From the Departments of Biochemistry and Molecular Biology and
Anatomy and Cell Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203
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ABSTRACT |
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Among the mass spectrometry-based in vitro stable isotope labeling methods, the proteolytic 18O labeling method (2) is particularly attractive because it has the least technical variations. In this method, the stable isotopic (18O atom) labeling of peptides is achieved concurrently with the proteolytic digestion of proteins. Therefore the yield of each isotopically labeled peptide depends only on the effectiveness of proteolytic digestion in both samples compared. In contrast, the other in vitro stable isotope labeling methods (such as the isotope-coded affinity tag method (3)) have the isotopic labeling and proteolytic digestion of proteins occurring at different steps, meaning that the yield of each isotopically labeled peptide has greater variability because it depends on both the yield of the isotope labeling and the effectiveness of the proteolytic digestion.
Trypsin has been the protease most utilized in proteolytic 18O labeling methods. However, it is known that trypsin generates a mixture of isotopic isoforms resulting from the variable incorporation of either one or two 18O atoms (18O1/18O2) into each peptide (4). This not only makes quantification of the peptides complicated but also increases the error in the experimentally determined 16O- and 18O-labeled peptides ratios. We recently reported a Lys-N-based technique, which is advantageous over tryptic digestion because it incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide (5). Lys-N is a metalloendopeptidase (peptidyl-Lys metalloendopeptidase, EC 3.4.24.20) that cleaves specifically peptidyl-lysine bonds (-X-Lys-) in proteins and peptides (6). In this initial study with several model proteins the unique single 18O atom incorporation property of Lys-N was shown to provide accurate quantification results. Other advantages of the Lys-N-based technique shown in this initial study include no enzyme-catalyzed 18O back exchange and production of a less complex protein digest.
Here we report the first biological application of the Lys-N-based proteolytic 18O labeling method, demonstrating the technique on cytokine/lipopolysaccharide (LPS)-treated versus untreated (control) human retinal pigment epithelium (ARPE-19) cells. Retinal pigment epithelium (RPE) performs many functions required for the neural retina to work properly, including transport of nutrients and removal of waste products into/from the photoreceptor cells, regeneration of bleached visual pigment, and phagocytosis of the outer segments of the rods and cones. ARPE-19 was developed as a spontaneous derived cell line using cells from a 19-year-old male donor and is an in vitro model system of RPE that is ideal for physiological and biochemical studies (7). Effects of cytokine treatment, including tumor necrosis factor and interferon-
, on cultured RPE and ARPE-19 cells have been studied extensively, and the induction of several proteins have been reported (811). In addition, several proteomic studies on RPE or RPE-derived cell lines have also been reported (1215). Therefore, ARPE-19 cells (±cytokine treatment) will serve as a good model system to evaluate the Lys-N-based proteolytic 18O labeling method.
The goal of this study was to evaluate the practicability of Lys-N-based proteolytic 18O labeling method with biological samples. The study resulted in identification of about 600 proteins along with semiquantitative information on their expressions in the cytokine/LPS-treated and untreated cells with a minimum detection accuracy of a 2-fold change. This method can thus be used as a general comparative proteomic technique applied to other systems. The results also provide a more complete description of the proteome in ARPE-19 cells and proteome changes resulting from cytokine/LPS treatment.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Cytokine/LPS Treatment, and Cell Harvest
Human retinal pigment epithelium (ARPE-19) cells were obtained from the American Tissue Culture Collection (Manassas, VA). Cells were cultured to 80% confluency in T-175 flasks as described previously (16) at 37 °C under 95% air and 5% CO2 in Dulbeccos modified Eagles medium:nutrient mixture F-12 (Hams) 1:1 (DMEM-F12) with 10% fetal calf serum, 2% L-glutamine, and 0.5% antibiotic/antimycotic. The cells, before harvesting, were either 1) treated in growth medium for 24 h with a combination of cytokines/LPS consisting of human tumor necrosis factor
(3.25 ng/ml, Upstate Biotechnology, Lake Placid, NY), human interferon-
(50 ng/ml, Upstate Biotechnology), and Escherichia coli LPS (10 µg/ml, Sigma-Aldrich, St. Louis, MO) or 2) untreated (control), keeping them in medium for 24 h. After 24 h, the medium was removed from the flask, and the cells were washed with PBS twice and DMEM-F12 once and harvested in DMEM-F12 by scraping the cells from the flask. The harvested cell suspension was centrifuged at 150 x g for 10 min at 4 °C, the supernatant was removed, and the cell pellet was stored at 80 °C until use.
Protein Extraction and S-Carbamidomethylation
The stored cell pellet was resuspended in 2.5 ml of 2% SDS in 50 mM Tris-HCl, pH 7.5, buffer and sonicated for 60 s. The resulting homogenate was centrifuged at 8,000 x g for 30 min at 4 °C, and the supernatant was recovered. The extracted proteins were reduced by adjusting the solution to 1 mM DTT and incubating for 2 h at 50 °C. An S-alkylation treatment was then done by adjusting the solution to 2.5 mM iodoacetamide and incubating for 1 h at 25 °C in the dark. After the S-carbamidomethylation, 4 times the volume of cold acetone (10 ml) was added to the protein solution and allowed to stand at 4 °C for 60 min in the dark to precipitate the proteins before centrifugation at 8,000 x g for 30 min at 4 °C. The resulting pellet was dried in a SpeedVac concentrator, redissolved in 25 µl of 100 mM pH 10.0 buffer consisting of glycine-NaOH and 8 M urea, and then diluted 8 times with 100 mM glycine-NaOH buffer (pH 10.0). The protein concentration was determined by the modified Bradford method (17). After protein quantification, protein concentrations of the untreated control and cytokine/LPS-treated sample were adjusted to be exactly equal in concentration (4 µg/µl) with 100 mM pH 10.0 buffer consisting of glycine-NaOH and 1 M urea and used for the experiments described below.
Protein Digestion by Lys-N
Validation Study
Two equal aliquots of the above prepared untreated control cell protein samples (200 µg of protein/50 µl for each) were dried in a SpeedVac concentrator. In one tube, 25 µl of [16O]water was added and mixed with 4 µg of Lys-N in 25µl of [16O]water. In another tube, 25 µl of [18O]water was added and mixed with 4 µg of Lys-N in 25 µl of [18O]water. The final concentration of glycine-NaOH buffer (pH 10.0) and urea should thus be 100 mM and 1 M, respectively, in both the reaction tubes. The resulting two reaction tubes were then incubated at 25 °C for 18 h to digest the proteins. After incubation, the two digests were mixed in a 1:1 ratio and analyzed by liquid chromatography-tandem mass spectrometry.
Comparative Proteomic Study
A 50-µl aliquot from each protein extract sample (treated and untreated, 200 µg of protein/50 µl for each) was dried in a SpeedVac concentrator. The protein pellet from the untreated control sample was redissolved in 25 µl of [16O]water and mixed with 4 µg of Lys-N in 25 µl of [16O]water. The protein pellet from the cytokine/LPS-treated sample was redissolved in 25 µl of [18O]water and mixed with 4 µg of Lys-N in 25 µl of [18O]water. The resulting two reaction mixtures were then incubated at 25 °C for 18 h to digest the proteins.
Cation Exchange Chromatography
The two digests (treated sample and untreated control) were then mixed in a 1:1 ratio and separated by strong cation exchange chromatography as follows. The cation exchange chromatography was performed using a strong cation exchange MicroBullet cartridge (12 µ, 300 Å, 2.0-mm inner diameter x 25 mm, Michrom BioResources, Inc., Auburn, CA) using a syringe pump (NP 70-2213 pump, Harvard Apparatus, Holliston, MA) as a solvent delivery system (18). The strong cation exchange column was first equilibrated with 1 ml of 5 mM ammonium formate adjusted to pH 3.2. An amount of 150 µg of protein of the mixed digest (37.5 µl) was diluted to 500 µl with 5 mM ammonium formate (adjusted to pH 3.2) and loaded onto the strong cation exchange column using a flow rate of 100 µl/min. The bound peptides from the strong cation exchange column were eluted stepwise with 200 µl each of the following eluents at a flow rate of 200 µl/min: 10, 40, 50, 80, 100, 150, and 500 mM ammonium formate (adjusted to pH 3.2). The unbound fraction and each of the stepwise eluents were collected and dried in a SpeedVac concentrator. Each dried fraction was then reconstituted in 20 µl of 0.1% TFA in [16O]water and analyzed by reversed-phase LC-MS/MS. Adjustment of ammonium formate solutions to pH 3.2 was done with formic acid.
LC-MS/MS Analysis
All LC-MS/MS analyses were done using an UltiMate nano-HPLC system (Dionex, San Francisco, CA) consisting of an isocratic pump, an autosampler, a gradient pump module, and a column switching module interfaced to a QStar quadrupole/time-of-flight mass spectrometer (Applied Biosystems-MDS Sciex, Foster City, CA) via a nano-electrospray ion source (Applied Biosystems-MDS Sciex) and a metal sprayer (New Objective Inc., Woburn, MA). Protein digests (5 µl) were injected into a reversed-phase C4 trapping column (300-µm inner diameter x 1 mm, Dionex) equilibrated with 0.1% formic acid, 2% acetonitrile (v/v) and washed for 5 min with the equilibration solvent at a flow rate of 10 µl/min using an isocratic pump that pumped the solvent through an autosampler. After the washing, the trapping column was switched in-line with the reversed-phase analytical column (0.075 x 50 mm, New Objective Inc.) packed in-house with Jupiter C18 media (10 µm, 300 Å, Phenomenex, Torrance, CA). A gradient pump module was used to produce a linear gradient of acetonitrile from 2 to 35% in aqueous 0.1% formic acid over a period of 100 min at a flow rate of 200 nl/min. After the gradient elution, the acetonitrile concentration was increased by a step change to 80% and maintained for 20 min at a flow rate of 200 nl/min. The column effluent was passed directly into the nanoelectrospray ion source attached to the metal sprayer on which 2,050 V of electrospray voltage was applied. The mass spectrometer was operated in a data-dependent MS to MS/MS switching mode with the three most intense ions in each MS scan subjected to MS/MS analysis. Survey MS spectra (from m/z 400 to 2,000) were acquired in the TOF analyzer with 1-s accumulation time. The three most intense ions in each survey TOF-MS analysis were sequentially selected in the first quadrupole mass analyzer and fragmented in the collision cell by collision-induced dissociation with nitrogen gas, and then the generated fragment ions were analyzed in the TOF analyzer using a 2-s accumulation time per precursor ion. The collision energy used for each precursor ion was dynamically selected based on its m/z value and charge state. Previously selected precursor ions were excluded for 60 s. AnalystQS software (version 1.0, Applied Biosystems-MDS Sciex) was used for instrument control, data acquisition, and data processing.
Protein Identification
Proteins were identified by comparing all of the experimental product ion spectra of the peptides to the Swiss-Prot database using the Mascot database search software (Matrix Science, London, UK). Only human proteins were searched. S-Carbamidomethylation of cysteine was set as a fixed modification. 18O labeling of the carboxyl terminus of the peptide and the oxidation of methionine (methionine sulfoxide) were set as variable modifications in the database search. Mass tolerances for protein identification on precursor and product ions were both set to 0.2 Da. Strict Lys-N specificity was applied while allowing for one missed cleavage. A minimum Mascot search score of 15 was used as the cutoff for a positive identification.
Calculation of Corrected 16O/18O-Peptide Ratios
Precursor peptide ions identified by the Mascot database search were manually extracted from the LC-MS/MS raw data on AnalystQS software, and the peak intensities of 16O- and 18O-labeled peptides were obtained from the extracted signal. The peak intensities for the 16O- and 18O-labeled peptides were corrected for 1) the effect of 5% H216O in the [18O]water and 2) the contribution of the M + 2 isotope of the 16O-labeled peptide to 18O-labeled peptide peak. A corrected 16O/18O peptide ratio was calculated as described previously (5).
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RESULTS |
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Theoretically the 16O- and 18O-labeled peptide ratios of all the generated peptides should be 1:1. Corrected ratios of 16O- and 18O-labeled peptides for 121 Lys-N peptides arising from 40 different proteins are given in Fig. 1. For most of the peptides the corrected 16O/18O peptide ratios were within the 0.52.0 range compared with the theoretical ratio of 1. However, 21 peptides (17% of 121 peptides) gave ratios below 0.5 or above 2.0, implying false results for 17% of the peptides for biological samples analyzed by this method under the conditions specified.
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It is instructive to examine product ion spectra of Lys-N peptides produced by the low energy collision-induced dissociation used in this work. Product ion spectra of two representative Lys-N peptides are shown in Fig. 2. The first peptide is KNQLTSNPENTVFDA. This peptide shows a typical spectrum obtained for a Lys-N peptide. As can be seen in Fig. 2a, the predominant fragment ions observed in the product ion spectrum were b-type ions. This is in contrast to the product ion spectra of tryptic peptides in which y-type ions are generally more predominant than b-type ions (19). This may be attributed to the preferential localization of positive charges on - and/or
-amino group(s) of the amino-terminal lysine residue for Lys-N-generated peptides. The second peptide is KYNQLLRIEEELGS (Fig. 2b). This peptide has a basic amino acid residue (arginine) in the middle of the peptide. Such peptides are known to produce complex product ion spectra arising, at least in part, from internal rearrangements of the b-type ions (20). As can be seen in the product ion spectrum (Fig. 2b), predominant ions were still b-type with y-type ions containing the arginine residue also present. The spectra presented and the fact that 1,046 peptides from 584 proteins were identified in the comparative proteomic study (see below) confirm the effectiveness of the Mascot database search software in identifying proteins from Lys-N-generated peptides. This is seen even though the fragmentation patterns of Lys-N peptides are different from those of tryptic peptides and for peptides that have a basic amino acid residue in the middle of the sequence.
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These analyses resulted in the identification of 584 proteins in the cells, listed in Supplemental Table I along with peptide sequences identified. Relative abundances for most of these proteins were determined except for 22 proteins that were identified solely by a single carboxyl-terminal peptide because the carboxyl-terminal peptide of a protein does not incorporate 18O atom by this method. A total of 1,046 peptides were quantified through manual data analysis to determine the ratio of each peptide in the two samples. Note that any corrected 16O/18O peptide ratios greater than 10 or smaller than 0.1 were recorded as ratios >10 or <0.1 because this method may not be accurate for these large -fold changes (5). Proteins were identified from various cellular compartments. The highest number came from the cytoplasm (19%) followed by 17% from the membrane, 13% from the nucleus, 7% from the mitochondrial, 2% from the endoplasmic reticulum, and 1% from lysosomes. Approximately 32% of proteins were from unknown locations. As expected, this method has the capability of identifying membrane proteins, which are generally difficult to identify by the commonly used 2D PAGE method. As far as we know this is the most comprehensive description of a proteome in RPE that has been reported.
The expression levels of 11 proteins were found to be greater than 2-fold in cytokine/LPS-treated cells compared with untreated control cells (16O/18O peptide ratio <0.5) (Table II). Among the 11 proteins, five proteins (indoleamine 2,3-dioxygenase (21), intercellular adhesion molecule-1 (22), interferon-induced guanylate-binding protein (23), plasminogen activator inhibitor-1 (24), and tryptophanyl-tRNA synthetase (25)) were previously known to be induced by cytokines, providing further evidence supporting the quantitative reliability of the technique. The other six proteins are not previously known to be induced by cytokines. Further work will need to be done to substantiate that these identified proteins are in fact induced by cytokines.
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DISCUSSION |
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Our validation study revealed that peptides generated by cleavages on one or more of the following amino acid sequences: 1) -Lys-X03-Lys-, 2) -Glu-Lys-, and 3) -Pro-Lys-, may not be accurately quantified due to presumed incomplete cleavage at these sites under the given digestion conditions. The recognition of amino acid sequences that have been shown to give inaccurate quantification results is significant because the inaccuracy of the technique can be reduced by excluding peptides generated from one or more of these sequences. The incomplete protein digestion for particular amino acid sequences is a drawback of this method. Efforts to achieve complete digestion of substrate proteins by Lys-N are in progress.
We must assume that our results contain false positive results (8%) based on the validation experiment. One effective way to reduce false positive results would be to perform a reverse labeling experiment in which proteins from the control and experimental sample are digested in H218O and H216O, respectively, and compare the results with the results presented (control and experimental sample were digested in H216O and H218O, respectively). However, such an experiment would require an enormous amount of effort to complete unless software that can accelerate the data interpretation process becomes available as discussed below.
In the present study, quantification of each peptide was performed manually without the help of computational tools. It was an extremely time-consuming process. It took an experienced mass spectrometrist more than 3 months to complete the quantification of all 1,046 positively identified peptides (4 h/day). The manual analysis included the following: 1) extraction of each 16O- and 18O-labeled peptide peak from the LC/MS/MS raw data based on Mascot database search results, 2) measurement of signal intensities of each 16O- and 18O-labeled peptide, and 3) calculation of corrected 16O- and 18O-labeled peptide ratio by incorporating the contribution of M + 2 isotope of the 16O-labeled peptide to the 18O-labeled peptide peak. Recently Halligan et al. (26) developed a stand alone computational tool that quantifies the mass spectra of 18O-labeled peptides from an ion trap instrument. Development of such software that can handle data produced by any type of commercial instruments will greatly facilitate the use of the proteolytic 18O labeling method in comparative proteomics.
The identified proteins are assumed to be relatively high abundance proteins. In this study, we separated peptides by strong cation exchange chromatography followed by reversed-phase chromatography. We observed many peptides that were not subjected to MS/MS analysis in the survey MS spectra because their signal intensities were not within the three most intense signal peaks. Therefore there was no sequence information for these peptides, indicating that more peptides could be analyzed with the development of better peptide separation techniques. This confirms the importance of achieving better peptide separation before mass spectrometric analysis to identify low abundance proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, July 5, 2005, DOI 10.1074/mcp.M500150-MCP200
1 The abbreviations used are: 2D, two-dimensional; LPS, lipopolysaccharide; Lys-N, peptidyl-Lys metalloendopeptidase; RPE, retinal pigment epithelium; DMEM, Dulbeccos modified Eagles medium.
* This work was supported in part by NEI, National Institutes of Health Grant RO3 EY014020 (to M M.), Grant P20 RR016741 from the IDeA Network of Biomedical Research Excellence program of the National Center for Research Resources, and Grant P20RR017699 from the Center of Biomedical Research Excellence program of the National Center for Research Resources. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, 501 North Columbia Rd., Grand Forks, ND 58203. Tel.: 701-777-2760; Fax: 701-777-2382; E-mail: mmiyagi{at}medicine.nodak.edu
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REFERENCES |
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