Metabolic Labeling of Human Primary Retinal Pigment Epithelial Cells

May 17, 2017 - Center for Genetic Medicine, Children's National Medical Center, ... human retinal pigment epithelial cell (hRPE) culture prepared from...
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Metabolic Labeling of Human Primary Retinal Pigment Epithelial Cells for Accurate Comparative Proteomics Yetrib Hathout,*,† Jessica Flippin,† Chenguang Fan,† Pinghu Liu,† and Karl Csaky‡ Center for Genetic Medicine, Children’s National Medical Center, Washington, D.C. and National Eye Institute, National Institutes of Health, Bethesda, Maryland Received December 27, 2004

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation. Keywords: metabolic labeling • SILAC • human RPE • comparative proteomics • MALDI-TOF-TOF • proliferating cells • resting cells

Introduction Comparative proteomics is a versatile experimental approach that can provide a global snapshot of the proteome status between two samples. There are several methods to quantify protein ratios between two samples.1 With all methods, mass spectrometry plays a crucial role for both identification and quantification of proteins. Two-dimensional gel electrophoresis (2-DG) in combination with software assisted image analysis is still the preferred approach to obtain a quick snapshot of the protein expression pattern and compare the proteome of two or several samples.2-4 However, the technique relies on measurement of protein spot intensities and sometimes deals poorly with accuracy in estimating relative protein abundance changes especially when the spots are saturated or not very well focused (e.g., basic proteins). Thus, alternative methods were developed for comparative proteomics. Isotope coded affinity tag (ICAT)5 and proteolytic labeling in 18O enriched water6 in combination with shot-gun proteomics7 provided excellent ways to quantify protein ratios between two samples. While these existing methods are being applied to different studies8-11 and tested for reproducibility and accuracy, new alternative methods are being developed and evaluated. The * To whom correspondence should be addressed: Tel: (202) 884-3136. Fax: (202) 884-6014. E.mail: [email protected]. † Children’s National Medical Center. ‡ National Institutes of Health.

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metabolic labeling strategy is gaining popularity, especially in tissue culture assays.12,13 The metabolic labeling strategy was first tested on microorganisms for comparative proteomics,12 then extended to eukaryotic cells including mammalian cells13 and was found to be useful for accurate comparative proteomics. Because labeled and unlabeled cells can be mixed before protein extraction and processing, variations that would result from sample handling are minimized. Thus, more recent proteomics studies have reported more accurate quantitative proteomic data when using metabolic labeling by stable isotopes.14-21 Furthermore, in a recent study 15N metabolic labeling was evaluated for even more complex organisms such as Caenorhabaditis elegans and Drosophila melanogaster by feeding them on 15N-labled E. coli and yeast.22 Complete labeling of the proteins in these multicellular organisms was achieved, providing a way for accurate comparative proteomic in these systems. In the present study, we conducted metabolic labeling, using both 13C6, 15N2-Lys and 13C6-Arg, of hREP cells that were isolated from the eye of an 81 year old donor. hRPE cells were studied because of the presumptive importance of these cells in the pathophysiology of age-related macular degeneration (AMD), the leading cause of blindness in patients over the age of 60 years living in western societies. We show that the use of 10% whole serum in the culture medium does not interfere with the incorporation of stable 10.1021/pr049749p CCC: $30.25

 2005 American Chemical Society

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isotope labeled amino acids into the cellular proteins. This observation allows one to avoid the use of dialyzed serum15,19 which may compromise growth of primary cultures. The method was then tested to measure differential protein expression between dividing and resting human primary RPE cells. The goal of this study was to evaluate metabolic labeling of human primary RPE cultures and establish a reliable quantitative method for future comparative proteomic studies using RPE cells generated from eyes with retinal degenerative diseases versus those generated from healthy eyes.

Materials and Methods Isolation and Culture of hRPE cells. A pair of human eyes from an 81 yr old donor was received through Heartland Lions Eye Bank, Columbia, MO. The eyes were autopsied within a few hours after death and shipped in sterile medium. The anterior segment from each eye was removed by cutting around the iris and removing the anterior segment. hRPE cells were harvested according to a previously published procedure23 with some modifications. The vitrous and the retina were carefully peeled away from the RPE-choroid-sclera using a fine forceps. Then, the RPE-choroid was carefully separated from the sclera and placed face up in small sterile Petri dish with 0.1% Dispase (Roche, Indianapolis, IN) prepared in Ca2+. Mg2+ free Hank’s balanced salt solution was added to completely cover the tissue. After incubation at 37 °C for 3 h with occasional shaking, the supernatant was carefully aspirated and transferred to a sterile centrifuge tube. The tube was then centrifuged at 1000 rpm for 5 min and the pellet resuspended gently in Dulbecco’s modified Eagle’s medium DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA) and 100 U/mL each of penicillin and streptomycin. The suspended cells were then transferred to T25 culture flasks and placed in an incubator at 37 °C and 5% CO2 under humidified atmosphere. Fluorescence Activated Cell Sorter (FACS) Assay. To check if the obtained cell cultures were of epithelial origin, aliquots were taken from each culture flask, trypsinized and processed for flow cytometry analysis using anti-cytokeratins CAM 5.2 (BD Biosciences, San Jose, CA) which are specific to Moll’s peptides #7 and #8.24 A negative control was performed using human fibroblast cultures. Metabolic Labeling of hRPE Cells. A suspension of RPE cells (about 150 000 cells) was incubated in custom-made DMEM/ F12 medium without Arg nor Lys (Atlanta Biologicals, Lawrenceville, GA) to which the following were added: 10% FBS (Invitrogen, Carlsbad, Ca), 100 U/mL each of penicillin and streptomycin, 13C6-Arg (147.5 µg/mL) and 13C6, 15N2-Lys (91.25 µg/mL) (Cambridge Isotope Laboratories, Inc., Andover, MA). In parallel, the same amount of hRPE cells were grown in the same media except that unlabeled L-Arg and L-Lys were added instead of isotopically labeled. The cells growing in labeled medium were sub-cultured to passage 4 then harvested before confluence (e.g., 75% confluence). Cells grown in unlabeled medium were grown to confluence and maintained in culture for an extra two weeks after confluence. Cell Fractionation and Protein Extraction. Both labeled and unlabeled cells were harvested separately and processed immediately for cytosolic protein extraction following the method described by Ramsby et al.25 Briefly, the cell monolayer were washed twice with PBS solution then treated with 5 mL of trypsin EDTA solution (Sigma, St Louis, MO) for 2 to 3 min at 37 °C. Then, 15 mL of DMEM/F12 medium containing 10%

research articles FBS was added to each flask to stop trypsin activity. The cell suspensions were centrifuged at 300 × g for 5 min, then the pellets were washed twice with PBS solution, followed by two washes with ice cold solution of NaCl (100 mM). Cells were pelleted by centrifugation at 300 × g between each wash. The pellets were then weighed and digitonin extraction buffer (10 mM PIPES, 0.015% digitonin, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, and 1 mM PMSF, pH 6.8) was added to the pellet (3.7 mL per gram of wet cells). Extraction of cytosolic proteins was carried out in ice by gentle shaking for 10 min. Cell debris and organelles were separated from the cytosolic fraction by centrifugation at 500 × g for 10 min. The pellets were stored at -80 °C for further fractionation and protein extraction. The supernatant containing cytosolic proteins was collected in clean polypropylene tubes and further centrifuged at 16 000 × g for 15 min to remove any residual debris. Aliquots were taken from the supernatant and protein concentration was measured using a Bio-Rad protein assay reagent following the manufacturer instruction (Bio-Rad, Hercules, CA). The cytosolic extracts were then aliquoted and stored at -80 °C until analysis. To check the degree of incorporation of stable isotope labeled amino acids in the proteins of hRPE cells, 75 µg of cytosolic proteins extracted from labeled hRPE cells were processed for 2-DG/mass spectrometry analysis. After desalting the protein sample against 10 mM Tris HCl pH 7 using p6 BioSpin columns, the protein solution was dried by centrifugation under vacuum then 180 µL of rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 50 mM DTT and 0.5% ampholite pH 3-10 was added to solubilize and denature the proteins. The first dimension electro-focusing was performed on IPG strips (11 cm, pH 3-10) using a Bio-Rad electrofocusing chamber (Bio-Rad, Hercules, CA) operated as follows: 12 h rehydration, 250 V for 15 min, 1000 V for 1 h and 10 000 V for 4 h). The second dimension SDS-PAGE was performed on criterion Tris-HCl gels (8 to 16%) pre-cast gels (Bio-Rad, Hercules, CA). Protein spots were visualized using Bio-Safe Coomassie stain (Bio-Rad, Hercules, CA). The gel was then scanned on GS800 densitometer (Bio-Rad, Hercules, CA) and imaged as TIFF file. Twenty protein-spots were randomly excised from the gel and processed for in-gel tryptic digestion as previously described by Jensen et al.26 with some modifications (e.g., reduction and alkylation steps were omitted). The resulting peptides from each protein spot were desalted using C18 ZipTip micropipet tips (Millipore Co., Bedford, MA) following the manufacturer’s User Guide and analyzed by mass spectrometry as described below. Comparative Proteomics between Dividing and Resting hRPE Cells. Equal amounts of proteins from labeled dividing hRPE cells and unlabeled resting hRPE cells (200 µg each) were mixed then processed for two-dimensional gel electrophoresis as described above except that large format gels were used (17 cm IPG strip pH 3-10 and 20 × 20 cm Tris HCl pre-cast gel 8-16%). A total of 156 spots were excised from the gel and digested with trypsin as described previously26 with some modification to the protocol (e.g., the reduction and alkylation steps were omitted). The in-gel digestion of the 156 spots was carried out into two 96 polypropylene well plates using a 12 channel pipettman to facilitate the multiple washing steps. After digestion, peptides were extracted and transferred to clean 96 polypropylene well plates then desalted using C18 ZipTip micropipet tips (Millipore Co., Bedford, MA). The peptides were Journal of Proteome Research • Vol. 4, No. 2, 2005 621

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Figure 1. Scatter plot showing cell size versus cytokeratin-staining intensity. hRPE culture contains a homogeneous cell population with more than 90% of them stained positive to cytokeratin while in fibroblast culture more than 97% of the cells stained negative to this same cytokeratin marker.

eluted from the ZipTip in 10 µL of acetonitrile/0.1% TFA (70: 30, v/v) and stored at -80 °C until analysis. Mass Spectrometry Analysis and Protein Identification. Typically 0.3 µL of peptide solution is mixed with 0.3 µL of matrix solution (50 mM R-cyano-4-hydroxynamic acid in acetonitrile/0.1% TFA (70:30, v/v)) on a well on the MALDI plate. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses were performed on a 4700 ABI TOF-TOF mass spectrometer (Applied Biosystems, Foster City, CA) equipped with Nd:YAG 200 Hz laser. The instrument was operated with delayed extraction in reflectron positive ion mode. A mixture of standard peptides (4700 Cal Mix from Applied Biosystems, Foster City, CA) was used to externally calibrate the instrument. Protein identification was carried out using the GPS explorer software (Applied Biosystems, Foster City, CA) by entering the mass list of unlabeled peptides. Most of the searches gave confidence scores based on Mascot criteria. Protein identity was further confirmed by checking if each assigned peptide has its labeled partner. If the peptide contains one Lys residue, the labeled and unlabeled peptides will be separated by 8 Da, while peptide containing one Arg residue will be separated by 6 Da. Peptides that contained more than one Arg or Lys residue or a combination of both were also detected and checked for consistency. Protein Ratio Measurements and Data Normalization. The raw mass spectrometry data (e.g., mass list and intensities) obtained for each spot were subjected to computational analysis using SAS software (SAS Institute, Inc., Cary, NC). Basically, the height of the mono-isotopic peak of unlabeled peptide was divided by the height of the mono-isotopic peak of the corresponding labeled peptide. For accurate protein ratio measurement, ratios were calculated from all the peptides detected per protein-spot then averaged. Normalization was performed using the ratio values of several nondifferentially expressed proteins whose ratio between dividing and resting cells were equal or close to 1.

Results and Discussion Characterization of RPE Cells. The cells isolated from the retina of autopsied eyes resulted in well growing colonies that displayed a mixture of fibroblast-like and epithelial-like morphology under the microscope (data not shown). This fibro622

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blastic phenotype observed in primary hRPE cell culture was previously reported by others.27-29 The hRPE which have very distinct morphology in vivo become rapidly fibroblastic and invasive when explanted in vitro.27-29 In our study, the isolated hRPE cells were able to grow well up to 12 passages. To further characterize these growing cells, FACS analysis was performed using antibody against cytokeratins 7 and 8 as marker24 to distinguish epithelial from fibroblastic origin. Figure 1 shows a Scatter plot of cell size versus cytokeratin-staining intensity. hRPE culture, prepared from an autopsied eye from an 81 year old donor contained a homogeneous cell population with more than 90% staining positive to cytokeratin while a human fibroblast cell line demonstrated only 3% of the cells staining positive to this same cytokeratin marker. These data suggest strongly that the isolated cells are of epithelial origin. Metabolic Labeling of hRPE Cells in Culture. To check the degree of incorporation of the exogenous stable isotope labeled amino acids (e.g., 13C6-Arg and 13C6, 15N2-Lys) in the proteins of hRPE cells, the cells were grown in the labeled medium as described above then harvested at passage 4 and 75% confluence. The cytosolic proteins were extracted and processed for 2-DG separation. Twenty protein spots were randomly chosen, excised, digested with trypsin and the resulting peptides analyzed by MALDI-TOF/MS. Since cells were labeled with both Arg and Lys and since trypsin cuts after these amino acid residues it would be expected to get an increase in mass in every single tryptic peptide that contains Arg or Lys or both. A peptide containing one Arg residue would show an increase in mass of 6 Da (e.g., 13C6-Arg), while a peptide containing one Lys residue would show an increase in mass of 8 Da (e.g., 13C6, 15N -Lys). An example of a spectrum obtained for a protein spot 2 corresponding to human triosephosphate isomerase is shown in Figure 2. Most of the major peaks seen in the spectrum (Figure 2) are 6 or 8 Da higher in mass than those expected for the tryptic digest of this protein. Inserts in Figure 2 show expanded regions of the spectrum providing more details about the relative intensity of labeled and unlabeled peptides. The average ratio between labeled and unlabeled triosephosphate isomerase was calculated based on 11 tryptic peptides and indicated that 94 ( 4% of the total protein was labeled. The degree of labeling with 13C6-Arg (98 ( 2%) was found slightly higher than that with 13C6, 15N2-Lys (93 ( 4%). This slight

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Figure 2. MALDI-TOF mass spectrum of tryptic digest of triosephosphate isomerase spot showing the degree of incorporation of stable isotope labeled amino acid in this protein. Primary hRPE cells were grown in a medium where 12C6-Arg and 12C6-Lys where replaced by 13C6-Arg and 13C6, 15N2-Lys. The cells were propagated to passage 4 in the labeled medium then the proteins were extracted from the cells and processed for 2-DG analysis. Protein spots were then excised, digested by trypsin and the resulting peptides analyzed by MALDI-TOF/MS in reflectron positive mode using the 4700 ABI TOF-TOF instrument. The expanded spectrum regions show the relative intensities of labeled peptide and unlabeled peptide along with the labeling percentage and the peptide sequences.

difference found between the incorporation of Lys and Arg is probably due to the difference in their amounts formulated for DMEM/F12 medium (Lys/Arg molar ratio ) 0.71). Overall, efficient labeling was obtained and similar degree of incorporation (∼94%) was found in 20 protein spots analyzed. This degree of incorporation reached a steady state and did not increase to 100% even after propagating the cells in the labeled medium for more than 4 passages (∼7 cell population doublings). Most importantly, the incorporation of these exogenous stable isotope amino acids was not impaired by the presence of 10% whole serum, the commonly used serum for the growth of most of the cells in vitro, thus avoiding the necessity of using dialyzed serum13,15,19 which is rather less common for cell and tissue cultures. In addition, dialyzed serum is devoid of low molecular weight components (e.g., dialyzed serum is prepared by dialysis of whole serum against 0.15 M NaCl using 10 000 molecular weight cut off cartridges) and could easily impair the growth of cells that necessitate low molecular mass growth factors (such as estrogen in epithelial breast cancer cells).21 Quantitative Proteome Profiling between Resting and Dividing hRPE Cells. A total of 200 µg of fully labeled proteins (e.g., cytosolic fraction) prepared from a 75% confluent hRPE culture (dividing cells) was mixed with 200 µg of unlabeled cytosilic proteins extracted from a fully confluent hREP culture (2 weeks post-confluent culture). The protein mixture was processed for 2-DG and a total of 156 spots were excised, digested with trypsin and analyzed by MADI-TOF-TOF/MS as described in Methods and Materials. High mass accuracy (better than 50 ppm) and high resolution (better than 7000) was routinely obtained for each MALDI-TOF-TOF/MS analysis permitting both accurate quantification and identification of the proteins. Typical results obtained from the analysis of three

different protein spots are shown in Figure 3 (top spectrum for actin, middle spectrum for 60S acidic ribosomal protein and bottom spectrum for alpha Crystallin B chain protein). The labeled and unlabeled peptides for actin were found to have almost similar intensities indicating that this protein was not differentially expressed between dividing and resting cells. On the other hand, 60S acidic ribosomal protein was up-regulated in dividing cells relative to resting cells while R-crytsallin B chain protein was down-regulated in dividing cells relative to resting cells. The 60S acidic ribosomal protein is involved in protein synthesis and its increased expression in dividing cells versus resting cells is in agreement with its function. The increased expression of R-crystallin B chain protein in resting cells versus dividing cells is in agreement with previous findings demonstrating that differentiating rat RPE-J cells express more of this protein than proliferating rat REP-J cells.30 Thus, crytsallin proteins seem to accumulate in differentiating RPE cells. R-crystallin B chain, is a major protein expressed in most mammalian lens and has a chaperone like activity and may prevent precipitation of denatured proteins and increase cell tolerance to stress.31 Overall, a total of 156 proteins were analyzed and their ratios in resting versus dividing cells were determined (see Supporting Information). We implemented a computational strategy using SAS to directly use the mass spectrometry raw data and obtain ratios measurements for all the protein spots analyzed. Since cells were labeled with both Arg and Lys and a 2-DG was used to separate the proteins, several peptide pairs (e.g., labeled and unlabeled) per protein spot were obtained (see example in Figure 3) thus providing more accurate and reliable quantification and identification as well. Labeled and unlabeled peptides were used to validate each other and confirm the protein identity (see Supporting Information). Journal of Proteome Research • Vol. 4, No. 2, 2005 623

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Figure 3. MALDI-TOF mass spectra of tryptic digest of three spots corresponding to actin (top spectrum), 60S acidic ribosomal protein (middle spectrum) and R-crystallin B chain protein (bottom spectrum) prepared from a 1:1 mixture of labeled (dividing cells) and unlabeled (resting cells) proteins. 200 µg of cytosolic proteins extracted from dividing hRPE cells grown in 13C6-Arg/13C6, 15N2-Lys labeled medium were mixed with 200 µg cytsolic proteins extracted form unlabeled resting RPE cells. The mixture was then processed for 2-DG analysis. Protein spots were then excised, digested by trypsin and the resulting peptides analyzed by MALDI-TOF/MS in reflectron positive mode using the 4700 ABI TOF-TOF instrument.

Figure 4. Differential protein expression profile between dividing and resting hRPE cells based on the analysis of 156 cytosolic proteins. The numbers on the top of each histogram correspond to the number of proteins that have the indicated ratio in the x axis.

The distribution of protein expression ratios (n ) 156) is shown in Figure 4. A total of 54 out of 156 cytosolic proteins analyzed were found unchanged in their relative expression between dividing and resting (ratio equal or close to 1). Therefore we used the average ratio values of these 54 proteins to calculate the normalization factor which was found equal to 0.98 ( 0.14. A partial list of proteins whose expression changed between dividing and resting cells is shown in Table 1. The theoretical MW/pI values and the number of peptides 624

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used to calculate the ratios are also included in the table. Proteins that were found highly expressed during cell division were primarily those involved in protein synthesis, cell cycle and cell proliferation. Proteins that were found highly expressed in resting cells versus dividing cells were mostly those involved in actin and cytoskeleton remodeling as well as chaperon proteins. The differential expression of these proteins seems to agree with their role and function thus supporting the data obtained. For example thymosin beta 4 and gelsolin are both known to bind to monomeric actin and prevent it from polymerizing. The fact that these two proteins are up-regulated in confluent RPE cells is in agreement with the recent study where it has been shown that the transition between polymerized actin (F-actin) and depolymerized actin (G-actin) depends on the cell density.32 Dense fibroblast cultures (e.g., confluent cells) have more of the depolymerized form of actin than dividing cells. Furthermore, we found that the combination of the resolving power of 2-DG electrophoresis with metabolic labeling can provide an excellent way to measure ratios of protein isoforms whose relative abundances change depending on the cell cycle. Indeed, the ratio of phophorylated/dephosphorylated cofilin-1 was found to fluctuate between dividing and resting hRPE cells (Figure 5). Both phosphorylated and unphosphoryalted cofilin-1 have been previously characterized and mapped on the 2-DG array of MCF-7 cells.33 The spot on the left (see Figure 5) corresponds to the phosphorylated form of cofilin-1, while the spot on the right corresponds to the dephophorylated form of

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Table 1. List of Proteins that Were Found Differentially Expressed, by at Least a Factor of 1.5, between Confluent and Dividing RPE Cells protein name and accession no.

ratio (resting/dividing)

peptide count

function

60S acidic ribosomal protein (P05387) 40S ribosomal protein S12 (P25398) Cofilin-1 phosphorylated form (P23528) 40S ribosomal protein SA (P08865) Purine nucleoside phosphorylase (P00491) Galectin-1(P09382) Eukaryotic translation initiation factor 5A (P10159) UDP-glucose 6-dehydrogenase (O60701) Ribose-phosphate pyrophosphokinase (P60891) Elongation factor 2 (P13639) RuvB-like 1 (Q9Y265) Stathmin (P16949) Isocitrate dehydrogenase (O75874) Thymosin β-4 (P62328) Alcohol dehydrogenase (P14550)

0.38 ( 0.03 0.38 ( 0.06 0.42 ( 0.04 0.44 ( 0.17 0.55 ( 0.03 0.56 ( 0.07 0.61 ( 0.07 0.64 ( 0.08 0.66 ( 0.05 0.66 ( 0.06 0.67 ( 0.08 0.67 ( 0.05 1.51 ( 0.16 1.57 ( 0.09 1.57 ( 0.13

5 6 6 8 8 7 6 15 12 14 7 6 18 3 14

Translationally controlled tumor protein (P13693)

1.64 ( 0.24

7

Myosin light polypeptide 6 (P60660) Annexin A4 (P09525)

1.70 ( 0.07 1.75 ( 0.04

8 17

Heat shock 27 kDa protein (P04792) Tropomyosin 1 R-chain (P09493) Phosphoserine aminotransferase (Q9Y617) Gelsolin (P06396) Myosin regulatory light chain 2 (P24844) Transgelin (Q01995) R-Crystallin B chain (P02511)

1.84 ( 0.10 1.91 ( 0.01 1.97 ( 0.40 2.30 ( 0.10 2.53 ( 0.07 2.57 ( 0.30 3.65 ( 0.18

12 17 14 16 5 9 7

protein synthesis protein synthesis cell cycle protein synthesis nucleotide synthesis cell differentiation and tissue construction. protein synthesis Biosynthesis of glycosaminoglycans nucleotide synthesis protein syntehsis play a critical role in nuclear events involved in cell proliferation carbohydrate metabolism inhibit actin polymerization Catalyzes the NADPH-dependent reduction of aldehydes. function not well defined but bind to tubulin in the cytoskeleton actin-modulating protein may be involved in budding of clathrin coated pits. chaperone protein actin-modulating protein L-phospho serine metabolism actin-modulating protein actin-modulating protein actin cross-linking protein chaperone protein

a

Values and standard deviation are determined using the number of peptide count indicated for each protein.

phosphorylated cofilin-1 in dividing cells than in resting cells (see bottom spectra in Figure 5). This is in agreement with the fact that cofilin-1 is an actin-depolymerization factor and gets phophoryalated or dephosphorylated depending on the cell cycle.34 Indeed, it has been demonstrated that LIM motifcontaining protein kinase 1 (LIMK1) regulates actin reorganization by phosphorylating and inactivating cofilin during prometaphase and metaphase cell cycle.

Conclusion Overall, labeling of human primary cell cultures using both C6-Arg and 13C6, 15N2-Lys yielded reliable data for quantitative differential proteomics. Since trypsin cuts after Arg and Lys residues, measurement can be obtained for every single peptide except for those that does not contain any Arg or Lys residues. Even though the stable isotope labeled amino acids are quite expensive, the cost/benefit is justified since accurate measurements are obtained thus avoiding the need for repetitive analysis and the use of other reagent which may also be quite expensive. In addition, if there is a need to decrease the amount of the stable isotope labeled amino acids, one can easily scale down the cell culture and use smaller flasks thus minimizing the cost without compromising the accuracy in the results. The metabolic labeling by stable isotope labeled amino acids was found reliable since protein ratios can be accurately measured between slightly confluent (75%) and completely confluent hRPE cells. In this preliminary study we studied the cytosolic fraction and we used 2-DG approach in combination with MALDI-TOF analysis as proof of principle. A SAS algorithm was used to obtain a profile ratio of 156 proteins directly from the text file raw data. For comprehensive comparative proteomics, we are developing a high-throughput analysis method using automated LC-MS and LC-MS/MS. However, for this shotgun approach a more automated way to measure peptide ratio is needed since thousands and thousands of pairs of peptides are generated. Thus far, only few laboratories (Mann

13

Figure 5. MALDI mass spectra showing the ratios of phosphorylated and unphosphorylated cofilin-1 in dividing (heavy peptide) versus resting (light peptides) hRPE cells. (A) zoomed-in portion of 2-DG array of the 1:1 mixture of the cytosolic proteins extracted from 13C6, 15N2-Lys/13C6-Arg labeled dividing RPE cells and from unlabeled resting hREP cells. (B) MALDI-TOF mass spectra of trysptic digest obtained from phosphryalated and unphosphoryalted colfilin-1. (C) expanded m/z region showing the isotopic distribution of cofilin tryptic peptide YALYDATYETK. The ratio of the unlabeled peptide (m/z 1337.7) to the 15N2, 13C6Lys labeled peptide (m/z 1345.7) are calculated from the peak heights. These ratios were found constant throughout other peptides and reflect the amount of the protein in dividing versus resting RPE cells

cofilin-1. In this study, using metabolic labeling with stable isotope amino acids, we were able to obtain the ratio between phosphorylated and dephosphorylated cofilin-1 in dividing versus resting RPE cells. The results indicate that there is more

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research articles and co-workers) have the ability to quantify large number of proteins in an automated way when using SILAC strategy. A SILAC-specific software (MSQuant) has been developed and was found reliable for quantifying ratios of large number of peptides.19 In this study, we demonstrated that one can fully label human primary RPE cells with stable isotope amino acids even in the presence of 10% whole serum. The use of 10% whole serum instead of dialyzed serum is important since dialyzed serum, which is prepared by a 10 kDa cut off dialysis membrane, is typically devoid of low molecular weight growth factors which are essential for the growth of many primary cell cultures. In addition whole serum is commonly used instead of dialyzed serum in a number of cell culture systems. However, the degree of labeling by stable isotope amino acids could still depend on the type of cells and the medium used to grow them. So, for each system, it is better to first check the degree of labeling, then carry out the comparative study.

Acknowledgment. We would like to thank Robert Smith for his generous donation to support this project. Financial support to Dr. Karl Csaky was provided by NIH/NEI intramural program. We also thank Dr. Nataly Strukinnova for her advice on RPE isolation and culture and Dr. Eric Hoffman for his helpful discussions. Supporting Information Available: Representative 2-DG of cytosolic fraction prepared from primary cultures of human retinal pigment epithelial cells isolated from an eye of 81 year old donor. (Figure S1) and spot nos., protein names and Swiss-Prot accession nos. (along with molecular weights, pI’s, and peptide counts (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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