An Optimized Strategy for ICAT Quantification of Membrane Proteins
2005; Elsevier BV; Volume: 5; Issue: 1 Linguagem: Inglês
10.1074/mcp.m500205-mcp200
ISSN1535-9484
AutoresClaire Ramus, Anne Gonzalez de Peredo, Cécile Dahout, Maighréad Gallagher, Jérôme Garin,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe work presented here focuses on the development of a method adapting isotope labeling of proteins with ICAT to the study of highly hydrophobic proteins. Conditions for the labeling of proteins were first established using two standard soluble proteins and iodoacetamidyl-3,6-dioxaoctanediamine biotin (PEO-iodoacetyl biotin). Results demonstrated the efficiency of the labeling in the presence of high concentrations of both SDS and urea. These conditions were then used to label a highly hydrophobic mitochondrial membrane protein, the adenine nucleotide translocator ANT-1, with PEO-iodoacetyl biotin and then with the cleavable ICAT reagent. The results presented here show that labeling of proteins with cleavable ICAT is possible and may even be improved in strong denaturing buffers containing both SDS at a concentration higher than 0.5% (w/v) and urea. These results open the possibility of applying the ICAT strategy to complex samples containing very hydrophobic proteins solubilized in urea-SDS buffers. The adaptability of the developed method is demonstrated here with preliminary results obtained during the study of membrane-enriched fractions prepared from murine embryonic stem cells. The work presented here focuses on the development of a method adapting isotope labeling of proteins with ICAT to the study of highly hydrophobic proteins. Conditions for the labeling of proteins were first established using two standard soluble proteins and iodoacetamidyl-3,6-dioxaoctanediamine biotin (PEO-iodoacetyl biotin). Results demonstrated the efficiency of the labeling in the presence of high concentrations of both SDS and urea. These conditions were then used to label a highly hydrophobic mitochondrial membrane protein, the adenine nucleotide translocator ANT-1, with PEO-iodoacetyl biotin and then with the cleavable ICAT reagent. The results presented here show that labeling of proteins with cleavable ICAT is possible and may even be improved in strong denaturing buffers containing both SDS at a concentration higher than 0.5% (w/v) and urea. These results open the possibility of applying the ICAT strategy to complex samples containing very hydrophobic proteins solubilized in urea-SDS buffers. The adaptability of the developed method is demonstrated here with preliminary results obtained during the study of membrane-enriched fractions prepared from murine embryonic stem cells. Membrane proteins are involved in a multitude of cellular processes such as signal transduction, cellular adhesion, ion transport, and drug resistance. Their identification provides clues to the understanding of cellular functions and mechanisms. Proteins bearing multiple transmembrane helices, which include many transporters and receptors, are very hydrophobic. Because such proteins are difficult to solubilize and are therefore often poorly represented as part of the total protein pool, their identification and quantification remain important challenges. Considering the biological and pharmacological importance of this protein class with over 50% of current drug targets being membrane proteins (1Drews J. Drug discovery: a historical perspective.Science. 2000; 287: 1960-1964Google Scholar), it appears necessary to develop new methods that will allow the study of changes in membrane protein expression. Whereas two-dimensional electrophoresis (2DE) 1The abbreviations used are: 2DE, two-dimensional electrophoresis; ANT-1, adenine nucleotide translocator; PEO-iodoacetyl biotin, iodoacetamidyl-3,6-dioxaoctanediamine biotin; c-ICAT, cleavable ICAT; LAPAO, 3-laurylamido-N,N′-dimethylpropylaminoxide; ES, embryonic stem; ESd, differentiated ES; Ccam, carbamidomethylated cysteine; Cpeo, cysteine modified with PEO-iodoacetyl biotin. 1The abbreviations used are: 2DE, two-dimensional electrophoresis; ANT-1, adenine nucleotide translocator; PEO-iodoacetyl biotin, iodoacetamidyl-3,6-dioxaoctanediamine biotin; c-ICAT, cleavable ICAT; LAPAO, 3-laurylamido-N,N′-dimethylpropylaminoxide; ES, embryonic stem; ESd, differentiated ES; Ccam, carbamidomethylated cysteine; Cpeo, cysteine modified with PEO-iodoacetyl biotin. can be used to identify and compare protein samples quantitatively (2Newsholme S.J. Maleef B.F. Steiner S. Anderson N.L. Shwartz N.W. Two dimensional electrophoresis of liver proteins: characterization of drug-induced hepatomegaly in rats.Electrophoresis. 2000; 21: 2122-2128Google Scholar, 3Tonge R. Shaw J. Middleton B. Rowlinson R. Rayner S. Young J. Pognan F. Hawkins E. Currie I. Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.Proteomics. 2001; 1: 377-396Google Scholar), this technique is not applicable for the study of highly hydrophobic proteins (4Santoni V. Molloy M. Rabilloud T. Membrane proteins and proteomics: un amour impossible ?.Electrophoresis. 2000; 21: 1054-1070Google Scholar). In this context, despite significant improvements (5Luche S. Santoni V. Rabilloud T. Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis.Proteomics. 2003; 3: 249-253Google Scholar), 2DE is being gradually replaced by multidimensional protein identification technology (MudPIT) (6Peng J. Gygi S.P. Proteomics: the move to mixtures.J. Mass Spectrom. 2001; 36: 1083-1091Google Scholar, 7Washburn M.P. Ulaszek R. Deciu C. Schieltz D.M. Yates III, J.R. Analysis of quantitative proteomic data generated via multidimensional protein identification technology.Anal. Chem. 2002; 74: 1650-1657Google Scholar). Such strategies appear better adapted to the analysis of hydrophobic proteins than 2DE as membrane suspensions can be used and the analysis focused on the surface-exposed portions of integral membrane proteins (8Wu C.C. MacCoss M.J. Howell K.E. Yates III, J.R. A method for the comprehensive proteomic analysis of membrane proteins.Nat. Biotechnol. 2003; 21: 532-538Google Scholar). However, whereas different approaches are being developed for the quantitative profiling of proteins using mass spectrometry, very few studies report quantitative analysis of membrane proteins using chemical labeling such as ICAT. In a typical ICAT experiment, samples to be compared are individually labeled by the light or heavy form of the ICAT reagent, resulting in the detection of a pair of peaks for each peptide in common between the two samples when analyzed by nano-LC-MS (9Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar). The peptides labeled by the light or the heavy ICAT isoforms co-elute on the liquid chromatography reverse phase column, and the ratios between the MS signal areas for the low and high mass components of these pairs of peaks provide an accurate measure of the relative abundance of the peptides and thus of the relative levels of protein expression in the original samples. In a classical ICAT analysis, all of the protein labeling and preparation steps are carried out in solution. This technique has been used for the quantitative analysis of a total yeast extract carried out by Han et al. (10Han D.K. Eng J. Zhou H. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Google Scholar). It is of note that during the course of the study by Han et al., among the hundreds of proteins analyzed, quantitative data concerning true membrane proteins were scarce because of the low abundance of these proteins and to the fact that ICAT labeling requires the presence of both exposed soluble regions of sufficient length to generate one or more tryptic peptides and a cysteine residue present in the extramembrane material to be accessible to the reagent. Consequently, during such large scale quantitative proteome analyses, hydrophobic proteins remain largely underrepresented. One strategy for studying membrane proteomes in depth is to apply specific methods, such as biological fractionation, to enrich the hydrophobic protein core present in the fraction of interest (11Ferro M. Salvi D. Brugiere S. Miras S. Kowalski S. Louwagie M. Garin J. Joyard J. Rolland N. Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana.Mol. Cell. Proteomics. 2003; 2: 325-345Google Scholar, 12Goshe M.B. Blonder J. Smith R.D. Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis.J. Proteome Res. 2003; 2: 153-161Google Scholar, 13Ferro M. Salvi D. Riviere-Rolland H. Vermat T. Seigneurin-Berny D. Grunwald D. Garin J. Joyard J. Rolland N. Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11487-11492Google Scholar). The present work was aimed at adapting the ICAT protocol to experimental conditions that would allow ICAT labeling of fully solubilized and denatured core membrane proteins. Therefore, we set up and tested new conditions that allow ICAT labeling in the presence of high concentrations of SDS. In this study, cysteine labeling with PEO-iodoacetyl biotin, a chemical reagent whose structure is close to that of the ICAT reagent, first allowed us to optimize conditions for the labeling of two cysteine-rich non-membrane proteins, ovalbumin and β-lactoglobulin, and ICAT labeling was then performed on the bovine mitochondrial ADP/ATP translocator, a highly hydrophobic 33-kDa protein containing six α-helical transmembrane domains. Finally our newly developed ICAT labeling technique was tested on an enriched membrane protein fraction prepared from murine embryonic stem cells. Ovalbumin (chicken) and β-lactoglobulin (bovine) proteins were from Sigma. The ADP/ATP translocator was isolated from bovine heart mitochondria as a carboxyatractyloside-carrier complex in the presence of 0.05% 3-laurylamido-N,N′-dimethylpropylaminoxide (LAPAO) according to the procedure described by Block et al. (14Block M.R. Zaccai G. Lauquin G.J. Vignais P.V. Small angle neutron scattering of the mitochondrial ADP/ATP carrier protein in detergent.Biochem. Biophys. Res. Commun. 1982; 109: 471-477Google Scholar). The recovered ADP/ATP translocator-enriched fraction (obtained in 50 mm Na2SO4, 10 mm Tris, pH 7.3, 1 mm EDTA, 0.05% LAPAO) was precipitated in acetone at −20 °C overnight. After this, the precipitate was washed at least twice in water before being solubilized in buffer U8S4 (50 mm Tris-HCl, pH 8.3, 5 mm EDTA, 8 m urea, 4% (w/v) SDS), and protein concentration was determined by a Bradford assay (15Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Google Scholar). 60 × 106 totipotent murine embryonic stem cells (ES cells) or early differentiated murine embryonic stem cells (ESd cells) were resuspended in 10 mm Tris, pH 7.5, complemented with an anti-protease mixture (Complete, Roche Diagnostics). Lysis was performed using a cell disrupter system (Constant Systems, Northants, UK) in one-shot mode at 350 bars. The resulting lysate was submitted first to a short centrifugation at 1000 × g at 4 °C to eliminate nuclei and intact cells. The postnuclear supernatant was ultracentrifuged for 1 h at 100,000 × g at 4 °C (in an MLA-80 rotor). For the enrichment of membrane proteins in the samples, pellets were first resuspended in an alkaline buffer (0.1 m Na2CO3, pH 11) and centrifuged for 45 min at 100,000 × g at 4 °C. The Na2CO3 pellets were resuspended in 0.1 m Na2CO3, 0.5 m NaCl, pH 11 before centrifuging for 45 min at 100,000 × g at 4 °C. Pellets, containing the insoluble proteins, were further washed in water and finally resuspended in buffer U8S4 (see above) to perform ICAT labeling. After protein concentration determination, equal amounts of each sample (ES and ESd preparations) were modified by ICAT cleavable reagents (Applied Biosystems, Framingham, MA) as described under “Isotope Labeling of Cysteines.” Depending on the experiment, proteins were solubilized using one of the following buffers: buffer U8S4 (see above), buffer U8S0.5 (50 mm Tris-HCl, pH 8.3, 5 mm EDTA, 8 m urea, 0.5% (w/v) SDS), buffer U8S0 (50 mm Tris-HCl, pH 8.3, 5 mm EDTA, 8 m urea), buffer U6S0.05 (50 mm Tris-HCl, pH 8.3, 5 mm EDTA, 6 m urea, 0.05% (w/v) SDS), or buffer U0S0 (50 mm Tris-HCl, pH 8.3, 5 mm EDTA). Protein disulfide bonds were reduced by 10 mm tributylphosphine for 30 min at 37 °C. Cysteine residues were then alkylated by either PEO-iodoacetyl biotin (Pierce) (45 min in the dark at 37 °C) or cleavable ICAT (c-ICAT, Applied Biosystems) (2 h in the dark at 37 °C). The alkylation reaction was quenched with DTT before loading labeled proteins on a 12% polyacrylamide gel and submitting them to electrophoresis (16Chua N.H. Electrophoresis analysis of chloroplast proteins.Methods Enzymol. 1980; 69: 434-436Google Scholar). After migration, proteins were colored by Coomassie staining. In the case of complex mixtures, 100 μg of proteins from the enriched membrane fraction of either ES or ESd cells were labeled with light and heavy c-ICAT, respectively, before mixing together. One-quarter of the resulting mixture was then loaded on a 12% SDS-PAGE gel and submitted to a short migration to fractionate the mixture into 10 bands, an adaptation of a method described by Ferro et al. (11Ferro M. Salvi D. Brugiere S. Miras S. Kowalski S. Louwagie M. Garin J. Joyard J. Rolland N. Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana.Mol. Cell. Proteomics. 2003; 2: 325-345Google Scholar). For Western blotting purposes, 20 μg of membrane proteins from differentiated stem cells were separated by SDS-PAGE and transferred onto a nitrocellulose sheet according to the standard method (17Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar). Immunodetection was carried out using a polyclonal antibody (diluted at 1:1000) raised against the bovine ANT-1 protein. The immune complexes were revealed by protein A-peroxidase (Pierce) at a dilution of 1:5000 and ECL reagent (Amersham Biosciences). Each gel band was excised and prepared for mass spectrometry analysis. Gel bands were destained by repeated cycles of incubation in 25 mm NH4HCO3 for 15 min and then with 50% (v/v) acetonitrile in the same buffer (25 mm NH4HCO3) for 15 min (18Rabilloud T. Kieffer S. Procaccio V. Louwagie M. Courchesne P.L. Patterson S.D. Martinez P. Garin J. Lunardi J. Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: toward a human mitochondrial proteome.Electrophoresis. 1998; 19: 1006-1014Google Scholar). After drying by vacuum centrifugation, the bands were incubated with a reducing solution (25 mm NH4HCO3 containing 10 mm DTT) for 30 min at 37 °C. Alkylation was performed with an alkylating solution (25 mm NH4HCO3 containing 55 mm iodoacetamide) for a further 30 min at 37 °C. Bands were then washed several times with the destaining solutions and finally in pure water for 15 min before being dehydrated with 100% acetonitrile. In-gel digestion was performed using trypsin (sequencing grade, Promega, Madison, WI) at a 1:20 protease to protein ratio in 25 mm NH4HCO3 for 5 h at 37 °C. Peptides were extracted from the gel using passive diffusion in the following solutions: 50% CH3CN, then 5% formic acid, and finally 100% CH3CN. The extract was dried by vacuum centrifugation; peptides were then resolubilized in an aqueous trifluoroacetic acid solution for MALDI-TOF/MS analysis. c-ICAT-labeled peptides were purified using the avidin column supplied in the c-ICAT kit (Applied Biosystems) according to the manufacturer’s instructions. The bound peptides were eluted using 30% CH3CN, 0.4% TFA. After drying, cleavage of the acid-labile moiety of the c-ICAT reagent was performed as recommended by the manufacturer (37 °C in 95% TFA for 2 h) before nano-LC-MS/MS analysis. Peptide mixtures were analyzed with a MALDI-TOF/MS mass spectrometer (Autoflex, Bruker Daltonics) in reflector mode. 0.5 μl of sample was mixed with the same amount of matrix solution (α-sinapinic acid saturated in 50% CH3CN, 0.1% TFA) and loaded on a target before analysis. The dried gel-extracted tryptic peptides were solubilized in 95% water (v/v) containing 2.5% acetonitrile and 2.5% trifluoroacetic acid for nano-LC-MS and nano-LC-MS/MS analysis (CapLC and Q-TOF Ultima, Waters, Milford, MA). The method consisted of a 50-min run at a flow rate of 200 nl/min using a two-solvent gradient: solvent A (2% acetonitrile, 97.9% water, 0.1% formic acid) and solvent B (80% acetonitrile, 19.9% water, 0.1% formic acid). The system includes a 300-μm × 5-mm PepMap C18 precolumn (LC-Packings, Dionex, Sunnyvale, CA) to concentrate peptides before injection onto a 75-μm × 150-mm C18 column (LC-Packings) directly coupled to the mass spectrometer. Proteins were identified from MS/MS data with Mascot 2.0 software (Matrix Science, www.matrixscience.com). Database searches were performed on the Swiss-Prot_Trembl data bank specifying several variable amino acid modifications: acetylation, oxidized methionine, ICAT light/heavy, and carbamidomethylated cysteines. Identification of all ICAT peptides was manually confirmed by inspection of MS/MS spectra. Quantification was done manually after integration of peaks for both isoforms of each labeled peptide identified on reconstituted chromatograms. Reconstituted chromatograms were obtained after extraction of a specific mass (±0.1 Da) from the nano-LC-MS data using MassLynx software (Waters). Membrane proteins are often difficult to extract from their native environment without encountering difficulties because of insolubility and protein loss through precipitation. High concentrations of detergents, such as SDS, allow the solubilization of most proteins, even highly hydrophobic ones, as the detergent mimics the native lipid bilayer environment of the proteins. However, in such conditions, the labeling of cysteine residues present on hydrophobic proteins with ICAT probes requires adaptation of the standard protocol that is not compatible with the presence of high concentrations of SDS (19Smolka M.B. Zhou H. Purkayastha S. Aebersold R. Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.Anal. Biochem. 2001; 297: 25-31Google Scholar). Fig. 1 underlines the major differences between the ICAT standard protocol and the strategy that was set up during the course of this work to obtain quantitative expression data on membrane proteins. Usually, protein samples are first labeled with ICAT in a saline buffer, mixed, and then submitted to trypsin digestion. In our modified method, as proteins are first labeled with ICAT in the presence of high amounts of SDS, their migration on a 12% polyacrylamide gel is necessary before trypsin digestion can be performed. During electrophoresis, excess ICAT and other buffer components incompatible with the tryptic digestion are separated from the protein samples. Thus, the in-gel treatment of the proteins replaces the cation exchange column used for cleaning and fractionation of the samples after “in-solution” trypsin digestion in the classical protocol. As the yield of in-gel alkylation with iodoacetamide has been shown to be close to 100% (20Herbert B. Galvani M. Hamdan M. Olivieri E. MacCarthy J. Pedersen S. Righetti P.G. Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when and how?.Electrophoresis. 2001; 22: 2046-2057Google Scholar), cysteine residues of the ICAT-labeled proteins were further in-gel alkylated with iodoacetamide to allow the identification of cysteine peptides that may have only partially reacted with ICAT. Similarly to the classical ICAT method, ICAT-labeled tryptic peptides were then concentrated by affinity chromatography on an avidin column prior to their analysis by nano-LC-MS and nano-LC-MS/MS. To set up experimental conditions offering the ability to label proteins with ICAT in highly denaturing buffers, optimization of the labeling conditions was first performed with ovalbumin and β-lactoglobulin, two soluble proteins often used as models for the evaluation of alkylation as they contain six and seven cysteine residues, respectively. Cysteine labeling was carried out using an ICAT analog, PEO-iodoacetyl biotin. This reagent is composed of a biotin affinity tag, a linker, and a specific thiol-reactive group. It reacts with cysteine residues by the same alkylation reaction as the one implicated in ICAT labeling (19Smolka M.B. Zhou H. Purkayastha S. Aebersold R. Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.Anal. Biochem. 2001; 297: 25-31Google Scholar). Being far less expensive than ICAT reagents, PEO-iodoacetyl biotin allowed us to test several buffer conditions for labeling. The effects of both a chaotropic agent (urea) and a detergent (SDS) were tested on the labeling of ovalbumin and β-lactoglobulin. During the labeling reaction, temperature was not allowed to exceed 37 °C to avoid protein carbamylation that would occur on lysine residues at high temperatures in the presence of urea (21Lippincott J. Apostol I. Carbamylation of cysteine: a potential artifact in peptide mapping of hemoglobins in the presence of urea.Anal. Biochem. 1999; 267: 57-64Google Scholar). After the labeling step, proteins were analyzed by SDS-PAGE (Fig. 2 ). When a protein is modified by PEO-iodoacetyl biotin, an increase in mass of 414 Da per cysteine is induced. Thus, complete alkylation of ovalbumin and β-lactoglobulin with this molecule should induce a mass shift of 2484 and 2898 Da, respectively. Taking advantage of this easily detectable evidence of modification, labeling was roughly estimated from the migration shift of the proteins on the SDS-PAGE gel at this stage of the work. Fig. 2 demonstrates the effect of different buffers during the labeling step. Protein labeling was first performed in the absence of both urea and SDS (lane 4, buffer U0S0): when compared with unmodified proteins (lane 5), no shift in mass is noticed for ovalbumin, whereas multiple bands appear for β-lactoglobulin, suggesting incomplete labeling. Although labeling efficiency was actually improved by the use of buffer U8S0 (lane 3) and was even better in buffer U6S0.05 (the classical ICAT conditions, lane 2), a slight band corresponding to unlabeled ovalbumin was reproducibly detected. Therefore we tried labeling conditions where a high percentage of SDS was included in the solubilization buffer, conditions that would be well adapted for solubilization of hydrophobic proteins. When 8 m urea and 0.5% SDS were used (lane 1, buffer U8S0.5), a complete mass shift was observed for both proteins, suggesting that complete labeling was achieved. The same result was obtained in the presence of 8 m urea and 4% SDS (not shown). To assess the labeling of ovalbumin and β-lactoglobulin by PEO-iodoacetyl biotin more accurately, protein gel bands (observed on Fig. 2) were excised and submitted to in-gel alkylation with iodoacetamide and tryptic digestion, and the resulting peptides were analyzed by MALDI-TOF/MS. This experiment confirmed the partial labeling by PEO-iodoacetyl biotin in the absence of SDS (buffer U8S0). In contrast, in the presence of SDS and urea (buffer U8S0.5), no cysteine peptide labeled with iodoacetamide could be detected (Fig. 3). The efficiency of the labeling and the identity of each PEO-iodoacetyl biotin-modified peptide were confirmed by nano-LC-MS and nano-LC-MS/MS (data not shown). Taken together, these results lead to the conclusion that, far from being detrimental, the presence of a high concentration of SDS, when associated with 8 m urea, enhances the labeling of these two soluble proteins, whereas the previous publications dealing with ICAT labeling insist on the importance of not exceeding 0.1% (manufacturer’s protocol) or even 0.05% SDS (19Smolka M.B. Zhou H. Purkayastha S. Aebersold R. Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.Anal. Biochem. 2001; 297: 25-31Google Scholar).Fig. 3Ovalbumin labeling by PEO-iodoacetyl biotin: estimation of reaction efficiency by MALDI-TOF/MS. After solubilization and labeling, ovalbumin was subjected to migration on a 12% SDS-PAGE gel. Bands were visualized by Coomassie Blue staining, excised, destained, reduced, alkylated by iodoacetamide, and submitted to in-gel trypsin digestion (as described under “Experimental Procedures”). The peptides obtained were analyzed by MALDI-TOF/MS to estimate the efficiency of PEO-iodoacetyl biotinylation. Peptide masses for cysteine peptides are shown. Underlined masses correspond to PEO-iodoacetyl biotin-labeled peptides, whereas masses of corresponding peptides containing a carbamidomethylcysteine modification are not underlined. a, unlabeled ovalbumin; b, ovalbumin labeled in buffer U8S0.5; c, ovalbumin labeled in buffer U8S0.View Large Image Figure ViewerDownload (PPT) We next tested these modified labeling conditions on a highly hydrophobic protein, the adenine nucleotide translocator ANT-1. This protein, containing four cysteines and six transmembrane domains (22Dahout-Gonzalez C. Brandolin G. Pebay-Peyroula E. Crystallization of the bovine ADP/ATP carrier is critically dependent upon the detergent-to-protein ratio.Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2353-2355Google Scholar), one of which contains a cysteine residue, can be considered as a model for multitransmembrane domain proteins. Because of its numerous transmembrane domains, ANT-1 is a highly hydrophobic protein and is poorly soluble in buffers that are classically used for ICAT labeling (6 m urea, 0.05% SDS). Therefore, ANT-1 was solubilized in the presence of 8 m urea using a range of SDS concentrations (1–4% (w/v)). The solubilized protein was then labeled with PEO-iodoacetyl biotin and loaded onto an SDS-PAGE gel. When ANT-1 was labeled in the presence of 8 m urea and 4% SDS, the four ANT-1 cysteine residue-containing peptides were detected by MALDI-TOF/MS analysis as peptides bearing the PEO adduct, and for all these peptides, no mass fragments corresponding to peptides alkylated by iodoacetamide were detected in the same spectrum (Fig. 4, a and b). Partial solubilization of ANT-1 resulting in a loss of material was observed when solubilization was performed in buffers with less than 4% SDS (not shown). In-gel alkylation, tryptic digestion, and MALDI-TOF/MS and nano-LC-MS analysis demonstrated that this loss of material was associated with a less efficient labeling reaction (data not shown). These observations indicate that, as expected, high SDS concentrations are necessary to obtain a good recovery and labeling of membrane proteins like ANT-1. Therefore, for all further experiments, the solubilization and labeling of ANT-1 were carried out in the presence of 8 m urea and 4% SDS. Analysis of the ANT-1 tryptic peptides by nano-LC-MS/MS confirmed the identity of all the peptides observed by MALDI-TOF/MS. Fig. 5 a shows the fragmentation spectrum of peptide T22-PEO (EFTGLGNCITK). A characteristic signature of the presence of a PEO-labeled cysteine is illustrated by the presence of the following m/z ions: 227, 270, 314, 375, and 449 specific for the fragmentation of the reagent itself. Subsequently analysis by nano-LC-MS was carried out to allow more accurate quantification of the labeling yield for each cysteine.Fig. 5Sequencing of the labeled ANT-1 T22 tryptic peptide by tandem mass spectrometry. T22 peptide (EFTGLGNCITK, z = 2) labeled with either PEO-iodoacetyl biotin or the c-ICAT light reagent was submitted to fragmentation by MS/MS. a, MS/MS spectrum of PEO-iodoacetyl biotin-labeled T22 tryptic peptide. b, MS/MS spectrum of c-ICAT light-labeled T22 tryptic peptide.View Large Image Figure ViewerDownload (PPT) The entire quantification process is illustrated in Fig. 4, c and d, for peptide T22 (EFTGLGNCITK). After generation of the reconstituted ion chromatograms for both forms of each peptide, carbamidomethylated or labeled with PEO-iodoacetyl biotin (see Table I), the elution peaks corresponding to each form were integrated, and the ratio of the calculated areas was used to determine relative abundance. We are aware that quantifying two distinct forms of the same peptide, each one being modified by a different alkylating reagent (PEO-iodoacetyl-biotin and iodoacetamide, respectively), may induce a bias in the quantification. However, we found that it was the most convenient way to evaluate labeling efficiency. Based on this approximation, the abundance of three of the cysteine-containing peptides of ANT-1 modified by PEO-iodoacetyl biotin relative to the abundance of the same peptides modified by iodoacetamide was calculated (Fig. 4e). The analysis of these peptides shows that the efficiency of labeling with PEO-iodoacetyl biotin was at least 80% when the reaction was carried out in the presence of 4% SDS and 8 m urea. Labeling of cysteine 56, present in peptide T8, could not be accurately quantified as it appeared in multiple peptide forms, resulting from miscleavage and posttranslational modification (trimethylation on residue Lys-51).Table ITheoritical masses of each cysteine peptide of ANT-1 under different labeling conditionsTryptic cysteine peptideMass of t
Referência(s)