Optimized Proteomic Mass Spectrometry Characterization of

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Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human µ-Opioid Receptor functionally expressed in Pichia pastoris cell lines Mònica Rosa, Joan Josep Bech-Serra, Francesc Canals, Jean Marie Zajac, Franck Talmont, Gemma Arsequell, and Gregorio Valencia J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00104 • Publication Date (Web): 19 Jun 2015 Downloaded from http://pubs.acs.org on July 6, 2015

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Journal of Proteome Research

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human µOpioid Receptor functionally expressed in Pichia pastoris cell lines. Mònica Rosa1, Joan Josep Bech-Serra2, Francesc Canals2, Jean Marie Zajac3, Franck Talmont3, Gemma Arsequell1, Gregorio Valencia1,*

1

Unit of Glycoconjugate Chemistry, Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC), Barcelona, Spain.

2

Proteomics Laboratory, Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, ProteoRed ISCIII, Barcelona, Spain.

3

Institut de Pharmacologie et de Biologie Structurale, Centre National de la

RechercheScientifique (CNRS) / Université de Toulouse, Université Paul Sabatier, Toulouse, France.

ACS Paragon Plus Environment


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ABSTRACT The human µ-opioid receptor (hMOR) is a class-A G protein-coupled receptor (GPCR) which is a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has recently been co-crystallized with an opioid antagonist and resolved by X-Ray diffraction; however further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Towards this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have succeeded to comprehensively characterize by MS analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein

(hMOR-c-myc-6-his) using an

optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin or proteinase K, followed by MALDITOF-MS or nanoLC-MS/MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.

KEYWORDS

Human Mu-Opioid Receptor (hMOR) • opioid receptors, ORs • G-Protein Coupled Receptor; GPCR • Membrane Protein • transmembrane protein • Proteomic analysis • Mass Spectrometry • MALDI-TOF-MS • protein structural biology • Orbitrap nanoLCMS/MS • membrane protein purification • transmembrane domains, (TMHs).


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INTRODUCTION

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes and make approximately 2% of the human genome. Based on their amino acid sequences GPCRs can be grouped in four major families: class A, B, C, and F (Frizzled), and numerous subfamilies. They are involved in a myriad of physiological functions in all kinds of tissues and mediate cellular responses to a variety of extracellular signals ranging from photons and small molecules to peptides and proteins.1,2,3 Important progress has been made in the study of GPCR presence and expression levels in tissues and in understanding GPCR activation.4 Also owing to their diverse and critical functions these receptors have become the target of more than 50% of the current therapeutic agents on the clinic which include more than 25 of the 100 top prescribed drugs with revenues in the range of billions of dollars each year. GPCR drug discovery has been very successful in the past decade (20002009) with the launch of 63 new GPCR drugs which represented about 24% of all drugs reaching the marked at this time. A number of them (i.e., taltirelin, ramatroban, bosentan, arprepitant and ramelteon) are first in class small molecule drugs targeting GPCRs.5 All this success has relied on high-throughput screening techniques of large compound libraries using cell-based assays to identify novel hits. Other drug discovery approaches using structure based drug design principles have been of little use owing to the poor structural information on GPCRs at the atomic level available at this time. Nowadays, with less than 20% of all GPCRs currently being drugged, this discovery area continues to be challenging with many clinically relevant GPCRs undrugged and over 100 orphan GPCRs for which the endogenous ligands remain unknown.6 In general, GPCR studies continue to be challenging because of the notorious difficulties encountered in their preparation purification and structural characterization



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by biophysical methodologies. These complications arise from the intrinsic nature of GPCRs of being integral membrane proteins with an extensive hydrophobic membrane spanning region containing seven transmembrane α-helices (TMHs) connected to intraand extracellular loops. In spite of this situation and after a gap from the first crystal structure of a class A GPCR rhodopsin receptor reported7 in 2000, different advances in techniques, tools and concepts8 have prompted a recent upsurge of 19 medium-to-highresolution structures of class A and one Frizzled GPCR. More recently, the first two crystal structures9 of class B GPCRs and the structures of two class C GPCRs have also been published.10These elegant structural achievements6 have been among the ones recognized with the 2012 Nobel Prize in Chemistry.11 At the same time only, a few number of GPCRs have had their primary protein sequences completely determined using proteomic analyses (>80% sequence coverage). Given its natural abundance and availability, the photoreceptor rhodopsin was the first GPCR to be completely mapped 12

with a sequence coverage including all seven TM domains. Complete peptide

mapping has been achieved for the following GPCRs:: the Tachykinin NK-1 receptor,13 the cannabinoid 2 (CB2) receptor,14 the Histamine H1 receptor,15 neurotensin16 and the human cannabinoid 1 (CB1) receptor.17 In all these studies sample preparation prior MS analysis which has been specifically devised for each of the receptors was identified as a bottleneck. The three classical opioid receptors (ORs), namely, µ, κ and δ-OR (MOR, KOR and DOR, respectively) and the related nociceptin/orphanin FQ peptide receptor (NOP) is a well-known subfamily of GPCRs which plays important roles in the central nervous system by modulating pain perception, mood and wellbeing.18, 19, 20 They are activated by binding of classical opioid alkaloids such as morphine and of a plethora of endogenous opioid peptides like the endorphins. Opioid alkaloids such as morphine


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produce a wide range of effects including analgesia, sedation, euphoria, respiratory depression and constipation. The majority of these positive and negative effects are mediated by the MOR receptor which makes it a privileged therapeutic target for the management of acute and chronic pain. Along with DOR and KOR, the MOR receptor belongs to the GPCR gamma subfamily of the class-A. They have a common transmembrane helical architecture and share about 70% sequence identity on these helices with more variations in the extracellular loops and very little similarity in their amino and carboxyl termini.19,20 The high-resolution crystal structures of chimeric forms of all of the four opioid receptors bound to different opioid antagonist ligands have recently been solved by Xray crystallography: the µ-opioid receptor bound to a morphinan antagonist,21 the human kappa-opioid receptor in complex with JDTic;22 the delta-opioid receptor bound to naltrindole23 and nociceptin/orphanin FQ receptor in complex with a peptide mimetic.24 These structures belong to modified and/or truncated receptor forms bound to antagonists rather than agonist ligands, thus, the specific receptor conformations that are stabilized by the binding of opioid ligands to selectively activate signaling pathways have not been captured by these recent crystal structures. To understand with greater chemical precision not only the structural features of these membrane proteins in their active state conformations but the functional mechanisms and structural dynamics of opioid receptors, other tools that complement the crystallographic techniques are required. Thus, for instance, the design and incorporation of informative molecular and chemical probes into the receptors to interrogate active signaling processes may be invaluable. In turn, to monitor the fate of these probes proteomic techniques may be the choice. Proteomic MS analyses of the traditional opioid receptors: mMOR, a mouse variant of MOR,25 hMOR,26 mDOR, a mouse variant of DOR,27 and rKOR, a rat variant



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of KOR28 have already been reported. However, the rather low sequence coverages (2737%) of these methods are not yet optimal for probe monitoring. Towards contributing to upgrade these approaches here, we report an improved proteomic MS analysis of the hMOR receptor. To obtain substantial amounts of functional and purified hMOR receptor we have used our already published high yield expression, isolation and purification system based on a C-myc-tagged/C-terminal 6His-tagged protein (hMOR-c-myc-6-his) in Pichia pastoris cells.29 After fine tuning a protocol to concentrate the purified protein sample, the receptor was in-solution digested with trypsin and analyzed by MALDITOF for confirmation of our previous proteomic analysis using the same and other hMOR constructs.26 In-gel digestions with chymotrypsin and examination of digested peptides by MALDI-TOF and nanocapillary liquid chromatography-tandem mass spectrometry, nanoLC-MS/MS (Orbitrap) techniques furnished a sequence coverage of up to 60%. Further in-gel digestions of hMOR with proteinase K and analysis by nanoLC-MS/MS have allowed us to identify new peptides that make most of the entire hMOR primary sequence (overall coverage, 82%). This includes six out of the seven transmembrane domains which are of special interest because these regions are involved in ligand-binding.30, 31, 32 We envisage that these optimized techniques should facilitate a full structural characterization of hMOR binding sites, help in the design of covalent probes and aid in future drug discovery programs which use structure based approaches to find more selective and novel analgesic drugs.

MATERIALS AND METHODS Reagents and Materials


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Unless otherwise specified, standard laboratory chemicals and buffers were purchased from Sigma-Aldrich (St. Louis, MO). EDTA (0.5 M) was from Invitrogen (Carlsbad, CA). Dithiothreitol (DTT) and micro-BCA assay kits were from Pierce (Rockford, IL). Mass spectrometry grade trypsin gold was obtained from Promega (Madison, WI). Chymotrypsin and proteinase K were from Sigma. Protease inhibitor cocktail tablets were procured from Roche Diagnostics (Mannheim, Germany). Iodoacetamide (IAA), methanol, acetonitrile, and water and HPLC grade solvents were from Sigma-Aldrich (St. Louis, MO). Expression of recombinant hMOR proteins and membrane isolation. Functional HMOR-c-myc-6-his (Supporting Information FigureS S1 and S2) was expressed in suspension-cultured Pichia pastoris cells and membrane proteins were prepared from whole-cell lysates as described.20 Briefly, cells expressing the mu-opioid receptor were harvested and broken during 30 min with glass beads in breaking buffer (Tris–HCl, 10 mM, pH 8) supplemented with protease inhibitors (benzamidine 1 µg/ml, pepstatin A 1 µg/ml, leupeptin 1 µg/ml, antipain 1 µg/ml, and aprotinin 1 µg/ml). The cell lysate was then centrifuged at 1000g for 15 min to remove unbroken cells and particulate matter. The supernatant was further centrifuged at 10,000g for 30 min to harvest a crude fraction. The resulting pellets were then stored at -80 °C in breakage buffer. Protein quantification measurements were performed as described previously.29 Solubilization Purification and Quantification of hMOR. Briefly, crude membranes prepared from P. pastoris expressing the hMOR-c-myc-6-his protein were initially washed three times with ice-cold buffer (100 mM NaH2PO4, 10 mM Tris–HCl pH 8, 20 mM β-mercaptoethanol). Samples containing of recombinant of hMOR-cmyc-6-his (12 mg/L culture) were solubilized in buffer containing 100 mM NaH2PO4,



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10 mM Tris-HCl pH 8, 20 mM β-mercaptoethanol, 0,1% SDS and 8 M urea for 1 h at room temperature with gentle agitation on a wheel. After centrifugation for 30 min at 100,000g, supernatant containing solubilized receptors was loaded on to a Ni-affinity column. The resin was washed with the preceding buffer. Proteins bound to the resin were subsequently eluted with a step imidazole gradient, ranging from 0 to 300 mM, in a 100 mM NaH2PO4, 10 mM Tris-HCl and 0,1% SDS buffer (Supporting Information Figure S3). The IMAC column eluted fractions were collected and monitored by SDS/PAGE. A protein quantification test Pierce Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, lL, USA) was used to quantify the protein amount in the isolated receptor sample, following the protocol described for the microplate procedure. Calibration curves were made from a BSA standard commercial solution diluted at several concentrations between 0.03 and 2 mg/mL in different wells. Samples were diluted in MilliQ H2O from 1/4 to 1/256 in several wells. Kit reagents were added, consisting on a mix of a tartrate/carbonate buffer, bicinchoninic acid (BCA) and copper sulfate. The reaction was incubated during 30 min at 37 ºC and quantified by measuring the absorbance at 562 nm on a SpectraMax® M5 multi-mode microplate reader (Molecular Devices). Data was analyzed and validated with Excel (Microsoft).

SDS-PAGE and Immunoblotting. Previous to gel electrophoresis, several procedures were applied to increase receptor concentration in protein samples as, among them, evaporation, centrifugation and protein precipitation of the samples. Samples were evaporated on SpeedVacTM concentrator (5301, Eppendorf, Germany) from 50 mL to 2 mL. Samples were concentrated by centrifugating on an Amicon® Ultra-15


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centrifugal filters (Millipore, USA) from 10 mL to 1,5 mL for 20 min at 4000g and 4 ºC. Protein precipitation was effected by overnight treatment of the samples with a 90% acetone aqueous solution and subsequently resuspension in a 100 mM Tris-HCl (pH 7,8) and 2 M urea buffer. The concentrated sample of purified receptor were separated by Laemmli SDS-PAGE electrophoresis with a Mini-PROTEAN®II (BioRad, USA) electrophoresis cell using a 6% stacking and 6-12% separating polyacrylamide gels. Electrophoresis was carried out at 25 ºC at a voltage of 120 V until the dye front reached the bottom gel (1-2h). Readyto-use protein molecular weight (MW) marker used was PageRulerTM prestained Protein Ladder (Thermo Scientific).Proteins were fixed on electrophoretic gels with a 50% EtOH and 7% AcOH solution and visualized by colloidal Coomassie Brilliant Blue G-250.The hMOR-c-myc-6-his protein major band was observed at a position just higher than 45 kDa, correlates well with the 47 kDa molecular weight of the receptor. Receptor band was excised with a sterile stainless steel scalpel and transferred to a 1.5 mL microcentrifuge tube. For detection of the receptor by immunoblotting analysis, proteins were transferred from electrophoretic gels to PVDF membranes (Millipore, Bedford, MA, USA) with semi-wet method at 15 V and 1,12 A for 20 min. The protein blots were visualized by enzyme immunodetection with a 1:10000 dilution of antibodies. Antibodies used were mouse anti-myc-tag antibody as primary antibody (clone 9E10, Sigma, St Louis, Missouri, USA) and with a secondary anti-mousecoupled antibody (Jackson Immunoresearch, West Grove, PA, USA). Membranes were revealed with a luminol kit Amersham ECL Prime (GE Healthcare Life Sciences) in photographic plates in darkroom (1 sec – 15 min). In-gel enzymatic digestion. Protein bands were excised from the gel and destained with EtOH. The gel piece was cut into small pieces to increase surface and put into a



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tube. The gel piece was treated with 10 mM DTT in 50 mM NH4HCO3 for 1 hour at 56 ºC and alkylated with 55 mM IAA in 50 mM NH4HCO3 for 30 min at rt in the darkness. Gel bands were dehydrated again with ACN and protein was digested with the following proteases, 0.1 µg trypsin (Promega), 0.2 µg chymotrypsin (Sigma) or 0.02 µg proteinase K (Sigma) in each of the corresponding buffers [100 mM NH4HCO3 buffer for trypsin and proteinase K, and 100 mM Tris (pH 8.0) with 10 mM CaCl2 buffer for chymotrypsin] with incubation times ranging from 30 min to o.n., and at temperatures of 30 ºC or 37 ºC. Digestions were stopped with ACN and peptides were extracted with a 0.2% TFA aqueous solution for 30 min at rt and the sample was ultrasonicated for 2 min (Supporting Information Figure S4 and Tables S1A and S1B). In solution enzymatic digestion. The sample of concentrated protein was diluted in 100 mM Tris-HCl buffer (pH 8.0) to a concentration of 0.5 mg/mL. The sample was treated with 10 mM DTT in 5 mM NH4HCO3 for 30 min at 56 ºC and alkylated with 55 mM IAA in 50 mM NH4HCO3 for 30 min at rt in darkness. Reaction was stopped by addition of 1 µL of 50 mM DTT. Afterwards, protein was digested with either 1 µg trypsin (Promega) in 25 mM NH4HCO3 buffer or 2 µg in chymotrypsin (Sigma, for protein sequence analysis grade) in 100 mM Tris (pH 8,0) and 10 mM CaCl2 buffer using several incubation times (from 1h to o.n.), and temperatures (30 ºC or 37 ºC). Enzymatic digestion was stopped acidifying with 1 µL of 20% TFA aqueous solution for 30 min at 37 ºC (Supporting Information Figure S4 and Tables S1A and S1B). MALDI-TOF MS. Peptide analysis by MALDI-TOF MS was performed as follows. First, digested peptides were desalted with C18ZipTip® (Millipore, USA) and eluted with 2 µL of 70% ACN in water (v/v) and 0,1% TFA. Samples were applied on a polished steel plate (MTP 384 target plate polished steel TF target, Bruker Daltonik GmbH, Bremen, Germany) mixed with DHB as matrix. Mass spectra were collected on


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a MALDI-TOF spectrometer (Bruker Autoflex III TOF/TOF, Bruker Daltonik, GmbH, Bremen, Germany) in positive reflector mode with an accelerating voltage of +21 kV. A nitrogen laser (λ=337 nm) operating at 50 Hz was used to irradiate samples at intensity sufficient to ionize the sample. Spectra were externally calibrated using a standard mixture of Substance P and Angiotensin IV. Mass spectra were recorded from 500 to 4300 Da under reflector mode. The mass spectrometry data obtained were processed with FlexControl (for acquisition of spectra) and FlexAnalysis (for spectra analysis) software packages (Bruker Daltonik GmbH, Bremen, Germany). Unprocessed spectra, with no baseline correction or smoothing, were used for mass/charge value labeling. Peak lists were generated using the SNAP algorithm embedded in the FlexAnalysis software, which labels only the monoisotopic peak of each peptide isotopic distribution. A signal to noise threshold of >5 was set for peak labeling. Peptidic masses were assigned using Biotools 3.2 software (Bruker Daltonik GmbH) and MASCOT™ software 2.3 (Matrix Science, London, UK) search engine and compared with theoretical digestions of the sequence of recombinant hMOR and Swiss-Prot 57.3 (Swiss Bioinformatics Institute, Geneva, Switzerland) database,27with taxonomy restricted to Homo Sapiens (20401 sequences) (Supporting Information Figure S18). Relevant search parameters were set as follows: enzyme, trypsin, chymotrypsin; fixed modification, cysteine carbamidomethylation (C); variable modification, methione oxidation (M); mass values, monoisotopic; maximum 5 missed cleavage; peptide mass tolerance, 100 ppm. The criteria for a positive identification was a Mascot score greater than the significant threshold at p90% after in solution digestion with trypsin and chymotrypsin.14,

17

Also a baculovirus system has been used for the full MS

coverage characterization (>80%) of the Histamine H1 receptor after in gel trypsin processing of samples.15 Finally, a receptor for neurotensin (NTS1) has been produced in E. coli and up to >80% peptide mapped after in solution trypsin and chymotrypsin digestion.16 As evidenced, no consensus has been reached for the expression systems of these receptors nor for the enzymes used on the proteomic analyses. Moreover, none of the opioid receptors are in this group and the most recent proteomic study for any of them has been for the rat variant of KOR with an 27% of sequence coverage.28 Many severe but well identified technological roadblocks are in the cause of this situation for the ORs and for most of the GPCRs. Finding a suitable cell expression system for these integral membrane proteins with an extensive hydrophobic membrane spanning region containing seven transmembrane α-helices yielding substantial amounts of the receptor comes in the first place. For quite some time we have being addressing the challenge of developing a high expression system for the hMOR. We have had reasonable success with different cell systems which include the methylotrophic yeast Pichia pastoris, the Chinese hamster ovary (CHO) cells and the human neuronal SH-SY5Y cells.29, 34-38 In


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the course of these studies and using the Pichia pastoris system we had also conducted the first and up to date single proteomic study of hMOR by MALDI-TOF-MS on tryptic digests of the protein obtaining a 27% of sequence coverage.26 To reproduce these early experiments and further optimize the proteomic methodologies for hMOR we have produced a new batch of pure receptor protein using the Pichia pastoris system and applied proteomic and MS methodologies for sequencing the receptor by following the flow chart of Scheme 1.

Expression of recombinant hMOR in Pichia pastoris cells Solubilization of membranes in SDS solubilization buffer (SB)

Purification of hMOR by Immobilized Ni-affinity chromatography (IMAC)

PROTEIN CONCENTRATION

In solution digestion

SDS-PAGE

Western Blot

MALDI-TOF MS

In gel digestion

MALDI-TOF MS

Orbitrap NanoLC-MS/MS

Orbitrap NanoLC-MS/MS

Scheme 1. Flow chart showing the methodology for hMOR-c-myc-6-his purification and preparation for LC-MS/MS in our protocol. The workflow combines large-scale protein expression, membrane protein solubilization, in-solution and in gel multienzyme digestion, and acquisition of high resolution full scan and fragmentation spectra by different mass spectrometry techniques.



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Expression, solubilization, purification and quantification of hMOR. To prepare a new batch of hMOR we have used our hMOR-c-myc-6-his construct in Pichia pastoris as expression system. Crude membranes were treated with solubilization buffer containing 8 M urea and 0.1% SDS and the solution applied to an IMAC chromatography column and receptor bound were eluted with an imidazole gradient in the 0-300 mM range (Supporting Information Figure S3). The identification of the purified hMOR receptor was performed by gel electrophoresis and Western blot detection using c-myc antibodies.

We have obtained a sample of pure hMOR

containing 20 µg/mL of protein as measured by the bicinchoninic acid assay (BCA). The results matched with the ones we have previously reported.36 The hMOR-c-myc-6-his samples obtained from column purification were not yet ready for proteomic analysis since they contained low protein concentration and high amount of surfactant (SDS) that can interfere in sample separation and ionization when using in line RP- LC separations and MS identification systems. Thus, to circumvent surfactant interference and to improve the low protein concentration of the sample, a simultaneous concentration and surfactant removal step were assayed. Sample Concentration. Attempts to simply concentrate the samples in a SpeedVac evaporator logically rised the protein but also the SDS concentration up to levels unsuitable to further process the samples by SDS-PAGE electrophoresis. Alternatively, precipitation of the protein with organic solvents such as acetone at -20 ºC and redissolution in buffer to a working concentration of about 2 mg/mL, allowed to reduce the SDS concentration in the samples. However, those solutions tended to become cloudy owing to protein aggregation as evidenced by electrophoresis. Best results were obtained by centrifugal ultrafiltration using Amicon® centrifugal filters which have been originally designed for rapid processing of aqueous biological solutions in


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volumes ranging from 15 to 70 mL.39 Ultrafiltration rendered hMOR concentrated samples with an improved electrophoretic behavior with no sign of aggregated bands. MS characterization of tryptic digestions of hMOR. In-solution digestions of hMOR and subsequent MALDI-TOF analyses allowed to confirm a rather low sequence coverage that can be theoretically predicted because tryptic cleavage sites on hMOR are mainly located at cytosolic and extracellular regions of the protein while the most part of this highly hydrophobic protein lacks polar Lys and Arg cleavage points. After attempts to improve the enzymatic digestions by modifying the reaction temperature and time and the addition of denaturing agents (urea, organic solvent) a 16% sequence coverage was achieved. The peptide sequences that were identified mainly belong to the C-terminal region of the protein (Supporting Information Figure S12). As shown in Table 1, four of the seven identified peptides: (168-176), (351-367), (374-384) and (385-404), matched with four of the seven peptides that we reported in our previous proteomic characterization attempt of hMOR that also resulted in a similar low sequence coverage.26 A representative MALDI-TOF mass spectrum is provided as Figure 1.



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Figure 1. Representative MALDI-TOF MS mass spectrum of tryptic peptides derived from the purified hMOR-c-myc-6-his receptor. Peptide mixtures obtained after in solution enzymatic digestions. Tryptic peptides were resolved in the range of m/z 5004300.

Some

of

the

receptor-derived

tryptic

peptides

identified

CFREFCIPTSSNIEQQNSTR

are:

[348–367],

DHPSTANTVDRTNHQLENLEAETAPLPLEQK

[374-404]

and

TNHQLENLEAETAPLPLEQK [385-404], all derived from the C-proximal terminus region. All the cysteines were detected as carbamidomethylated cysteines (CM). Table 1: Tryptic digestions of hMOR and peptides identified by MALDI-TOF MS.

Measured Theoretic Mass al Mass (M+H)+ (M+H)+ Position 1086.636 1086.576 168 - 176

Missed cleavages Sequencea 0 YIAVCHPVK

2474.124

2474.119 348 - 367

1

C*FREFC*IPTSSNIEQQNSTR

Regio n TM3+ IC2 C-term

2010.928

2010.919 351 - 367

0

EFC*IPTSSNIEQQNSTR

C-term -0.976

787.484

787.453 368 - 373

1

IRQNTR

C-term -2.033


GRAVY Indexb 0.800 -0.790

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1212.643

1212.560 374 - 384

0

DHPSTANTVDR

C-term -1.455

3468.741

3468.699 374 - 404

1

DHPSTANTVDRTNHQLENLEAETAPLPLEQK C-term -1.190

2275.160

2275.156 385 - 404

0

TNHQLENLEAETAPLPLEQK

C-term -1.045

a) Carbamidomethyl (C*) modifications in bold; b) GRAVY index calculated at: ttp://web.expasy.org/protparam/ MS characterization of chymotryptic digestions of hMOR. To improve hMOR characterization the use of a second protease like chymotrypsin that complements the action of trypsin seemed the next option. Owing that chymotrypsin mainly targets aromatic hydrophobic residues (Tyr, Phe and Trp) the theoretical cleavage points of this enzyme on hMOR are mainly located almost exclusively in the transmembrane domains. In this case in-gel digestions techniques were chosen to facilitate isolation of the receptor from buffer salts prior enzymatic digestions. Also a small set of experiments with gels prepared with different proportions of acrylamide/bisacrylamide (6% to 12%) were also performed to achieve more efficient digestions and improve extraction of longer peptides from gels after digestion. No significant progress could be detected with any of the gels rather than the difficulties of working with fragile and more sticky materials to conclude that best experimental results came from standard conditions. The eluted peptides from these gels where analyzed with both MALDI-TOF and nanoLC-MS/MS (Orbitrap) techniques. As predicted, the combined peptide sequences identified using both techniques (Table 2 and Figure 2 and Supporting Information Figure S6) correspond to the N-terminal and transmembrane domains (TM1, TM2, TM3, TM4 and TM5). As seen in Figure 2 some of the peptides have been identified by both MS methods but many by only one of them thus illustrating their value as complementary techniques. The overall sequence coverage reached using chymotrypsin was up to 60% (Supporting Information Figure S13).



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Table 2. Chymotryptic digestions of HMOR and peptides identified by MALDI-TOF MS.

1552.808

Theoretical Mass (M+H)+ 1552.674

2294.173

Measured Mass (M+H)+

Position

Missed cleavages

Sequence

Region

GRAVY index

018 – 032

2

AYSSC*SPAPSPGSWV

N-term

-0.073

2294.083

032 - 052

4

VNLSHLDGNLSDPC*GP NRTDL

N-term

-0.624

1860.862

1860.927

059 - 076

5

C*PPTGSPSMITAITIMAL

N-term+TM1

1.017

797.348

797.434

096 - 101

2

VRYTKM

IC1

-0.717

854.337

854.408

126 - 132

3

QSVNYLM

TM2

0.114

1122,574

1122.565

131 - 140

3

LMGTWPFGTI

EC1

0.830

1491.769

1491.803

133 - 145

4

GTWPFGTILC*KIV

EC1+TM3

1.054

903.449

903.533

138 - 145

3

GTILC*KIV

TM3

1.812

1107.589

1107.634

169 - 178

3

IAVC*HPVKAL

TM3+IC2

1.410

1856.006

1856.086

176 - 191

4

KALDFRTPRNAKIINV

IC2+TM4

-0.388

2004.026

2004.023

179 - 194

4

DFRTPRNAKIINVC*NW

TM4

-0.613

1855.003

1855.012

181 - 195

4

RTPRNAKIINVC*NWI

TM4

-0.307

1389,717

1389.719

189 - 200

5

INVC*NWILSSAI

TM4

1.358

1049.534

1049.504

213 - 221

1

RQGSIDC*TL

EC2

-0.289

1297.636

1297.621

213 - 223

2

RQGSIDC*TLTF

EC2

-0.045

1129.661

1129.713

255 - 264

5

GLMILRLKSV

TM5

1.240

1694.860

1694.824

323 - 337

5

C*IALGYTNSC*LNPVL

TM7

1.007

1446.694

1446.683

339 - 349

3

AFLDENFKRC*F

TM7+C-term

-0.218

1878.888

1878.896

339 - 352

4

AFLDENFKRC*FREF

TM7+C-term

-0.543

1323.692

1323.636

350 - 360

1

REFC*IPTSSNI

C-term

-0.100

a) Carbamidomethyl (C*) modifications in bold; b) GRAVY index calculated at: ttp://web.expasy.org/protparam/


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L G G R D S

D

T

C P D S L N G D L H S L N V W S G P S P A P S C S S Y A L A D T C N R N P G S A 41 31 N T 11

51

A A S S

L C

Extracellular

61 P P T G

L S P S

W

I

M A L Y S I V C V V G L 81 F G N F L V M Y V 91 I V R

I Y A T K 101 M T K

C

S D I

1 F

S H

G Q R

G

G 131 M L V Y N S Q F P L 121 S T T L A D A L A 111 A N L

Y

Intracellular

T F

T

M I T A I T

Membrane

P

221 T

T

I F N

Y 211 K

T

T

I

T L C A 141 K I V M I S F I V M D Y Y N 151 G L P 201 I M F T S A S S I I L F T W N L C C T 161 191 V MS N I V D I R K Y I A A N V C P R 171 T H R P F V D K A L 181

I P

T V

T

W Y W E N 231 L L K I C V F 241 I F A F I M P V L I I 251 T V C Y G L M I L L S V

R

hMOR chymotrypsin digestion Sequence coverage: 60 % G

G G D V A S N L D E E S 411

I L

K Q E L 401

H I P I W T C 291 I V V F A V V L V M V R 281 I T R R L R N D K

K 261

E 271

R

K S

M L

H H H 421 H H H

K A I 301 I Y V

S G

P E 311 T T F Q T V S W 321 H F C I A L G Y T N S C 331 L N P V L Y A F L D 341 E

C 351 F

I P

N

E R F K R C F

T S S N I E Q 361 Q N S T

MALDI-TOF MS chymotryptic fragments nanoLC-MS/MS chymotryptic fragments Superimposition (MALDI-TOF MS and nanoLC-MS/MS)

Q P L P A T E A E L N E L Q H N T R D V T N A T S P H D R T N 371 391 381

R

I

R

Figure 2. Two dimensional graphical representation on hMOR-c-myc-6-his sequence of chymotrypsin digested peptides identified by MALDI-TOF MS and nanoLC-MS/MS.

Similar MS techniques combined with in-gel digestions of trypsin and chymotrypsin have been previously reported in the literature for the proteomic analysis of an engineered mouse variant of MOR expressed in a human embryonic kidney (HEK) 293 cell system.25 In this case a 37% overall sequence coverage was obtained. Interestingly, although the human and mouse variants of MOR share a sequence similarity of 93%, none of the reported peptides matched with our findings. Most probably the divergent results are due to the use of different expression systems. In the case of the mouse variant and because of the human expression system, the MOR receptor was expressed as a glycoprotein bearing other post-translational modifications that may alter the proteolytic performance of the digestion enzymes and thus the masses of the peptides.



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Using the same human cells expression system, this same research group has also reported two more proteomic analyses on opioid receptors. One, on a mouse variant of DOR that was performed on trypsin in-gel digestions using MALDI-TOF-MS methodologies and arrived at a 28% of sequence coverage.27 The other, on a rat mouse variant of KOR which used sequential trypsin in-gel digestion and cyanogen bromide and MALDI-TOF MS procedures that yielded 27% of sequence coverage.28 MS characterization hMOR digested with proteinase K. A robust proteolytic enzyme less frequently used in proteomics because of its substrate inespecificity is proteinase K. This protease has been applied to the proteomic analysis of membrane proteins40-42 including the mass spectrometric characterization of a GPCR.43 Proteinase K is an endoprotease that cleaves peptide bonds at the carboxylic side of aliphatic, aromatic, and hydrophobic amino acids. Exposed hydrophilic domains of membrane proteins are targets for this enzyme. Owing to substrate inespecificity the use of proteinase K in proteomic studies cannot be aided by peptide mass theoretical predictions like in the case of trypsin and chymotrypsin. This difficulty is usually overcome by applying nanocapillary liquid chromatography-tandem mass spectrometry for the separation and MS/MS identification of peptides from the digested protein. In our hands, proteinase K digestions in conjunction with nanoLC-MS/MS (Orbitrap) techniques have been conclusive for the optimal characterization of hMOR (see Supporting Information Figures S7, S8, S9A, S9B, S10 and S11). The assignation of identified peptides by nanoLC-MS/MS of proteinase K digested hMOR-c-myc-6-his functional receptor mapped along the whole sequence of the protein is shown in Figure 3A. Also portrayed in more detail are the overlapping peptides corresponding to the TM4 domain (Figure 3B). An example of the results obtained on ion detection and MS/MS sequencing is provided in Figure 4 (4A and 4B) for the sequence:


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AIGLPVMFM*AT (199-209) (M*, methionine oxidation) which is one of the identified peptides at the same TM4 domain. With these experiments we were able to reach a 70% of sequence coverage which is illustrated as the two dimensional hMOR serpentine diagram presented in Figure 5.



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A)


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B)

Figure 3 (Figures 3A and 3B). A) Assignation of identified peptides by nanoLCMS/MS of a proteinase K digested sample of hMOR-c-myc-6-his functional receptor mapped along to the sequence of the protein. See text for more details on experimental conditions. B) Overlapping Proteinase K-derived peptides of the TM4 region of the hMOR-c-myc-6-his functional receptor are here shown. Each rectangle in grey corresponds to a single, identified peptide. The rectangles in red, inside the grey rectangle, correspond to the detected y- and b-ions (lower and upper red rectangles, respectively) for each peptide.



Figure 4 (Figures 4A and 4B). LC-MS analysis of a Proteinase K digestion-derived peptide from the TM4 region using Orbitrap NanoLC-MS/MS instrument.

RT: 0.00 - 109.99 51.82

100 95 90 85 80 75 70 65 Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 55 50 45 40 35 30 35.28

25 20

41.14

15 10 5 2.31

0

8.18

0

10

18.46

44.74

27.49 29.70

52.38 59.36 20

30

40

50 60 Time (min)

74.93 79.92 70

80

91.22 90

103.65 100

Figure 4A. Extracted ion chromatogram for Proteinase K digestion-derived peptide AIGLPVMFM*AT (199-209) (M*, methionine oxidation) with precursor ion (M+2H)2 at m/z = 583.8049.


Page 27 of 41

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Figure 4B. Tandem mass spectrum sequence annotation of the hMOR-c-myc-6-his TM4 peptide AIGLPVMFM*AT (199-209) obtained from proteinase K digestion (M*, methionine oxidation) with precursor ion (M+2H)2+ at m/z = 583.8049; peptide (M = 1165.5953) using Orbitrap nanoLC-MS/MS). Mass spectra of the parent ion for the peptide (corresponding to RT = 51.82 min) is shown in the inset. The standard y- and bion series shown represent the complete sequencing of the peptide containing AIGLPVMFM*AT where M* designates an oxidized methionine. See text for more details on experimental conditions. m/z measured (Da)a 1165.5953

m/z theoretical. (Da)b 1165.5875

Error (ppm)c

zd

6.65

2

Peptide sequence AIGLPVMFM*AT


Positione (199-209)

Scoref 67.2


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Page 28 of 41

a) Measured mass to charge ratio in Thomson; b) theoretical mass to charge ratio in Thomson; c) error between measured and theoretical mass in ppm; d) charge state of the spectrum; e) position of sequence; f) Mascot score.

L G G R D S

D

T


51

A A S S

L C

61 P P T G

Extracellular

L S P S

W

Membrane

I M A L Y S I V C V V G L 81 F G N F L V M Y V 91 I V R

C

1 D

221 T

T F

S

F

S H

G Q R

D I

Y 211

G

G 131 M L V Y N S Q F P L 121 S T T L A D A L A 111 A N L

F I Y I T N A

T K 101

Y

Intracellular

P

T

M I T A I T

K

T

I

L C 141 K I V I S I

T

A

T

M F V M D Y Y N 151 G L P 201 I M F T S A S S I I L F T W N L C T 161 191 C V MS N I V D I R K Y I A A V C P R N 171

T

H

T K M

P V K A L 181

I P

T V

T

W Y W E N 231 L L K I C V F 241 I F A F I M P V L I I T V251 C Y G L M I L R L

R S V

F D

K A L I 301 I Y V H I P I W T C 291 I V V F A V V L V M V R 281 I T R L R R N D K

K 261

E 271

R

K S

M L

H H H 421 H H H

P

S G

P E 311 T T F Q T V S W 321 H F C I A L G Y T N S C 331 L N P V L Y A F L D 341 E

C 351 F

I P

N

E R F K R C F

T S S N I E Q 361 Q

hMOR Proteinase K digestion Sequence coverage: 70 %

nanoLC-MS/MS proteinase K fragments

N S T

G


I L

K Q E L 401


R

I

R

Figure 5. Two dimensional graphical representation on hMOR-c-myc-6-his sequence of proteinase K digested peptides identified by nanoLC-MS/MS.


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Owing to the high coverage yielded by proteinase K and the good complementarity of identified peptide between chymotrypsin and proteinase K, the overall sequence coverage finally observed was up to 82% (349 identified amino acids out of a total of 424). This is the first human opioid receptor that is proteomically peptide mapped with a high coverage yield. As Figure 6 shows, except for TM6, an almost virtually complete sequence coverage of the hMOR hydrophobic TM domains was achieved which is an essential finding given the structural and functional importance of the TM domains for opioid receptor activation.

L G G R D S

D

T


51

A A S S

L C

61 P P T G

Extracellular

Membrane

Intracellular

L S P S

W

P

T F

T

C

1

221 T S

F

S H

G Q R

D I

G

G M I 131 M L T A I N V Y T I S Q F P M A L L 121 S T Y S I V T L A C V V D A L A G L 81 111 F G A N L N F F L I Y I V M Y V N 91 A T I V T R K 101 Y M T K

Y 211 K

T

I

L C 141 I V S I

K

A

T

T

M F V M D Y Y N 151 G L P 201 I M F T S A L S S I I F T W N L C T 161 191 C V MS N I V D I R K Y I A A V C P R N 171 T H R P F V D K A L 181 I

I P

T V

T

W Y A W E I K N 231 301 I L L Y V K I C V H I F P I 241 I F W T A F C 291 I M I V P V F V L A I I V V 251 T V L V C M V R 281 Y G L M I T R I L L R R R N L S V

H H H 421 H H H

Sequence coverage: 82% (349/424) G


I L

K Q E L 401

E 311 T T F Q T V S W 321 H F C I A L G Y T N S C 331 L N P V L Y A F L D 341

E 271 K S

R M L

hMOR digestion overlaid

D K

K 261

P

S G

E

C I

351 F

P

N

E R F K R C F


● Trypsin only ● Chymotrypsin only ● Proteinase K only ● Superimposition of enzymes

P L P A T E A E L N E L Q H N T R D V T N A T S P H D R T N 371 391 381

Q

R

I

R

Figure 6. Two dimensional serpentine representation of hMOR-c-myc-6-his sequence coverage of peptides identified by MALDI-TOF-MS and nanoLC-MS/MS after trypsin (in orange), chymotrypsin (in blue) and proteinase K (in pink) enzymatic digestions.



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Superimposed enzymatic digestions (trypsin, chymotrypsin and proteinase K) are shown in green as a result the trypsin (orange) trypsin-derived peptides cannot be appreciated because they are overlaid by other identified peptides from other enzymatic digestions. CONCLUSIONS The mass spectrometric characterization of opioid receptors and other GPCR membrane proteins has always been hampered by their hydrophobic properties and relatively low natural abundance. In the present work we have addressed these challenges for the hMOR by using a highly productive expression system of functional receptor and a multienzymatic bottom up MS-based proteomic method. A key methodological feature for preparing suitable hMOR samples for MS analysis has been the use of our own previously reported hMOR-c-myc-6-his expression system in Pichia pastoris cells implemented with a sample concentration step based on AmiconTM centrifugal filters prior to proteomic analysis. Other advantageous operational features have been to fine tune enzymatic digestion conditions by working in gel rather than in solution modalities, increase digestion times and the temperature on the digestion protocols, and use multiple endoprotease enzymes. Trypsin digestions of our hMOR construct have afforded low sequence coverage (16%) after MALDI-TOF-MS analyses matching our previously published data. In-gel digestions of chymotrypsin and simultaneous monitoring by MALDI-TOF MS and nanoLC-MS/MS allowed a 60% of coverage owing that these MS techniques provided a good proportion of complementary sequence information on the N-terminus and TM motives. Additional proteinase K digestions in conjunction with nanoLC-MS/MS have been conclusive for the optimal characterization of hMOR rendering and overall sequence coverage of 82% that included most of the residues located at six out of the seven transmembrane domains


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(TM1, TM2, TM3, TM4, TM5 and TM7) and the extracellular and intracellular loops of hMOR. This is the first MS based proteomic study comprehensively covering the primary sequence of a human opioid receptor. These results add a new example to the group of six GPCRs that have already been proteomically determined at >80% sequence coverage. We envisage that these results will contribute in the intricate progress towards understanding hMOR receptor activation essential for future drug discovery and development.



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ASSOCIATED CONTENT Supporting Information. Amino acid sequence hMOR-c-myc-6-his (Figure S1); Recombinant fusion protein used (Figure S2); purification of the human mu-opioid receptor (hMOR-c-myc-6-his) (Figure S3); predictions of cleavage sites (trypsin and chymotrypsin) of hMOR-c-myc-6-his (Figure S4, and Tables S1A and S1B); nanoLCMS/MS of digested samples of hMOR-c-myc-6-his functional receptor protein (Figure S5, S6, S7 and S8); other MS/MS spectra from proteinase K digested sample of hMORc-myc-6-his protein (Figures S9a, S9B, S10, S11, S12 and S13); MALDI-TOF MS identified peptides of digested sample of hMOR-c-myc-6-his functional receptor protein (Figures S14, S15, S16, S17 and S18). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Dr. Gregorio Valencia. Instituto de Química Avanzada de Cataluña (IQAC-CSIC), Jordi Girona 18-26, E08034-Barcelona (Spain); Phone: + 34934006113. Fax: +34932045904. E-mail: [email protected].

ACKNOWLEDGEMENTS This work was supported by a grant from the Fundació La Marató de TV3 (Pain, project reference 070430-31-32-33) to GV and GA. This work was supported by the


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French Centre National de la Recherche Scientifique (FT and JMZ). The Proteomics Laboratory at VHIO belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.

ABBREVIATIONS AcOH, acetic acid; ACN, acetonitrile; BCA, bicinchoninic acid; BHK, Baby Hamster Kidney cells; BSA, bovine serum albumin; C-, carboxy; C*, carbamidomethylated cysteine; CHO, Chinese Hamster Ovary cells; CM, carbamidomethylated; C-myc, EQKLISEEDL peptide tag; CNBr, cyanogen bromide; DHB, 2,5-dihydroxybenzoic acid; DOR, delta-opioid receptor; DTT, dithiothreitol; ECL, extracellular loop; EtOH, ethanol; FA, formic acid; GPCR, G protein-coupled receptor; His6, hexa-histidine peptide

tag;

HRP,

horseradish

peroxidase; HPLC,

High-performance

liquid

chromatography; id. Internal diameter; IAA, iodoacetamide; ICL, intracellular loop; IM, Imidazole; IMAC, Immobilized Metal Affinity Chromatography; KOR, kappa-opioid receptor; LC, liquid chromatography; LTQ-FT MS, linear ion trap-Fourier transform mass spectrometer; M*, oxidized methionine; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MOR, mu-opioid receptor; m/z, mass-to-charge ratio; MS/MS, tandem mass spectrometry; N-, amino; o.n., overnight; PBS, phosphate-buffered saline; rt, room temperature; PMSF, phenylmethylsulfonyl fluoride; PNGase F, Peptide-N-Glycosidase F; PVDF, polyvinylidene difluoride; rt, room temperature; RT, retention time; SB, solubilization buffer; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM, Standard Error of the Mean; SDS, Sodium dodecyl sulfate; TFA, trifluoroacetic acid; TM, transmembrane; TMHs, transmembrane α-helices.



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TABLE OF CONTENTS/ABSTRACT GRAPHIC

L G G R D S

D

T


51

A A S S

L C

61 P P T G

Extracellular

L S P S

W

I

M A L Y S I V C V V G L 81 F G N F L V M Y V 91 I V R Y

Intracellular

T F

T

M I T A I T

Membrane

P

T

221 T

C

S D I

G

G 131 M L V Y N S Q F P L T S 121 T L A D A L A 111 A N L F I Y I T N A T K 101 K M

1 F

S H

G Q R

Y 211 K

T T L C A 141 K I V M I S F I V M D Y Y N 151 G L P 201 I M F T S A L S S I I F T W N L C T 161 191 C V MS N I V D I R K Y I A A V C P R N 171 T H R P F V D K A L 181

T

I

I P

T V

T

W Y W E I K A N 231 301 I L L Y V K I C V H I F P I 241 I F W T A F C 291 I M I V P V F V L A I I V V 251 T V L V C M V R 281 Y G I T L M R I L L R R R N L S V

H H H 421 H H H

Sequence coverage: 82% (349/424) G


I L

K Q E L 401

E 311 T T F Q T V S W 321 H F C I A L G Y T N S C 331 L N P V L Y A F L D 341

E 271

R

K S

M L

hMOR digestion overlaid

D K

K 261

P

S G

E

C 351 F

I P

N

E R F K R C F


● Trypsin only ● Chymotrypsin only ● Proteinase K only ● Superimposition of enzymes



R

I

R

Optimized Proteomic Mass Spectrometry Characterization of

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