Identification and Relative Quantitation of an Orphan G-Protein

TECHNICAL NOTE pubs.acs.org/jpr

Identification and Relative Quantitation of an Orphan G-Protein Coupled Receptor SREB2 (GPR85) Protein in Tissue Using a Linear Ion Trap Mass Spectrometer Masatoshi Yuri, Masashi Hiramoto, Masanori Naito, Mitsuyuki Matsumoto, Shun-ichiro Matsumoto, Shuji Morita, Keitaro Mori, Hiroyuki Yokota,* and Toshio Teramura Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan ABSTRACT: SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate. KEYWORDS: G protein coupled receptor, SREB2, GPR85, linear ion trap mass spectrometer, proteotypic peptide

’ INTRODUCTION The superfamily of G protein-coupled receptors (GPCRs) is a large group of membrane receptor proteins that mediate a variety of important cellular signal transduction events. Over 90% of GPCRs are expressed in the central nervous system (CNS), and half of these are orphan receptors for which endogenous ligands have yet to be identified.1 Recently, the SREB (Superconserved Receptor Expressed in Brain) family was reported to be composed of orphan GPCRs expressed in the CNS.2,3 This family consists of three members, termed SREB1 (GPR27), SREB2 (GPR85), and SREB3 (GPR173). An intriguing feature of the SREB family is its high degree of sequence conservation throughout vertebrate evolution. Surprisingly, the primary amino acid sequence of SREB2 is identical between humans, rats, and mouse.2,4,5 Moreover, mammalian SREB2 shares 94% amino acid identity with its zebrafish homologue,2 which is unexpectedly higher than other GPCRs.6
8 The extremely high evolutionary conservation suggests that the CNS functions of SREB are of indispensable importance in the vertebrate CNS, although these functions are not yet known. To further investigate these functions, our group generated a SREB2-overexpressing transgenic (Tg) mouse and showed that SREB2 was involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia.9 Quantitative RT-PCR analysis showed that SREB2 mRNA expression was 2-fold higher in the forebrain of the Tg mouse than in a wild type (WT) littermate. However, we were unable to detect SREB2 protein using immunochemical methods such as Western blotting and r 2011 American Chemical Society

immunohistochemistry because of the lack of availability of specific antibodies. All attempts to prepare suitable specific antibodies against SREB2 have been unsuccessful, probably due to its extraordinary evolutionary conservation in mammals and its seven-transmembrane structure. In this article, we use SREB2-overexpressing cultured cells to identify SREB2 proteotypic peptides, which have specific sequences and are readily observable by mass spectrometry (MS).10 Using the proteotypic peptide, we detected SREB2 directly in tissues and compared the protein expression levels of SREB2 in Tg and WT mice using its stable isotope-labeled analog as an internal control.

’ MATERIALS AND METHODS Materials and Reagents

Tris (2-carboxyethyl) phosphine, strong cation exchange (SCX) cartridge column purification systems of ICAT reagent kit containing POROS 50HS column (50 μm, 4.0  15 mm) and buffers (buffer-Load, buffer-Elute and buffer-Clean) were purchased from Applied Biosystems (Foster City, CA). Iodoacetamide was obtained from Sigma (St. Louis, MO), sequence grade modified trypsin from Promega (Madison, WI), TCI Opti-Guard Fit ODS (1.0  15 mm) from Tokyo Chemical Industry (Tokyo, Japan), stable isotope-labeled peptide GP*TPPTLLGIR (P* denotes Received: December 16, 2010 Published: March 14, 2011 2658

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Journal of Proteome Research [13C5, 15N1] proline) from ThermoElectron GmbH (Sedanstrasse, Germany), and PolySULFOETHYL A column from Poly LC Inc. (Columbia, MD). All other reagents were of analytical grade. Preparation of Membrane Fraction of SREB2 Overexpressing CHO Cells

SREB2-overexpressing Chinese hamster ovary (CHO) cells were prepared as described previously.2 Cells were homogenized in 20 mM HEPES buffer (pH 7.8) using a Polytron homogenizer. The membrane fraction was obtained after centrifugation twice at 40 000 g for 20 min. Aliquots were stored at
80 °C. Preparation of Forebrain Membrane Fractions of the SREB2Overexpressing Tg and WT Mice

TECHNICAL NOTE

consisted of 20 mM potassium phosphate buffer (pH 3.0)/25% acetonitrile (SCX-solvent A), and 20 mM potassium phosphate buffer (pH 3.0)/25% acetonitrile/350 mM KCl (SCX-solvent B). The flow was split just before the injector and adjusted at the flow rate of 0.2 mL/min. The column was initially equilibrated at 0% B for 10 min, and the separation was performed over a linear gradient of 0% B to 100% B over 60 min. UV detection was performed at 210 nm. Fractions (0.2 mL each) were collected between 15 and 65 min, and desalted with the method described above. For quantitation samples, this fractionation step was omitted to improve the recovery rate for the SREB2 peptides. RP-LC
MS/MS Analysis

Membrane fraction (115 μg of protein) was resuspended in 80 μL of 20 mM HEPES buffer (pH 7.8), 80 μL of 0.1% SDS/ 50 mM Tris-HCl (pH 8.5), and 2 μL of 50 mM tris (2carboxyethyl) phosphine. After boiling for 15 min and cooling to room temperature, 65.5 μg of iodoacetamide in 20 μL of acetonitrile was added, and sample was incubated at 37 °C for 2 h in the dark. Subsequently, 20 μg of trypsin in 80 μL of 0.05% SDS was added, and the sample was incubated overnight at room temperature. As an internal control, 100 fmol of stable isotopelabeled synthetic peptide (GP*TPPTLLGIR) was added to the digested peptide mixture.

The desalted peptide mixture was dissolved in 100 μL of RPsolvent A, 10 μL of this solution was injected into an online trap column (0.3  5 mm, Pepmap C18, 5 μm, 100 Å; LC-Packings, Amsterdam, Netherlands). After concentration on the trap column, the peptide mixture was separated with a Picofrit column (0.075  150 mm, New Objective, MA) packed in house with a Capcellpack C18 MG (Shiseido, Tokyo, Japan) using two Nanospace SI-1 pumps (Shiseido) coupled with an Accurate microsplitter (AC-100-VAR, LC-Packings). The LC effluent was directly interfaced with the nanoelectrospray ion source on a LTQ linear ion trap mass spectrometer (Thermo Fisher, Waltham, MA). The mobile phases were the RP-solvents A and B. For protein identification, separation was performed at a flow rate was 200 nL/min by a linear gradient of 10
50% B over 35 min, then 90% B over a further 10 min. For quantitation, separation was performed by a linear gradient of 12
35% B over 69 min, then 90% B over a further 8 min. For CHO cells, a two-event scan was applied. The first was a full MS scan (m/z 400
1500) and the second was a datadependent product ion scan for the most intense ion from the first event. Dynamic exclusion (2 min duration) was used to acquire product ion spectra from low-intensity ions. For tissues, a three-event scan was applied. The first was a full MS scan (m/z 400
1500) and the second was a product ion scan for the doubly charged ion of the peptide GPTPPTLLGIR [M þ 2H]2þ (m/z 561.7). The third scan was carried out as a product ion scan either for the doubly charged ion of the peptide LLVDEFK [M þ 2H]2þ (m/z 489.1) (for identification), or for the stable isotope-labeled peptide GP*TPPTLLGIR [M þ 2H]2þ (m/z 564.7) (for quantitation). Analyses were performed in triplicate.

Removal of SDS and Excess Reagents

Protein Identification

SREB2 Tg mice were generated by overexpressing mouse SREB2 gene (the whole fourth exon encoding its entire intronless open reading frame, ∼4.5 kb from the start codon) driven by the mouse CaMKII promoter (∼8 kb) as described previously.11 The expression construct was injected into the pronuclei of fertilized eggs from DBF1 mice. The Tg mice generated were maintained on a C57BL/6 background. In this study, we used heterozygotes of the F3 or F4 generations, and comparisons were made with littermate WT mice. Tails were used for genotyping, of the SREB2 Tg mice, which was performed by PCR using the primers 50 -GCACGAGGGCCTGTAGTACC-30 and 50 -CATCCAGACAGCGGCTGTTA-30 . All experiments were performed in compliance with the regulations of the Animal Ethics Committee of Astellas Pharma Inc. Brains were harvested from neonatal (seven days postnatal) male SREB2 Tg mice or their WT littermates. Forebrain half-hemispheres (without cerebellum) were homogenized, and their membrane fractions were prepared as described above. Protein Digestion with Trypsin

SDS and excess reagents were removed with the ICAT kit. After dilution with 3 mL of buffer-Load, the digested peptide mixture was injected into an SCX column and then washed with 1 mL of buffer-Load prior to peptide elution with 0.5 mL of bufferElute. The eluted solution was evaporated in a vacuum, redissolved with 0.1 mL of RP-solvent A (containing 0.1% formic acid/2% acetonitrile), and then injected into a TCI Opti-Guard Fit ODS column for desalting. After washing with 0.5 mL of RP-solvent A, peptides were eluted with 0.2 mL of RP-solvent B (containing 0.1% formic acid/90% acetonitrile), and evaporated in a vacuum. Fractionation with SCX Column Chromatography

The desalted sample was dissolved in 0.1 mL of 20 mM potassium phosphate buffer (pH 3.0)/25% acetonitrile, and separated with PolySULFOETHYL A column (2.1  100 mm, 5 μm, 300 Å) using a Hewlett-Packard (Palo Alto, CA) HP1050 HPLC system (LC-pump and UV detection). The mobile phases

Protein identification was performed using Mascot software (Version 1.9.01, Matrix Science Inc. London, UK). An in-house customized database based on the NCBI nonredundant protein sequence database (‘nr’ FASTA file) download from ftp://ftp. ncbi.nih.gov/blast/db/FASTA on first Apr 2005. Only human, mouse, rat and bovine sequences were extracted from ‘nr’ file, and partial protein sequences were filtered out using NCBI EntrezGene (ftp://ftp.ncbi.nih.gov/gene/DATA/gene2accession.gz). In total, 331213 protein entries in the database were actually searched. DTA files for each MS/MS spectrum were generated by Bioworks 3.1 software (Thermo Fisher) from the raw data for a peptide mass range of m/z 400
4000 with a minimum ion count of 50. The Mascot search parameters were as follows. Peptide tolerance was 2.0 Da and MS/MS tolerance was 0.8 Da (average mass). Fixed modification of carbamidomethyl (Cys), and variable modifications of oxidation (Met), acetylation (N-terminal of protein), and pyro-glu (Glu and Gln) were 2659

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Figure 1. Product ion spectra of tryptic peptides from membrane fraction of SREB2-overexpressing CHO cells. (A) Product ion spectrum of GPTPPTLLGIR (m/z 561.7, Mascot score: 82). (B) LLVLDEFK (m/z 489.1, Mascot score: 62). The accession number of mouse SREB2 (GPR85) is NP_659503.1 in the NCBI Refseq protein database.

TECHNICAL NOTE

Figure 3. Product ion spectrum of SREB2 proteotypic peptide (GPTPPTLLGIR). (A) Product ion spectrum of the proteotypic peptide in the SREB2 Tg mouse forebrain. This spectrum was obtained by subtracting the averaged product ion spectrum of the preceding elution (68.6
68.7 min) and the following elution (71.8
72.0 min) from that of the target elution (69.9
70.0 min). (B) Product ion spectrum of the stable isotope-labeled synthetic analog, GP*TPPTLLGIR (P* denotes [13C6, 15N1] proline). The m/z shifts of the precursor ion (m/z 561.7 to 564.7) and b2þ fragment ion (m/z 256.1 to 262.1) were observed due to the isotopic labeling of the second proline residue.

point boxcar smoothing was applied for each chromatogram. The relative concentration of SREB2 was estimated from AUCs of EIC for the endogenous peptide (m/z 561.7 > 475.3 ( 0.5, and 561.7 > 433.8 ( 0.5) and for the isotope-labeled synthetic peptide (m/z 564.7 > 475.3 ( 0.5 and 564.7 > 433.8 ( 0.5).

’ RESULTS Figure 2. SREB2 amino acid sequence. The seven transmembrane (TM) domains are underlined. The two tryptic peptides identified from CHO cell membrane fraction are shown in bold and underlined. The identified peptides exist in the cytoplasmic loop between transmembrane domains 5 and 6.

selected, and up to three missed trypsin cleavages were allowed. Peptide identification criteria was P < 0.05 (ions score >47), where P was the probability that the observed match was a random event. Estimation of Expression Ratio of SREB2 Protein between Tg and WT Mice

Extracted Ion Chromatogram (EIC) data were processed by Qual Browser software (Version 1.4, Thermo Fisher). A seven

Identification of SREB2 Proteotypic Peptides

We attempted to identify SREB2 from the forebrain membrane fraction of the SREB2 Tg mice, which should contain higher amounts of SREB2 protein than wild type mice. However, peptides derived from SREB2 were undetectable even in the Tg mice samples by a simple 2D LC
MS/MS analysis using a datadependent product ion scan mode. To overcome this problem, we tried to identify proteotypic peptides in advance. This procedure should be performed using samples containing higher amounts of SREB2 than those in tissues from the SREB2 Tg mice. So, for this purpose, we used CHO cells overexpressing the SREB2 gene. Tryptic peptides obtained from the membrane fraction of the cells were analyzed with 2D LC-MS/MS in a data-dependent product ion scan mode. We identified two SREB2-specific peptides (GPTPPTLLGIR (m/z, 561.7, 2þ) 2660

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Journal of Proteome Research and LLVLDEFK (m/z, 489.1, 2þ)) using the Mascot search engine (Figure 1). Both peptides were located in the long cytoplasmic loop between transmembrane domains 5 and 6 of SREB2 (Figure 2). Identification and Relative Quantitation of SREB2 in Tissues

We attempted to identify SREB2 directly from the forebrain membrane fraction of the SREB2 Tg mice with product ion scanning of the peptides. By manually inspecting product ion spectra, we found characteristic product ions of the proteotypic peptide, GPTPPTLLGIR, at the corresponding retention time of the peptide determined from the analysis of the SREB2-overexpressing CHO cells. However, many other product ions derived from coeluted non-SREB2 peptides, whose precursor ions have similar m/z with that of GPTPPTLLGIR, made it impossible to identify specific peptides in the spectrum using Mascot (data not shown). To clean the spectrum, the averaged product ion spectrum of the preceding and following elutions, which did not include product ions derived from SREB2, were subtracted from the target spectrum. After the background subtraction, GPTPPTLLGIR became the top hit peptide in the spectrum by Mascot (Mascot score: 27). The processed product ion spectrum, shown in Figure 3A, was identical with that derived from the CHO cells (Figure 1A). To confirm the identification further, the stable isotope-labeled synthetic analog, GP*TPPTLLGIR (P* denotes [13C6, 15N1] proline), was also analyzed. The retention time and product ion spectra of the endogenous and synthetic peptides were identical (Figure 3B), indicating that SREB2 was directly identified in tissues.

TECHNICAL NOTE

We tried to compare SREB2 expression between the forebrain membrane fractions of the Tg and WT mice. For this purpose, we selected two product ions of the GPTPPTLLGIR peptide (the doubly charged, dehydrated y9 ion (m/z 561.7 > 475.3 ( 0.5) and the doubly charged y8 ion (m/z 561.7 > 433.8 ( 0.5)), because of their high intensity and selectivity (Figure 3A). The stable isotope-labeled synthetic analog, GP*TPPTLLGIR, was used as an internal control. The relative concentration of the SREB2 peptide was estimated by comparison of the AUC of endogenous peptide EICs with that for internal standard (GP*TPPTLLGIR, m/z 564.7 > 475.3 ( 0.5 and 564.7 > 433.8 ( 0.5) on 1D LC
MS/MS analysis. The EICs for the peptides in the analyses of the Tg and littermate WT mice are shown Table 1. Protein Expression Ratio of SREB2 in the Forebrain Membrane Fractions of the Tg Mouse and WT Littermates ratio of AUCsa 433.8 ( 0.5

product ion sample

WT

Tg

475.3 ( 0.5 WT

Tg

1 0.25 0.69 0.14 0.64 2 0.20 0.61 0.20 0.74 3 0.13 0.60 0.20 0.70 Mean ( SEM 0.19 ( 0.03 0.63 ( 0.03 0.18 ( 0.02 0.69 ( 0.03 95% confidential interval 0.11
0.27 0.57
0.70 0.13
0.23 0.63
0.76 Tg/WT ratio 3.3 3.8 a

Data are expressed as ratios of the AUCs of the product ions and the internal control.

Figure 4. EICs for the proteotypic peptide GPTPPTLLGIR and its internal control in the forebrain membrane fractions of Tg and WT mice. EICs (Extracted ion chromatograms) for m/z 561.7 > 433.8 ( 0.5 of endogenous peptide GPTPPTLLGIR and m/z 564.7 > 433.8 ( 0.5 of stable isotopelabeled synthetic peptide GP*TPPTLLGIR. The y-axis (intensity of ions) is fixed at 400. AUCs of EICs are calculated from the indicated gray areas. The analyses were performed in triplicate. E, endogenous peptide; S, synthetic peptide for internal control; Tg, SREB2 transgenic mouse; WT, wild type littermate. 2661

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Journal of Proteome Research in Figure 4. From the EICs, the protein concentration of SREB2 in the forebrain of the Tg mouse was estimated to be approximately three times higher than in the WT littermate (Table 1).

’ DISCUSSION The identification and quantification of low-abundance membrane proteins, such as GPCRs, is desirable because their functions and relationships with disease are very important. SREB2, an orphan GPCR expressed in the CNS, was not amenable to evaluation by immunochemical methods because of a lack of availability of specific antibodies. In these circumstances, MS-based analysis becomes a powerful tool for protein identification and quantification.12,13 Many authors have reported analyses of various proteins with MS analysis using proteotypic peptides.10,14
18 However, analysis of GPCRs with MS-based approaches is often challenging because of their low abundance and highly hydrophobic nature. Rhodopsin has been identified and quantified in the rod outer segment, where the protein exists in large amounts.19 The human cannabinoid CB2 receptor is fully characterized, but this required production of large amounts of protein from baculovirus cells overexpressing the CB2 gene with an epitope tag. 20 GPCR proteins are sometimes included in the large protein lists made by shotgun profiling using MS. In the case of shotgun analysis, however, identification of each protein is not usually confirmed. SREB2 was included in one such protein list produced following shotgun analysis of the postsynaptic density fraction,21 but its identification was not individually confirmed, and the identified peptide sequence of SREB2 was not represented. To identify SREB2 protein in tissues, we tried to search for SREB2 proteotypic peptides by analyzing SREB2 overexpressing CHO cells, which contained relatively high amounts of SREB2. With the aid of the resultant proteotypic peptide, GPTPPTLLGIR, SREB2 was identified directly in tissues and the identification was further confirmed with the synthetic analog of its proteotypic peptide. To our knowledge, there have been few reports of MSbased identification of low abundant orphan GPCR proteins directly in tissues. We estimated that SREB2 protein expression in the forebrain of the Tg mouse was three times higher than in the WT littermate. This result can validate the Tg mouse after other validation methods were unsuccessful. For more accurate evaluation of SREB2 expression, selected reaction monitoring (SRM) measurements with a triple quadrupole mass spectrometer should be useful because making an antibody against SREB2 is difficult owing to its high homology between mammals and the seven transmembrane structure common to GPCRs. In the case of the SRM, the precursor and product ions of the proteotypic peptide (GPTPPTLLGIR, m/z 561.7 > 475.3 and 561.7 > 433.8) selected in this report might be useful.

’ AUTHOR INFORMATION Corresponding Author

*Hiroyuki Yokota, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Tel.:þ81 29-863-6465. Fax: þ81 29-852-5444. E-mail: [email protected].

’ ACKNOWLEDGMENT We wish to thank Mari Masumoto, Akiko Ohtsu, Misaki Mito, Ikue Sato, Narumi Kamata, and Kumiko Fukumaru for their technical assistance.

TECHNICAL NOTE

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