Absolute Quantification of the G Protein-Coupled Receptor Rhodopsin

University, Bozeman, Montana 59717, and Thermo Finnigan Corporation, San Jose, California 95134. Methods for the absolute quantification of a membrane...
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Anal. Chem. 2003, 75, 445-451

Absolute Quantification of the G Protein-Coupled Receptor Rhodopsin by LC/MS/MS Using Proteolysis Product Peptides and Synthetic Peptide Standards David R. Barnidge,*,† Edward A. Dratz,‡ Therese Martin,† Leo E. Bonilla,§ Liam B. Moran,§ and Arnold Lindall†

Neuromics Incorporated, Minneapolis, Minnesota 55414, Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, and Thermo Finnigan Corporation, San Jose, California 95134

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments. Integral membrane proteins represent a difficult analytical challenge as a class due to their hydrophobic nature and because they often occur in relatively low copy number. Integral membrane proteins perform a variety of vital roles in cell membranes ranging from ion channels and transmembrane pumps to mediators of signal transduction. In particular, G protein-coupled receptors (GPCRs) represent a large superfamily in this class with an estimated 600 different GPCR genes encoded in the human * Corresponding author. Curresnt address: Mayo Proteomics Research Center, Mayo Clinic, Rochester, MN 55905. Phone: (507) 266-4777. E-mail: [email protected]. † Neuromics Inc. ‡ Montana State University. § Thermo Finnigan Corp. 10.1021/ac026154+ CCC: $25.00 Published on Web 01/01/2003

© 2003 American Chemical Society

genome. Each GPCR protein is thought to have a similar framework structure but different binding pockets to accommodate specific ligands.1 GPCRs are also referred to as seven transmembrane receptors (7TMRs) due to the seven membranespanning helices indicative of these proteins. GPCRs are the targets of a large fraction of the therapeutic drugs currently on the market and are also a major focus for new drug development.2 Much of the information on the structure and function of GPCRs has been derived from rhodopsin, a prototypical GPCR that is the light receptor in the retinas of organisms from mammals to insects. The ligand in dark-adapted rhodopsin is 11-cis-retinal that is bound to rhodopsin by means of a Schiff base linkage to a lysine residue in the binding pocket of rhodopsin. The 11-cis-retinal undergoes a cis to trans conformational change upon absorption of a photon, transforming the retinal into an agonist that induces the G protein activating state of rhodopsin, called metarhodopsin II, triggering a signaling cascade that ultimately results in the detection of light by the organism.3 Just as rhodopsin has been used as a model GPCR for structural determination, it has also been used as a test bed for analytical methods designed to isolate and characterize membrane proteins. Examples of methods include the proteolysis of rhodopsin for structural characterization4-7 and more recently the proteolysis of rhodopsin to generate peptides for structural evaluation by mass spectrometry.8-10 Mass spectrometry (MS) has been used to characterize rhodopsin and its posttranslational (1) Sadee, W.; Hoeg, E.; Lucas, J.; Wang, D. AAPS PharmSci 2001, 3, E22. (2) Debouck, C.; Metcalf, B. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 93207. (3) Okada, T.; Ernst, O. P.; Palczewski, K.; Hofmann K. P. Trends Biochem. Sci. 2001, 26, 318-324. (4) Hargrave, P. A.; McDowell, J. H.; Curtis, D. R.; Fong, S. L.; Juszczak, E. Methods Enzymol. 1982, 81, 251-256. (5) Pober, J. S. Methods Enzymol. 1982, 81, 236-239. (6) Dratz, E. A.; Miljanich, G. P.; Nemes, P. P.; Gaw, J. E.; Schwartz, S. Photochem. Photobiol. 1979, 29, 661-670. (7) Miller, J. L.; Dratz, E. A. Vision Res. 1984. 24, 1509-1521. (8) Kraft, P.; Mills, J.; Dratz, E. Anal. Biochem. 2001, 292, 76-86. (9) Ball, L. E.; Oatis, J. E., Jr.; Dharmasiri, K.; Busman, M.; Wang, J.; Cowden, L. B.; Galijatovic, A.; Chen, N.; Crouch, R. K.; Knapp, D. R. Protein Sci. 1998, 7, 758-764. (10) Barnidge, D. R.; Dratz, E. A.; Sunner, J.; Jesaitis, A. J. Protein Sci. 1997, 6, 816-824.

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modifications,11-13 but MS has not been used to quantify rhodopsin. Membrane proteins are commonly quantified by techniques other than mass spectrometry, such as western blot analysis, radioligand assays, or immunoassays. While these techniques provide valuable information on the levels of membrane proteins, and can be extremely sensitive, they often lack the specificity and reproducibility that can be provided by mass spectrometry. Although mass spectrometry has been used for the absolute quantification of peptides for some time,14 there has been limited usage of proteolytic peptides for the express use of quantifying the parent protein. Noteworthy examples of the use of LC/MS to quantify proteins through the use of proteolytic peptides include the work by Barr et al., who demonstrated that LC/MS/MS could be used in place of immunoassay to carry out absolute quantification of apolipoprotein A-1,15 and the recent work by Jeppson et al., who established an IFCC-approved method for the measurement of glycated hexapeptide derived from the proteolysis of the β-chain of hemoglobin as a measure of the glycemic state of persons with diabetes.16 At present, much work is being done in the area of proteomics where, by definition, all the proteins that are detectable are examined at a given time. Many of the LC/MS/MS techniques utilized in proteomics incorporate stable isotopes, either chemically17-23 or in the growth media,24 to derive quantitative information on protein concentrations. These techniques give information on the relative concentration of many proteins in one sample versus another through the use of proteolytic peptides but do not provide absolute concentration levels due to the lack of true standards. Here we demonstrate that proteolysis, followed by the monitoring of a specific proteolytic fragment using tandem mass spectrometry, can be used to perform absolute protein quantification of a specific membrane protein. The starting concentration of rhodopsin in rod outer segments (ROS) used for these experiments was determined using a spectrophotometric assay. After the concentration of rhodopsin was calculated, aliquots of (11) Papac, D. I.; Oatis, J. E., Jr.; Crouch, R. K.; Knapp, D. R. Biochemistry 1993, 32, 5930-5934. (12) Papac, D. I.; Thornburg, K. R.; Bullesbach, E. E.; Crouch, R. K.; Knapp, D. R. J. Biol. Chem. 1992, 267, 16889-16894. (13) Lee, K. A.; Craven, K. B.; Niemi, G. A.; Hurley, J. B. Protein Sci. 2002, 11, 862-874. (14) Desiderio, D. M.; Kai, M. Biomed. Mass. Spectrom. 1983, 10, 471-479. (15) Barr, J. R.; Maggio, V. L.; Patterson, D. G., Jr.; Cooper, G. R.; Henderson, L. O.; Turner, W. E.; Smith, S. J.; Hannon, W. H.; Needham, L. L.; Sampson, E. J. Clin. Chem. 1996, 42, 1676-1682. (16) Jeppsson, J. O.; Kobold, U.; Barr, J. R.; Finke, A.; Hoelzel, W.; Hoshino, T.; Miedema, K.; Mosca, A.; Mauri, P.; Paroni, R.; Thienpont, L.; Umemoto, M.; Weykamp, C. Clin. Chem. Lab. Med. 2002, 40, 78-89. (17) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (18) Cagney, G.; Emili, A. Nat. Biotechnol. 2002, 20, 163-170. (19) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-2586. (20) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057. (21) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214-1221. (22) Ji, J.; Chakraborty, A.; Geng, M.; Zhang, X.; Amini, A.; Bina, M.; Regnier, F. J. Chromatogr., B. 2000, 745, 197-210. (23) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842. (24) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6596.

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ROS were digested and the resulting proteolytic peptides were subjected to quantitative analysis using LC/MS/MS. Recoveries were calculated for digests of low-picomolar amounts of rhodopsin. Our results show that synthetic peptides with the same amino acid sequence as a cleavage product from the protein of interest can be used to generate quantitative data on the absolute amount of the parent protein that is directly applicable to many current research needs in cell biology, protein chemistry, and clinical chemistry. EXPERIMENTAL SECTION Analysis of Rhodopsin Concentration in ROS Membrane Suspensions. ROS used for the experiments were prepared under dim red light to maintain the dark-adapted state for spectrophotometric quantification and to avoid possible phosphorylation of serine residues by endogenous kinases. The concentration of rhodopsin in ROS was determined using difference spectra (unbleached - bleached) at 498 nm using the established extinction coefficient for rhodopsin (40 000 L M-1 cm-1). Absorbance readings for unbleached ROS were obtained by thawing the material at room temperature followed by solubilization of the membrane in 4% lauryldimethylamine N-oxide (LDAO) (Sigma Chemical, St. Louis, MO), with each operation performed in dim red light. The solubilized ROS were then photobleached for 15 min using a high-intensity light source, followed by measurement of absorbance at 498 nm to record the bleached background absorbance value. All spectral measurements were carried out on a Beckman DU 65 single-beam scanning spectrophotometer. All data were acquired at 498 nm after background subtraction using a blank LDAO detergent solution. Digestion of Rhodopsin in ROS Membranes or after Detergent Solubilization. After the concentration of rhodopsin in the ROS membrane suspensions was determined, the suspensions were dispensed into two different aliquots, 2.58 and 5.16 pmol/µL. Aliquots of ROS membranes were either solubilized in a membrane solubilization buffer (6 M urea, 2% SDS, 100 mM TRIS, pH 8.5) or diluted in 50 mM TRIS, pH 8. Urea/SDS solubilized-ROS were reduced with 3 mM DTT for 30 min at 60 °C, alkylated with 6 mM iodoacetamide for 30 min at room temperature in the dark, and then diluted to lower the level of urea to 1 M and SDS to 0.33% prior to digestion to minimize inactivating the trypsin. Unsolubilized ROS membranes were not reduced and alkylated prior to digestion. Aliquots containing 5 pmol/µL rhodopsin were brought to a volume of 100 µL with 50 mM TRIS, pH 8, and digested with methyllysine-modified trypsin (Promega, Madison, WI) for 12 h at 37 °C at an enzyme-torhodopsin ratio of 1:100. After 12 h, a second 1:100 amount of trypsin to rhodopsin was added and allowed to incubate for 4 h more. The samples were acidified to a final concentration of 0.5% TFA after digestion to stop the reaction and centrifuged at 20000g for 30 min prior to analysis. LC/MS/MS Conditions. Peptide separation was performed using a 1.0 × 50 mm C18 column containing 5-µm bead size, 120-Å pore size TARGA media (Higgins Analytical, Mountain View, CA) run at 150 µL/min on a Thermo Finnigan Surveyor HPLC system (San Jose, CA). A linear gradient was used starting at 100% H2O with 0.1% v/v acetic acid added and ending with 80% ACN with 0.1% v/v acetic acid added over a period of 8 min (total run time 17 min). A sample volume of 10 µL was loaded on column via an

autosampler from a 96-well sample tray. A number of shorter runs (6 min) were also made using a 2.1 × 50 mm C8 column at a flow rate of 350 µL/min to examine higher throughput capacity, and the data from these shorter runs were comparable to the longer runs (data not shown). The synthetic peptide standard TETSQVAPA was made in two forms, an unlabeled form (MW 902.44) and a labeled form with three deuterium atoms on the methyl group of each alanine (average MW 908.43). A Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer was set up to run a single reaction monitoring (SRM) experiment. This experiment uses the selectivity of the triple quadrupole mass spectrometer by first allowing only ions with a specific mass, that of the intact peptide or parent ion, to be transmitted from the first quadrupole to the second quadrupole in the mass spectrometer (i.e., collision cell). The intact peptide ions are fragmented in the collision cell producing daughter ions. In an SRM experiment, only a single daughter ion is transmitted through the third quadrupole, resulting in the signal that ultimately is used for quantitation. In the case of the synthetic peptide ion TETSQVAPA, the parent-daughter transition of the singly charged parent peptide [TETSQVAPA]H+ to the b ion [TETSQVA]H+ (m/z 903.44-717.34) was optimized for signal intensity during direct infusion of the unlabeled standard. Optimized conditions were used for quantification, and the same conditions were used for the isotopically labeled peptide. The following parameters were used for the TSQ Quantum: spray voltage, 4.5 kV; sheath gas, 39 (arbitrary units); auxiliary gas, 3 (arbitrary units); capillary temperature, 350 °C; capillary offset, 35 V; tube lens offset, 214 V; collision energy, 26 V; collision gas pressure, 1.5 mTorr. Synthetic Peptide Standards. All peptides were synthesized on a Perseptive Biosystems instrument using standard FMOC chemistry. Crude peptide extracts were purified using HPLC to >80% purity and analyzed by amino acid analysis on a Pickering 5200 derivatizer. The net peptide content for the unlabeled peptide TETSQVAPA was 85.95%, and the labeled peptide was found to be 85.59%. All recoveries reported reflect these values for peptide concentration. Linear Response of Standards. Standard curves were made from the unlabeled synthetic peptide to evaluate the use of external standards for quantifying the native tryptic peptide cleaved from rhodopsin. The curves were made to span the range of the known amount of rhodopsin digested in each sample. Figure 1 shows standard curves for both the unlabeled peptide and labeled peptide. The standards covered a range from 100 fmol/µL to 8 pmol/µL and included a total of five standard concentrations within the range with a total of 10 µL of each standard loaded on column per injection. For each analysis, standard curves were placed at the beginning and end of the run to bracket the unknowns resulting in a total of two injections per point. Both 1/X and equal weighting operations were used to acquire the R2 values that were 0.994 or greater for the standard curve. RESULTS AND DISCUSSION Digestion Efficiency. Digestion of the parent protein into cleavage peptides must be complete in order to quantify the amount of a specific protein in a sample using a peptide cleavage product and appropriate synthetic peptide standards. To assess the efficiency of the tryptic digest protocol, a synthetic peptide mimicking the last 14 residues of rhodopsin (TTVSKTETSQVA-

Figure 1. Linear regression from the standard curve generated using the synthetic peptide TETSQVAPA (top) and the synthetic internal standard containing two deuterated alanines (bottom). The concentration ranged from between 100 fmol/µL to 8 pmol/µL and included a total of five standard concentrations. A total of 10 µL of each standard was loaded on column per injection with a total of two injections per concentration. Each standard curve was injected at the beginning and the end of the run to bracket the unknowns.

PA) and containing the same tryptic cleavage site as Lys 339 in the native protein was synthesized. The surrogate peptide was digested using the same digestion protocol for the ROS, as described in the Experimental Section, and analyzed using LC/ MS/MS. The undigested surrogate synthetic peptide and the digested peptide were monitored in SRM mode. Figure 2 shows SRM spectra monitoring the intact peptide TTVSKTETSQVAPA (bottom) and its cleavage product TETSQVAPA (top) after digestion with trypsin. The figure shows the complete absence of the starting peptide TTVSKTETSQVAPA and the presence of the product tryptic fragment TETSQVAPA. The tryptic cleavage product was quantified from two different experiments that were run on two different days using the same standard curve made from dilutions of a synthetic version of the tryptic peptide. Results from this experiment are shown in Table 1. Recoveries were calculated using the known amount of surrogate synthetic peptide standard TTVSKTETSQVAPA in the digestion, compared to the amount of the tryptic peptide found from the standard curve. The data in Table 1 illustrate that the digestion efficiency was quite good with recoveries ranging from 90% to 128% for the two Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

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Figure 2. LC/MS/MS in SRM mode (as described in the text) scanning for the unlabeled tryptic fragment TETSQVAPA, monoisotopic MW 902.43 (top) and the intact precursor synthetic peptide TTVSKTETSQVAPA, monoisotopic MW 1418.73 (bottom) from a tryptic digest of the precursor peptide used as a surrogate digestion substrate. Table 1. Recoveries of the TETSQVAPA Tryptic Peptide from Digestion of the TTVSKTETSQVAPA Precursor Peptide Used as a Surrogate Digestion Substrate concn, µmol/L sample type

by weight

from std curve

recovery, %

digest of the precursor peptide exp 1 digest of the presursor peptide exp 2

5.00 5.00 5.00 5.00 5.00 5.00

6.41 5.30 5.30 4.51 4.60 6.02

128 106 106 90 92 120

} }

CV, % 11 17

experiments. The CV values were also good with values of 11% and 17% for experiments 1 and 2, respectively. The mass spectrometer was also used to monitor for the peptide TETSQVAPA in the starting material, and none was observed (data not shown). These results led us to the conclusion that, for this model system, the digestion was near 100% completion and that a 1:1 molar ratio of starting material to cleavage product was confirmed. Digestion of Rhodopsin in ROS. Digestion of ROS to generate the C-terminal tryptic fragment TETSQVAPA from rhodopsin was approached using two techniques: digestion of rhodopsin in urea/SDS solubilized ROS and digestion of rhodopsin in intact ROS membranes. The first approach started with solubilization of the proteins in ROS using urea and SDS, mild sonication, reduction and alkylation, dilution of solubilizing agents to avoid trypsin denaturation, and finally digestion with trypsin. Similar steps are typically carried out to digest complex mixtures of proteins in order to maximize the number of peptides produced 448 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

Figure 3. Western blot of trypsin digested rhodopsin (triplicate samples lanes A-C), undigested rhodopsin control (lane D), and a blank (lane E).

from the proteins present. In the second approach ROS membranes were simply diluted in buffer and digested with trypsin in situ. These two approaches were evaluated to see whether membrane proteins with epitopes on the extracellular side of the membrane might be digested efficiently without having to solubilize the membrane, thus decreasing sample preparation time. The C-terminal tryptic peptide of rhodopsin is located on the cytoplasmic side in ROS disk membranes, which is external, and therefore makes a good model for quantifying the cleavage of external epitopes. Following digestion, samples were split and analyzed by western blot and LC/MS/MS. An example of a western blot of solubilized ROS digested with trypsin is shown in Figure 3. The figure demonstrates that the digestion was successful in quantitatively cleaving the C-terminal fragment from rhodopsin, since there was no evidence of antibody staining using the ID4 monoclonal antibody that recognizes the C-terminus of rhodopsin in western blots.25 The same yield of peptide was seen in the mass spectrometer for digestions of intact ROS membranes, indicating that the C-terminal epitope was efficiently cleaved without solubilization (data not shown). The results from the western blot analyses established that the C-terminal portion of rhodopsin was cleaved and the free peptide was not bound sufficiently to the nitrocellulose membrane to be detected.

Table 2. Recovery of the Rhodopsin Tryptic Peptide TETSQVAPA Determined Using an External Standard Curve concn, µmol/L sample type solubilized rhodopsin exp 1 solubilized rhodopsin exp 2

Figure 4. LC/MS/MS SRM chromatograms from the digestion products of rhodopsin in ROS membranes. The top trace monitors the native peptide from rhodopsin, monoisotopic MW 902.43, and the bottom trace monitors the isotopically labeled synthetic peptide, monoisotopic MW 908.43. The retention times of the native and isotopic standard are essentially identical.

Using the conditions stated previously, the other portion of digested material was subjected to analysis. Figure 4 shows two SRM ion chromatograms detecting the tryptic C-terminal peptide TETSQVAPA (top) digested from rhodopsin and the homologous isotopically labeled peptide spiked into the sample (bottom). The peak shape for the peptide eluting from the column was outstanding, with peak widths at fwhm approaching 3 s. Approximately 10 scans were taken across each peak, enabling good ion statistics to be attained. Similar results for peak width and reproducibility were found with the larger bore column with shorter run times (data not shown). Reproducibility in the retention times was excellent, with CV values less than 0.5% across 32 injections. Internal versus External Standards. Each experiment was carried out in triplicate on two different days. Quantification of the tryptic peptide TETSQVAPA released from rhodopsin was performed using two different approaches. The first approach compared the peak area of the peptide originating from rhodopsin to an external standard curve created from the equivalent synthetic peptide. The other approach compared the peak area of the peptide originating from rhodopsin to the peak area of an isotopically labeled internal standard peptide that was spiked into the sample prior to processing. For the results shown here, only the digestions done on solubilized ROS were spiked with internal standard peptide. Table 2A describes the amount of rhodopsin present in the ROS membranes used for digestion, the amount found by LC/ MS/MS, along with recoveries and CV values. It is assumed that the concentration of rhodopsin determined by visible spectroscopy is the correct value, since this methodology for calculating the concentration is well established. The average recoveries for experiment 1 and experiment 2 were 15% apart on average, with very similar CV values. Table 2B shows the recoveries for native rhodopsin where ROS membranes were digested in situ. The difference in the recoveries on average for experiment 1 compared to experiment 2 is 5%; however, the CV values are different, with

from vis spectra

from std curve

recovery, %

(A) Digested from Solubilized ROS 5.16 4.60 89 5.16 5.16 100 5.16 4.42 98 2.58 2.91 113 2.58 2.93 113 2.58 3.10 120

} }

6.1

3.6

(B) Digested from Native ROS Membranes native ROS 5.16 3.93 86.8 membranes 5.16 3.90 86.2 exp 1 5.16 3.92 86.6 native ROS 2.58 2.74 121 membranes 2.58 1.92 85 exp 2 2.58 1.59 70

}

CV, %

}

0.4

28

Table 3. Recovery of the Rhodopsin Tryptic Peptide TETSQVAPA from Digestion of Detergent-Solubilized ROS Membranes Determined Using an Isotopically Labeled Internal Standard Peptide and SRM Monitoring concn, µmol/L sample type

by weight

from internal std

recovery, %

solubilized rhodopsin exp 1 solubilized rhodopsin exp 2

5.16 5.16 5.16 2.58 2.58 2.58

4.98 4.48 4.34 3.87 4.10 4.05

110 87 84 150 159 157

} }

CV, % 17

4.1

experiment 1 having a value of 0.4% and experiment 2 having a value of 28%. The average overall recovery for the digests of solubilized ROS and native ROS was 97% with an overall CV of 16% using an external standard. Results from the use of an isotopically labeled internal standard are shown in Table 3. The average recovery using the internal standard for experiments 1 and 2 was 124% with CV values ranging from 17% for the first experiment to 4.1% for the second and an overall CV of 28%. The overall average recovery for rhodopsin from all the experiments performed was 106% with a CV of 24%. Replicate injections of the same standard peptide solutions, as well as for one experimental sample, gave CVs of 5% or better (data not show). Replicate injections were not carried out due to lack of material; however, it must be noted that identical experiments performed on different batches of ROS gave similar results when analyzed on different mass spectrometers (data not shown). The discrepancies observed in precision and accuracy for recoveries can likely be attributed to the variability in sample handling, most notably the dispensing of sample and buffers. The variability observed for sample concentrations determined by external standard curve versus internal standard is assumed to be due to errors in volumes delivered for internal standard. Internal standard methods are generally employed to overcome matrix-dependent or time-dependent responses of an analytical detector. Here, the high recoveries for the external standard method demonstrate that the calibration curve is valid for the Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

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Figure 5. LC/MS/MS SRM chromatograms from HEK cell membranes digested with trypsin. The top trace originated from ∼1.0 × 10-6 HEK cells expressing opsin and the bottom trace originated from approximately the same number of wild type HEK cells. This experiment illustrates the specificity of the LC/MS/MS SRM method for the peptide TETSQVAPA from a sample containing all the membrane proteins from HEK cells where opsin is a very small fraction of the total protein. The concentration of opsin determined in the sample was 74 µg, which is in good agreement with published values.26

digest matrix in which the peptides are analyzed and an internal standard is not necessary. If this technique is attempted in more complicated matrixes or if ballistic chromatography protocols are employed, matrix affects might manifest themselves and internal standards would then be required. If many different analytes are targeted simultaneously in the same runs, internal standards may also be of great value. Since the same ROS membrane stock was used for all the experiments, it is difficult to attribute discrepancies in the recoveries to incorrect values for the starting concentration of rhodopsin. To ensure that the concentration of rhodopsin was accurate, rhodopsin concentrations were also determined after addition of cis-retinal to the dark-adapted membranes under dim red light to regenerate endogenous bleached rhodopsin. The results were compared to samples where the rhodopsin was first fully photobleached and then regenerated with cis-retinal in the dark, followed by absorbance spectroscopy. The values derived using these regeneration experiments were nearly identical to the concentration determined using unbleached ROS, indicating that undetectable amounts of bleached rhodopsin were present in the ROS membranes used for this study. The recoveries for rhodopsin digested in native ROS membranes were comparable to the recoveries calculated from solubilized ROS, supporting the use of external epitopes on membrane proteins as targets for quantification following proteolysis. This is important since cleaving only the external epitopes from membrane proteins for quantification greatly reduces the complexity of peptide mixtures, which can translate into a more robust analysis. 450

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Digestion of Membranes Isolated from Cloned Opsin Expressed in HEK 293 Cells. To evaluate the specificity of the methodology in a whole-cell system where rhodopsin was expressed at relatively low levels compared to total cell protein, experiments were performed on HEK 293 cells expressing cloned bovine opsin (rhodopsin without the retinal) and compared to wild type HEK 293 cells.26 The wild-type cells do not express opsin but do contain the same background matrix of proteins in the cell line. Thus, these cells serve as an ideal control for determining specificity of the method. Figure 5 shows two SRM experiments from opsin-expressing cells (top) and wild type cells (bottom) illustrating the specificity of method. The peak observed in the top spectrum in Figure 5 is from cell membranes digested with trypsin (total cell membranes were isolated using ultracentrifugation from ∼1 × 107 lysed cells), and the peak representing the tryptic peptide TETSQVAPA is clearly visible. The spectrum in the bottom of Figure 5 is taken from cell membranes digested with trypsin from the same number of wild type HEK 293 cells not expressing opsin. No peak is evident in the lower spectrum, indicating that the method is specific for the identification of opsin. A total concentration of 74 µg of opsin was calculated for the opsinexpressing HEK 293 cells using an external standard curve, which is in good agreement with published values26 for this number of cells under the cell culture conditions used. (25) Molday, R. S.; MacKenzie, D. Biochemistry 1983, 22, 653-660. (26) Reeves, P. J.; Thurmond, R. L.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11487-11492.

CONCLUSIONS Overall the data demonstrate that a proteolytic peptide from an integral membrane protein may be used as a representation of that protein’s concentration in order to perform absolute quantification by LC/MS/MS. Using the model membrane protein rhodopsin, complete tryptic digestion was accomplished yielding a 1:1 molar ratio of cleavage peptide to starting protein. Also, a surrogate digestion substrate peptide was shown to quantitatively produce efficient digestion for the tryptic cleavage site essential for the generation of the proteolytic peptide used for quantification of rhodopsin. In addition, digestion of rhodopsin in ROS was shown to be complete by western blot using a monoclonal antibody that recognizes the C-terminal portion of rhodopsin where the tryptic fragment is located. Synthetic peptides having the same amino acid sequence as the C-terminal tryptic peptide from rhodopsin were used as standards, enabling absolute quantification to be performed and recoveries to be calculated. No modifications to the mass spectrometer, or the HPLC, were needed for the concentrations used in these experiments, making the method directly applicable to a high-throughput environment for other membrane proteins with similarly accessible proteolytic cleavage peptides. In the case of membrane proteins, many of the issues surrounding protein solubility are avoided since a soluble proteolytic peptide may be chosen to represent the intact protein. The number of specific proteases and chemical cleavage options currently available make it likely that a quantifiable proteolytic peptide can be obtained from almost any protein. In addition, use of a peptide as a standard instead of an intact protein creates a situation where, at least in theory, any protein can be quantified as long as a proteolytic fragment can be quantifiably obtained and

a synthetic peptide analogue can be made. Recently, broad-based proteomics approaches have been described that perform relative quantification on a large number of proteins in whole tissues or organisms, in response to a specific drug, stress, or genetic modification. Current proteomics techniques have the benefit of identifying a large number of proteins with perturbed expression levels; however, they provide relative levels of protein only and have limited dynamic range and sensitivity. The technique employed in this paper is quite complimentary to the global expression level searches in that it provides absolute quantification of a limited number of targets with greater sensitivity, dynamic range, precision, and speed. Indeed, selected peptide fragments found in the global search methods can be labeled and used as the quantification standards for the present method. Because of the relatively short analytical cycle times of the present method (minutes versus hours for the global search experiments), the levels of a specific set of proteins can be measured kinetically at many time points after the onset of a stress or acute drug dosing to perhaps more clearly elucidate regulatory pathways. ACKNOWLEDGMENT The authors thank Dr. P. J. Reeves and Dr. H. G. Khorana for kindly providing the HEK 293 cells expressing opsin, Dinesha Waleck at the University of Minnesota Microchemical facility for synthesizing the peptides used in this study, and John Butler and Dr. Ian Jardine for use of the mass spectrometer.

Received for review September 20, 2002. Accepted November 22, 2002. AC026154+

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