BSA labeled with the light ICAT reagent (12C) and the heavy ICAT reagent (13C), respectively. As can be seen, the peak heights for various BSA peptides were approximately in proportion to the relative amounts of BSA in the two samples. Figure 1B demonstrates the reconstructed ion chromatograms from LC-MS/MS analysis of a BSA tryptic peptide (sequence: LKPDPNTLCDEFK) labeled with light-heavy ICAT reagent in 1:1 ratio. The area under the entire envelope was used for measuring the ICAT ratio. Figure 1C shows data from LC-MS/MS showing a histogram of the 12C/13C ratios for all BSA peptides when a 1:1 ratio was utilized. For 53 peptides, the mean ratio was 1.04 and the standard deviation was 0.19.


Figure 1
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  		Fig. 1. A: matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra of bovine serum albumin (BSA) tryptic peptides from 1:1 mixing (top) and 1:2 mixing (bottom) of light-heavy isotope-coded affinity tag (ICAT) reagent-labeled BSA. Dashed boxes highlight some pairs of typical ICAT spectra containing 2 identical peptide peaks, 1 labeled with light reagent (left) and 1 labeled with heavy reagent (right), both of which have a mass difference of 9 amu. The relative intensities of these pairs correlate well with the mixing ratio (quantitative errors of 15.5 and 7.5% for 1:1 mixing and 1:2 mixing, respectively). Numbers above each peak indicate mass-charge ratio (m/z) and area under the peak (in parentheses). B: reconstructed ion chromatograms from liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of a BSA tryptic peptide (sequence: LKPDPNTLCDEFK) labeled with light ICAT reagent (top) and heavy ICAT reagent (bottom) in 1:1 ratio. A "reconstructed ion chromatogram" shows the peak height for an individual peptide collected from multiple spectra over time during elution from the HPLC column. The area under the entire envelope (gray area) was used for the quantification of ICAT ratio. C: histogram demonstrating LC-MS/MS ICAT ratios of 1:1 mixing of light-heavy ICAT reagent-labeled BSA.

 
Comparison of IMCD Samples with Inner Medullary Non-IMCD Samples
To analyze the IMCD proteome and its response to vasopressin, it is necessary to isolate IMCD cells from the renal inner medulla of rat. This is done by a low-speed centrifugation technique utilized previously in proteomic analyses of the inner medulla (15, 34) (described in METHODS). This technique yields purified IMCD cells in one sample and the residual cell types from the inner medulla in the other sample (termed non-IMCD cells). Figure 2 shows an immunoblot characterization of these cell fractions. As can be seen, for four different preparations labeled A–D, the collecting duct marker AQP2 was strongly enriched in the IMCD fraction. The AQP2 band density ratio for the IMCD fraction vs. the non-IMCD fraction was 259 ± 131 (mean ± SD; n = 4). The descending limb of Henle/vasa recta marker AQP1 was strongly de-enriched in the IMCD fraction. The band density ratio for the IMCD fraction vs. the non-IMCD fraction was 0.09 ± 0.10 (mean ± SD; n = 4). Thus the IMCD purification was successful.


Figure 2
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  		Fig. 2. Immunoblots demonstrating the quality of inner medullary collecting duct (IMCD) and non-IMCD sample preparation. AQP, aquaporin.

 
Quantitative LC-MS/MS Analysis of IMCD vs. Non-IMCD Proteome Using ICAT
An initial experiment (Fig. 3) was carried out to test the ability of ICAT to quantify protein abundance differences in biological tissues. This experiment compared IMCD samples (250 µg of protein pooled from 4 animals; labeled with 12C reagent) vs. non-IMCD inner medullary cell samples (250 µg of protein pooled from 4 animals; labeled with 13C reagent). Table 1 gives the 12C/13C ratios for proteins (n = 44), for which two or more unique peptides were identified. Figure 4 shows a plot of the correlation of ratios obtained with ICAT in this study vs. those obtained by DIGE in our previous study using the same technique for separating IMCD tubules from non-IMCD tubules. The specific proteins plotted in Fig. 4 are indicated in Table 1. In general, most of the 17 proteins identified in both the ICAT and DIGE studies changed in the same direction in both studies. Lack of correlation for the four proteins that changed in opposite directions potentially can be attributed to posttranslational modifications, which can produce changes in DIGE because of shifts in the position of the spots in the 2-D gels while not affecting the ratio obtained by ICAT. Full results for all proteins identified regardless of the number of peptide sequences found (n = 89) are presented in Supplemental Table S1 (available at the Physiological Genomics web site).1 All single-peptide identifications were checked by manually observing the associated spectra. A summary of the types of proteins identified are presented in Fig. 5. IMCD-to-non-IMCD abundance ratios using ICAT were in the range 0.03–7.69 (Supplemental Table S1), similar to the range seen in previous studies using DIGE (15).


Figure 3
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  		Fig. 3. Flow diagram of ICAT procedure in the IMCD vs. non-IMCD study.

 

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  		Table 1. Proteins identified and quantification of protein abundances from IMCD vs. non-IMCD samples

 

Figure 4
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  		Fig. 4. Scatter graph illustrating correlation between ICAT and differential in-gel electrophoresis (DIGE) results of the IMCD vs. non-IMCD studies (n = 17, r = 0.45, P value = 0.07). Grp58, glucose-regulated protein, 58 kDa; GST-Pi, glutathione S-transferase, pi; Hbß, hemoglobin beta chain complex; Hsp60, heat shock 60-kDa protein 1; Hsp70, heat shock 70-kDa protein; LDH-A, lactate dehydrogenase A; Tgm2, transglutaminase 2.

 

Figure 5
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  		Fig. 5. Pie chart showing overall types of proteins identified in the IMCD vs. non-IMCD study. ER, endoplasmic reticulum.

 
To test further the fidelity of the ICAT quantification in this experiment, we carried out semiquantitative immunoblotting for 10 of the proteins for which we could obtain suitable antibodies (Fig. 6). The immunoblots shown were carried out using aliquots of the same samples used in the ICAT experiment, except that the immunoblotting samples were not pooled. Thus each lane corresponds to an IMCD or a non-IMCD sample from a different animal. In general, the immunoblotting results paralleled the ICAT results, although the specific IMCD-to-non-IMCD ratios differed in several cases. In general, we conclude from comparison with DIGE data and immunoblotting data that ICAT can successfully identify and quantify differences in protein abundance in tissue samples.


Figure 6
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  		Fig. 6. Immunoblot confirmation of IMCD vs. non-IMCD ICAT results. aMean ± SE: *significantly different (n = 8; 4 IMCD and 4 non-IMCD). bMean ± SE: **based on 1 ICAT ratio value. Regular font indicates ratio value >1, and bold font indicates ratio value <1.

 
Quantitative LC-MS/MS Analysis of Response to Long-Term dDAVP Administration in IMCD from Brattleboro Rats
To identify IMCD proteins whose abundances are altered in response to long-term elevations of circulating vasopressin levels, we carried out experiments using Brattleboro rats, which have no endogenous vasopressin. Brattleboro rats were infused with either the V2R-selective vasopressin analog dDAVP (5 ng/h for 3 days in osmotic minipumps) or vehicle (for 3 days). Urine output and urine osmolality as a function of infusion period are shown in Fig. 7. As typically seen, the urinary output fell and the urinary osmolality rose substantially in response to dDAVP infusion. The animals were euthanized after 3 days, IMCD suspensions were prepared, and a portion of these samples were used for semiquantitative immunoblotting to confirm the action of the infused dDAVP (Fig. 8). There was a marked increase in the abundance of AQP2 in accord with previous observations (7). In addition, there was a marked increase in the abundance of the {gamma}-subunit of the ENaC and a trend toward an increase in ß-ENaC, similar to the changes previously recorded in the cortical and outer medullary collecting ducts (9). Thus we conclude that the infused dDAVP was effective in stimulating the expected long-term responses in the IMCD.


Figure 7
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  		Fig. 7. Urine output and urine osmolality (mosmol/kgH2O) between the vasopressin analog (dDAVP) and control groups. *Significantly different from the control group (P value < 0.05).

 

Figure 8
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  		Fig. 8. Immunoblots of AQP2, ß-epithelial Na channel (ß-ENaC), and {gamma}-ENaC confirming the action of the infused dDAVP. Normalized band densities are shown as means ± SE. *Significantly different from the control group (P value < 0.05).

 
We used the same samples to carry out ICAT analysis of the dDAVP response using a Thermo Finnigan LCQ LC-MS/MS system. This experiment compared IMCD proteins from dDAVP-treated animals (400 µg of protein pooled from 4 animals) with IMCD proteins from vehicle-infused animals (400 µg of protein pooled from 4 animals), as summarized in Fig. 9. A total of 165 proteins were identified that had high-quality spectra for which 13C/12C ratios could be determined (full results for all proteins identified are presented in Supplemental Table S2). Table 2 summarizes the proteins with dDAVP-control (13C/12C) ratios that were significantly different from unity based on observations in three or more quantifiable spectra corresponding to the same protein. Figure 10 shows a classification of these proteins based on the Collecting Duct Database (CDDB) identifiers (24) (http://cddb.nhlbi.nih.gov/cddb/). Cytoskeletal proteins and linkers/molecular motors (n = 7), biosynthetic proteins (n = 5), and proteins involved in energy metabolism (n = 4) are the major types of proteins that appeared to respond to the long-term vasopressin action.


Figure 9
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  		Fig. 9. Flow diagram of the ICAT analysis of response to long-term dDAVP administration in IMCD from Brattleboro rats.

 

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  		Table 2. Proteins identified that were significantly increased or decreased in abundance* in response to long-term dDAVP administration in IMCD from Brattleboro rats (n = 33)

 

Figure 10
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  		Fig. 10. Pie chart illustrating types of proteins that significantly changed in abundance as a result of long-term vasopressin action; a classification of these proteins is based on the Collecting Duct Database (CDDB) identifiers (24).

 
To confirm the ICAT ratios, immunoblots were performed on as many proteins as possible for which validated antibodies are available. Figure 11A shows immunoblotting results for 16 selected proteins quantified by ICAT. As can be seen, the immunoblots confirmed the direction of change determined by ICAT in 14 of 16 cases. The two proteins for which the direction of change was not verified (RACK1 and Rap1) were proteins that have only one ICAT ratio value. The proteins that significantly changed in abundance based on immunoblotting were cathepsin D (increased), GAPDH (increased), heat shock 70-kDa protein (increased), Rap1 (decreased), and syntaxin-7 (increased). As can be seen in Fig. 11B, there was a general correlation between the magnitudes of abundance change, as determined by immunoblotting and ICAT, which improved when proteins identified on the basis of only one peptide were excluded.


Figure 11
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  		Fig. 11. A: confirmatory immunoblots of the ICAT analysis of response to long-term dDAVP administration in IMCD from Brattleboro rats. aMean ± SE: *significantly different (n = 8; 4 dDAVP and 4 control). bMean ± SE: significantly different from 1.00 based on observations in 3 or more quantifiable spectra (*) and based on 1 ICAT ratio value (**). Regular font indicates ratio value >1, and bold font indicates ratio value <1. B: scatter graph illustrating correlation between ICAT and Western blot ratios (dDAVP-control). Black boxes represent proteins identified from 2 or more unique peptides (n = 12), and white boxes represent proteins identified from 1 unique peptide (n = 4). Correlation between ICAT and Western blot ratios was significant (r = 0.72) when proteins identified from 2 or more peptides were analyzed; however, the correlation was not significant (r = 0.34) when every protein was analyzed. Myosin IIA, myosin heavy chain, nonmuscle IIA; RACK1, receptor of activated protein kinase C 1.

 
Figure 12 shows a bioinformatic network representing the relationships between the core signaling pathway downstream from V2R occupation in IMCD demonstrated by previous studies (2, 3, 5, 8, 13, 14, 18, 23, 29, 36) and the five proteins regulated in response to long-term dDAVP administration that were validated by immunoblotting in this study (see above). The connections between the newly identified proteins and the core network were generated through manual and computer-aided literature searching (IPA and MetaCore; see METHODS). Supplemental Table S3 describes the interactions between parent nodes and child nodes in the bioinformatic network. Supplemental Table S4 demonstrates protein names and references documenting the presence of the individual proteins in IMCD.


Figure 12
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  		Fig. 12. Bioinformatic network representing the core signaling pathway downstream from V2 vasopressin receptor (V2R) occupation in IMCD demonstrated by previous studies (labeled in gray) and the 5 proteins regulated in response to long-term dDAVP administration that were validated by immunoblotting in this study (labeled in red). Open nodes (white background) represent IMCD proteins chosen to connect the core network with the red nodes. Supplemental Table S3 describes the interactions between parent nodes and child nodes in the bioinformatic network. Supplemental Table S4 demonstrates protein names and references documenting the presence of each protein in IMCD. Edge labels: A, activation; D, degradation; E, expression; I, inhibition; LO, translocation; P, phosphorylation; PP, protein-protein interaction; R, release; and T, transcription.

 
Protein Identifications in Flow-Through Fractions of Biotin-Avidin Affinity Purification in dDAVP Infusion Experiment
The flow-through samples from the biotin-avidin affinity purification step containing nonlabeled IMCD peptides were analyzed by an LTQ linear trap tandem mass spectrometer to expand the number of the IMCD proteins identified. Supplemental Table S5 shows IMCD proteins identified with two or more unique peptides (n = 630).

IMCD Proteome Database
With this study, we have now completed five distinct studies revealing elements of the IMCD proteome (1, 15, 16, 34). To provide a resource making these data generally available, we have created an IMCD Proteome Database that includes all proteins (presently, n = 848) identified in IMCD cells in these studies. This database is accessible at http://dir.nhlbi.nih.gov/papers/lkem/imcd/index.htm. The database will be updated further as new proteins are identified and is limited to proteins identified by MS in freshly isolated IMCD cells using high-stringency filters to avoid false-positive identifications. Figure 13 represents the distribution of proteins currently available in the IMCD Proteome Database categorized by the CDDB identifiers (24).


Figure 13
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  		Fig. 13. Bar graph representing the distribution of 848 proteins in the IMCD Proteome Database categorized by the CDDB identifiers (24).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have used LC-MS/MS-based mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. We present a WWW-based IMCD Proteome Database containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). Because 157 proteins are present in both lists, the current total count of proteins in the IMCD Proteome Database is 848. An important initial goal in this study was to validate the use of ICAT for large-scale quantification of proteins in isolated IMCD cells from kidney. We ultimately used ICAT to identify proteins that are increased or decreased in abundance in response to the long-term infusion of the V2R-selective agonist dDAVP. ICAT has been most successful in the quantitative proteomic study of regulatory processes in yeast (11), in which very large numbers of cells can be harvested. For example, in a study of the response of the yeast proteome to salt stress, a total of 800 µg were used for the starting material (25). In our initial studies, we found that similar amounts of kidney protein must be analyzed to identify all but the most abundant proteins. Thus ICAT (as applied in the present study) is relatively lacking in sensitivity, a factor that limits its practicality in some types of experiments, e.g., those involving prefractionation that may yield relatively small amounts of protein for analysis, or analysis of very small tissue elements such as those that may be harvested from developing embryos. In addition, the method is limited by the fact that it depends on labeling of cysteines. Consequently the method will be blind to many proteins that do not have cysteine moieties in tryptic peptides in a size range that is visible to the mass spectrometer. This includes AQP1 and AQP2, for example, proteins that are of considerable physiological importance in the renal inner medulla. Furthermore, as illustrated in Fig. 1C, the method is subject to considerable error even for relatively abundant proteins. Nevertheless, ICAT performed successfully in quantification of many relatively abundant proteins in the whole cell analyses presented in this paper, matching well with results from either immunoblotting or DIGE analysis.

One potential advantage of ICAT and LC-MS/MS in general over DIGE and other 2-D gel-based methods is the ability to quantify integral membrane proteins. In the present study, 9 of the total of 165 proteins (5.5%) were integral membrane proteins in the dDAVP infusion study, while 7 of 89 proteins (7.9%) were integral membrane proteins in the experiment in which we compared IMCD vs. non-IMCD cell fractions. In contrast, our previous studies (15) using DIGE for quantification identified 2 integral membrane proteins out of a total of 125 proteins (1.6%). Thus our results indicate that the combination of ICAT and LC-MS/MS indeed gives a greater yield of integral membrane proteins than does DIGE. Overall, we believe that 2-D DIGE and ICAT with LC-MS/MS are complementary methods that, when used in combination, will give a much higher yield of successfully identified and quantified proteins than either technique alone.

Another important issue addressed by our study is the need to isolate a tissue fraction that is as homogeneous as possible from the perspective of cell type. As illustrated in Table 1, many proteins are differentially expressed in IMCD and non-IMCD elements of the renal medulla. Attempts to quantify protein changes in response to a physiological perturbation based on analysis of whole inner medulla may therefore be reflective of IMCD cells or of non-IMCD cells. Furthermore, responses in IMCD cells may be masked by opposite changes in other cell types.

An important objective of the current study was to identify proteins in IMCD cells of rat whose abundances change in response to a long-term (3 day) infusion of the vasopressin analog dDAVP. As illustrated in Table 2, some proteins increased and some proteins decreased in abundance in response to dDAVP. The protein list in Table 2 can be considered a presumptive list of proteins regulated in response to long-term dDAVP administration. Members of this list can be considered targets for further hypothesis-driven investigation. The functional classification of these proteins was annotated using a terminology based on that of the CDDB (24) (http://cddb.nhlbi.nih.gov/cddb/). As shown in Fig. 10, cytoskeletal proteins and linkers/molecular motors, biosynthetic proteins, and proteins involved in energy metabolism appear to be the major types of proteins that responded to the long-term vasopressin action. Sixteen of the proteins that were quantified by ICAT analysis were investigated further by semiquantitative immunoblotting, which confirmed the direction of change demonstrated by ICAT in 14 of 16 proteins. The proteins that significantly changed in abundance based on immunoblotting were cathepsin D, GAPDH, heat shock 70-kDa protein (Hsp70), Rap1, and syntaxin-7. The responses were analyzed further by carrying out network analysis incorporating the core signaling pathway downstream from V2R occupation in IMCD demonstrated by previous studies (2, 3, 5, 8, 13, 14, 18, 23, 29, 36) and the five proteins validated by immunoblotting as described above. The functional interactions between proteins were culled from the literature through manual and computer-aided searching (IPA and MetaCore).

Overall, this study adds to the number of proteins known to populate the "IMCD Proteome." A long-term goal of our studies is to identify as many members of the IMCD proteome as possible to provide a database of information that will facilitate systems biological analysis (mathematical modeling) of cellular processes in IMCD cells. The database as it exists currently is available at http://dir.nhlbi.nih.gov/papers/lkem/imcd/index.htm and reflects IMCD proteins identified from five distinct studies (Refs. 1, 15, 16, and 34 and this study).

The remainder of the DISCUSSION will focus on the component of the network described in Fig. 12. The existing portion of the network (nodes indicated in gray) describe well-documented elements of vasopressin signaling demonstrated in prior papers. One objective of proteomics studies such as this one is to generate new hypotheses that can lead to critical experiments regarding signaling pathways. The new proteins, indicated in red, constitute hypothetical extensions of the existing network, linked to the existing network directly or via additional IMCD proteins indicated by uncolored nodes. All proteins in Fig. 12 have been specifically and unequivocally demonstrated to be expressed in the IMCD (IMCD Proteome Database, previous paragraph).

Syntaxin-7
Syntaxins are so-called t-SNARE proteins that, together with SNAP23 or SNAP25 and a synaptobrevin isoform, form a heterotrimeric coiled-coil SNARE complex that plays a critical role in vesicle fusion (20). Previous studies (26, 27) have demonstrated two syntaxins expressed in the IMCD, viz., syntaxin-3 and syntaxin-4, both of which are plasma membrane syntaxins. Subsequently, several endosomal syntaxins including syntaxin-7, syntaxin-12, and syntaxin-13 were demonstrated in AQP2-containing vesicles in IMCD cells (1). In the present study, the presence of syntaxin-7 in IMCD was confirmed, and its abundance was found to be upregulated in response to dDAVP infusion in Brattleboro rats. Syntaxin-7 is thought to be localized to either the early (31) or late (35) endosomal compartment. As previously described, AQP2 is regulated by vasopressin through separate processes that separately regulate exocytosis and endocytosis of the water channel (22). Conceivably, upregulation of syntaxin-7 abundance could be a component of the process regulating endocytosis.

Rap1
Rap1 is a small Ras-like GTP-binding protein that has been implicated in several regulatory processes in cells including activation of the MAP kinase pathway and mobilization of intracellular calcium through activation of calcium-induced calcium release channels in the endoplasmic reticulum (12). Rap1 is the downstream target of Epac, a guanine nucleotide exchange factor (GEF) that binds to and activates Rap1. Epac is a direct target for cAMP, which activates it. Hence, we can hypothesize that cAMP-induced calcium mobilization may be mediated by Epac and Rap1 as previously demonstrated in pancreatic ß-cells (19). This hypothesis is directly testable, since Epac-selective cAMP analogs are now commercially available. In the present studies, immunoblotting demonstrated an apparent decrease in Rap1 protein abundance in response to dDAVP, an effect that could attenuate the proposed role of Epac and Rap1. Rap1 has been demonstrated previously to be present in AQP2-containing vesicles in IMCD cells (1).

GAPDH
An increase in the IMCD abundance of GAPDH was demonstrated in the present study in response to dDAVP infusion, consistent with the prior studies showing an increase in GAPDH mRNA in response to dDAVP in the inner medulla (2). GAPDH is often considered a housekeeping protein and it is often used to normalize results from mRNA or protein measurements. However, our results indicating that GAPDH abundance can be regulated suggests that other normalizing measures should be sought. GAPDH is known as a glycolytic enzyme, but a variety of other functions have been demonstrated including a catalytic role in membrane fusion (10, 33). Thus increases in GAPDH abundance could be highly relevant to the regulation of AQP2 trafficking. GAPDH has been demonstrated to be a binding partner for tubulin, which inhibits GAPDH-catalyzed membrane fusion activity (10).

Hsp70
This study also demonstrated a dDAVP-induced increase in HSP70 expression in the IMCD, confirming previous results from DIGE-based studies (34). HSP70 is an abundant molecular chaperone. It has been demonstrated to be increased in abundance in cultured MDCK cells (6) in response to increased tonicity, leading us to speculate that the increase in HSP70 expression in the present study is a response to altered inner medullary tonicity rather than to dDAVP itself.

Cathepsin D
This is a renin-like proteolytic enzyme that was also demonstrated to be upregulated in response to dDAVP in the IMCD, confirming the findings of DIGE-based studies (34). This protein has also been demonstrated to be transcriptionally regulated by p53 (32), a protein that has been implicated recently in IMCD signaling in association with vasopressin escape (17).


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: M. A. Knepper, NIH, 10 Center Dr., Bldg. 10, Rm. 6N260, Bethesda, MD 20892-1603 (e-mail: knep{at}helix.nih.gov).

1 The Supplemental Material for this article (Supplemental Tables S1–S5) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00214.2005/DC1. Back


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
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