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• W2099693553 abstract "Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 μg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins. Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 μg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins. Since the last decade, MS has become one of the most powerful techniques in proteomics. Two-dimensional (2-D) 1The abbreviations used are: 2-D, two-dimensional; DIMAL-K, difference in mass analysis using labeled lysine (K); LPS, lipopolysaccharide; VIME, vimentin; SPARC, secreted protein acid and rich in cysteine; VCA1, vascular cell adhesion protein 1; C3L1, chitinase-3-like protein-1, chain 1; CO3A, complement C3 α chain; CO3B, complement C3 β chain; B2MG, β2-microglobulin; NGAL, neutrophil gelatinase-associated lipocalin precursor; Me, acetamidine moiety or Nε-lysine; Et, propionamidine moiety on Nε-lysine; AOP2, antioxidant protein 2; CYPHA, cyclophilin A. PAGE in combination with MALDI-TOF analysis provides a sensitive method for protein identification. An important challenge in proteomics deals with quantification of relative expression levels of individual proteins in two different biological samples. In this context, quantification by mass spectrometry has recently become a major alternative strategy to analysis based on densitometric or fluorescence technologies (1Patton W.F. A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics.Electrophoresis. 2000; 21: 1123-1144Google Scholar, 2Moritz B. Meyer H.E. Approaches for the quantification of protein concentration ratios.Proteomics. 2003; 3: 2208-2220Google Scholar, 3Unlu M. Morgan M.E. Minden J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.Electrophoresis. 1997; 18: 2071-2077Google Scholar, 4Tonge 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). Despite extensive work accomplished in the field of quantification, only few strategies have been associated with the widely used 2-D PAGE/MALDI. Quantification by mass spectrometry requires the production of differentially labeled peptides from two sets of proteins. The difference in expression levels is obtained by measuring the relative intensities of MS signals of the mixed labeled peptides. Different strategies have already been described (for reviews, see Refs. 2Moritz B. Meyer H.E. Approaches for the quantification of protein concentration ratios.Proteomics. 2003; 3: 2208-2220Google Scholar and 5Hamdan M. Righetti P.G. Modern strategies for protein quantification in proteome analysis: advantages and limitations.Mass Spectrom. Rev. 2002; 21: 287-302Google Scholar, 6Flory M.R. Griffin T.J. Martin D. Aebersold R. Advances in quantitative proteomics using stable isotope tags.Trends Biotechnol. 2002; 20: 23-29Google Scholar, 7Sechi S. Oda Y. Quantitative proteomics using mass spectrometry.Proteomics Suppl. 2004; 9: S41-S46Google Scholar, 8Conrads T.P. Issaq H.J. Hoang V.M. Current strategies for quantitative proteomics.Adv. Protein Chem. 2003; 65: 133-159Google Scholar). These strategies differ in 1) the pre- or postdigestion labeling, 2) the choice of the labeled function (thiol, primary amine, or carboxylic acid), 3) the use of stable isotopes or alternative chemicals, and 4) the type of MS analysis (LC-MS/MS or MALDI-TOF MS). Predigestion labeling allows for greater quantification accuracy than postdigestion labeling. Indeed introducing the chemical modification early in the experimental procedure reduces differential loss of proteins during further biochemical steps and guarantees accuracy of the quantification. Predigestion labeling often involves stable isotope incorporation through either metabolic labeling or chemical labeling on a specific residue of proteins. Metabolic labeling, such as the stable isotope labeling by amino acids in cell culture (SILAC) method, is introduced in vivo early in the process (9Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Accurate quantification of protein expression and site-specific phosphorylation.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6591-6596Google Scholar, 10Ong S.-E. Blagooev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Google Scholar) allowing for greater quantification accuracy than postdigestion labeling. However, metabolic labeling can only be used for cultured cells grown in controlled media. Chemical labeling with stable isotopes was mainly introduced by selective alkylation of cysteine residues with either a light or a heavy reagent. The most widely used methodology is based on a class of reagents referred to as ICATs (11Gygi 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, 12Hansen K.C. Schmitt-Ulms G. Chalkley R.J. Hirsch J. Baldwin M.A. Burlingame A.L. Mass spectrometric analysis of protein mixture at low levels using cleavable 13C-ICAT and multi-dimensional chromatography.Mol. Cell. Proteomics. 2003; 2: 299-314Google Scholar). In the same manner, cysteine residues of a mixture of proteins can be alkylated with d0- or d3-acrylamide (13Sechi S. Chait B.T. Modification of cysteine residues by alkylation: a tool in peptide mapping and protein identification.Anal. Chem. 1998; 70: 5151-5158Google Scholar, 14Gehanne S. Quantitative analysis of two-dimensional gel-separated proteins using isotopically marked alkylating agents and matrix-assisted laser desorption/ionisation mass spectrometry.Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698Google Scholar, 15Sechi S. A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis.Rapid Commun. Mass Spectrom. 2002; 16: 1416-1424Google Scholar) or by using the HysTag strategy (16Olsen J.V. Andersen J.R. Nielsen P.A. Nielsen M.L. Figey D. Mann M. Wisniewski J.R. HysTag—a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain.Mol. Cell. Proteomics. 2004; 3: 82-92Google Scholar). Very recently, isotope-coded protein labeling of free amino groups in intact proteins using succinimide activation was described (17Schmidt A. Kellermann J. Lottspeich F. A novel strategy for quantitative proteomics using isotope-coded protein labels.Proteomics. 2005; 5: 4-15Google Scholar). This contrasts with previously reported approaches based on lysine derivatization such as the global internal standard strategy (GIST) in which labeling is performed after enzymatic cleavage of protein samples (18Chakraborty A. Regnier F.E. Global internal standard technology for comparative proteomics.J. Chromatogr. A. 2002; 949: 173-184Google Scholar, 19Ji J. Charkraborty A. Geng M. Zhang X. Amini A. Bina M. Regnier F.E. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides.J. Chromatogr. B Biomed. Sci. Appl. 2000; 745: 197-210Google Scholar, 20Wang S. Regnier F.E. Proteomics based on selecting and quantifying cysteine containing peptide by covalent chromatography.J. Chromatogr. A. 2001; 924: 345-357Google Scholar, 21Wang S. Zhang X. Regnier F.E. Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates.J. Chromatogr. A. 2002; 949: 153-162Google Scholar). Beardsley and Reilly (22Beardsley R.L. Reilly J.P. Quantitation using enhanced signal tags: a technique for comparative proteomics.J. Proteome Res. 2003; 2: 15-21Google Scholar) have also proposed a method called QUEST (quantification using enhanced signal tags) in which efficient amidination of N-terminal and ε-amino groups is performed on peptides using S-methyl thioimidate reagents. We propose here a highly efficient strategy for differential quantification of proteins from different physiological states that we call difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential amidination of lysine residues in complex mixtures of intact proteins (before the digestion step) using the two closely chemically related reagents S-methyl thioacetimidate and S-methyl thiopropionimidate (22Beardsley R.L. Reilly J.P. Quantitation using enhanced signal tags: a technique for comparative proteomics.J. Proteome Res. 2003; 2: 15-21Google Scholar). The mixture of differentially labeled proteins was separated on 2-D gels, and identification and relative quantification of individual proteins were performed in a single MALDI-TOF mass analysis. The relative expression of individual proteins in each state was deduced from the difference in signal intensities of differentially lysine-labeled tryptic peptides separated by multiples of 14 Da. We applied DIMAL-K to quantify the variation of protein secretion in astrocytes secretomes (20 μg) following exposure to a proinflammatory treatment. S-Methyl thioacetimidate and S-methyl thiopropionimidate were synthesized according to Beardsley and Reilly (22Beardsley R.L. Reilly J.P. Quantitation using enhanced signal tags: a technique for comparative proteomics.J. Proteome Res. 2003; 2: 15-21Google Scholar). Ovalbumin (chicken egg), myoglobin (horse skeletal muscle), cytochrome c (horse heart), lysozyme (chicken egg), and lipopolysaccharide (type 055:B5) were from Sigma. Swiss mice were obtained from Elevage Janvier (Le Genest-Saint-Isle, France). Primary cultures of mouse striatal astrocytes were prepared as described previously (23el-Etr M. Cordier J. Glowinski J. Premont J. A neuroglial cooperativity is required for the potentiation by 2-chloroadenosine of the muscarinic-sensitive phospholipase C in the striatum.J. Neurosci. 1989; 9: 1473-1480Google Scholar). Briefly striata from 18-day-old Swiss mouse embryos were mechanically dissociated in PBS supplemented with 33 mm glucose (PBS-glucose). Cells were seeded (0.5 × 106 cells/ml) in 100-mm (15 ml/dish) culture dishes previously coated with poly-l-ornithine (1.5 μg/ml, Mr 40,000). The culture medium consisted of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and F-12 nutrient supplemented with 30 mm glucose, 2 mm glutamine, 13 mm NaHCO3, 5 mm HEPES buffer (pH 7.4), penicillin-streptomycin (100 IU/ml and 100 μg/ml, respectively), and 10% Nu-serum (BD Biosciences). The culture medium was changed every 3 days, and cells were maintained for 21 days at 37 °C in a humidified atmosphere containing 5% CO2. At this stage, cultures were shown to be highly enriched in astrocytes (more that 98% of glial fibrillary acidic protein-positive cells) that had formed a confluent monolayer and devoid of neuronal and microglial or endothelial cells (assessed by the lack of microtubule-associated protein 2 and isolectin B4 immunostaining, respectively) (24Lafon-Cazal M. Adjali O. Galéotti N. Poncet J. Jouin P. Homburger V. Bockaert J. Marin P. Proteomic analysis of astrocytic secretion in the mouse.J. Biol. Chem. 2003; 278: 24438-24448Google Scholar). Cells were washed six times with the serum-free culture medium. This washing procedure efficiently eliminated all serum proteins because we did not detect any bovine albumin, the major serum protein, in the astrocyte-conditioned medium. Cells were then covered with a minimal volume of the serum-free medium (6 ml/dish) for 18 h at 37 °C and 5% CO2 in the presence or absence of lipopolysaccharide (LPS) (1 μg/ml). Astrocyte-conditioned media were harvested and centrifuged successively at 200 × g (5 min), 1,000 × g (10 min), and 20,000 × g (25 min) to remove non-adherent cells and debris. Samples (about 20 μg of protein/dish) were precipitated for 2 h with 10% ice-cold TCA. Precipitates were then washed three times with diethyl ether, dried, and solubilized in 6 m urea. Protein concentration in conditioned media and cell extracts was determined by the bicinchoninic acid method (25Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150 (Correction (1987) Anal. Biochem. 163, 279): 76-85Google Scholar). 150 μg of labeling reagent (S-methyl thioacetimidate or S-methyl thiopropionimidate synthesized according to Beardsley and Reilly (22Beardsley R.L. Reilly J.P. Quantitation using enhanced signal tags: a technique for comparative proteomics.J. Proteome Res. 2003; 2: 15-21Google Scholar)) dissolved in 10 μl of 200 mm NaHCO3 (pH 8.7) was added to each standard protein solution (30 μg in 10 μl of either 6 m urea, 6 m urea plus 4% CHAPS, or 6 m urea plus 1% SDS (or a solution of proteins secreted by the astrocytes (about 20 μg of protein in 10 μl of 6 m urea)). The mixture was stirred for 90 min at room temperature and precipitated with 10% ice-cold TCA during 2 h. Centrifugation (38,000 × g for 30 min) was then performed. Precipitates were washed three times with diethyl ether and dried. Proteins were resuspended in 350 μl of isoelectrofocusing medium containing 7 m urea, 2 m thiourea, 4% CHAPS, ampholines (preblended, pI 3.5–9.5, 8 mg/ml, Amersham Biosciences), 100 mm DTT, 0.2% Tergitol NP7 (Sigma), and traces of bromphenol blue (26Laoudj-Chenivesse D. Marin P. Bennes R. Tronel-Peyroz E. Leterrier F. High performance two-dimensional gel electrophoresis using a wetting agent Tergitol NP7.Proteomics. 2002; 2: 481-485Google Scholar). Proteins were first separated according to their isoelectric point along non-linear IPG strips (pH 3–10, 18 cm long) using the IPGphor apparatus (Amersham Biosciences). Sample loading for the first dimension was performed by passive in-gel reswelling. After the first dimension, the IPG strips were equilibrated for 10 min in a buffer containing 6 m urea, 50 mm Tris-HCl (pH 6.8), 30% glycerol, 2% SDS, and 10 mg/ml DTT and then for 15 min in the same buffer containing 15 mg/ml iodoacetamide instead of DTT. For the second dimension, the strips were loaded onto vertical 10–15% gradient SDS-polyacrylamide gels. The gels were silver-stained according to the procedure of Shevchenko and Wim (27). Gels to be compared were always processed and stained in parallel. Gels were scanned using a computer-assisted densitometer. Spot detection, gel alignment, and spot quantification were performed using Melanie 5 software (Amersham Biosciences). Quantification of proteins was expressed as volumes of spots. To correct for variability resulting from silver staining, results were expressed as relative volumes of all spots in each gel. Data are the means of values from four gels originating from experiments performed on different sets of cultured astrocytes. 10 μg of standard protein labeled with either S-methyl thioacetimidate or S-methyl thiopropionimidate were solubilized in 20 μl of 1% formic acid, and 2 μl were transferred in a tip emitter (Protana, Odense, Denmark). Nanoelectrospray mass spectrometry was performed off line on a Q-TOF mass spectrometer (QSTAR Pulsar-i, Applied Biosystems, Foster City, CA) fitted with a Protana nanospray inlet system. Spectra were recorded using Analyst QS software (Applied Biosystems). Parameters were adjusted as follows: ion spray voltage, 900 V; curtain gas, 25; declustering potential, 45–75 V; focusing potential, 265 V; declustering potential 2, 15 V. Proteins of interest were excised and digested in-gel using trypsin (sequencing grade, Promega, Charbonnières, France) as described previously (27Shevchenko A. Wim M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar). Digested proteins were dehydrated in a vacuum centrifuge, solubilized in 10 μl of formic acid (2%), desalted using C18 ZipTips (Millipore, Bedford, MA; elution with 10 μl of 0.1% TFA in 60% acetonitrile), and concentrated to a 2-μl volume. 0.3 μl was mixed with the same volume of α-cyano-4-hydroxy-trans-cinnamic acid (10 mg/ml in 0.1% TFA in 50% acetonitrile), deposited on a 384-well MALDI target using the dry droplet procedure (28Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.Anal. Chem. 1988; 60: 2299-2301Google Scholar), and air-dried at room temperature. Analyses were performed using an UltraFlex MALDI-TOF/TOF mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) operating in the reflectron mode with a 20-kV accelerating voltage and a 70-ns delayed extraction. Mass spectra were acquired in the automatic mode using the AutoXecute™ module of Flexcontrol™ (Bruker-Franzen Analytik) (laser power ranged from 30 to 70%, 500 shots). Spectra were analyzed using FlexAnalysis™ software (Bruker-Franzen Analytik) and calibrated internally with the autoproteolysis peptides of trypsin (m/z 842.51, 1045.56, and 2211.10). Peptides were selected in the mass range of 900–4,000 Da. Peptide mass fingerprint identification of labeled proteins was performed by searching against the Swiss-Prot Databases using an in-house Mascot software package (Matrix Science) with Arg-C enzyme specificity and no trypsin missed cleavages. Acetimidoyl and propionimidoyl were set as fixed lysine modifications for searches on single labeled samples or as variable lysine modifications for searches on mixed labeled samples. A mass tolerance of 50 ppm was allowed for identification. Quantification was performed on pairs of identified labeled peptides by measuring the intensity of the 12C isotopic peaks. Only good quality signals (resolution > 8,000, signal to noise ratio > 15, and no overlap) were selected. Two inverse labeling experiments were performed for each sample: either LPS-treated samples were labeled with S-methyl thioacetimidate, and the untreated ones were labeled with S-methyl thiopropionimidate, or inversely LPS-treated samples were labeled with S-methyl-thiopropionimidate, and the untreated ones were labeled with S-methyl-thioacetimidate. Statistics given in Table II were calculated from a Fasta-formatted Swiss-Prot Database (November 23, 2004 update) by using home-made lua scripts (www.lua.org). These scripts were written to run on various platforms (Win, MacOSX, Linux, and UNIX) and can be obtained freely on request ([email protected]).Table IINumber (percentage) of human and mouse proteins and tryptic and Arg-C peptides that contain cysteine or lysine residues annotated in the Swiss-Prot Database (164,201 entries November 23, 2004)ProteinsTryptic peptidesArg-C peptidesHumanMouseHumanMouseHumanMouseTotal number11,6388,446675,765451,915339,921228,813Cys-containing (%)11,241 (96.6)8,171 (96.7)103,948 (15.4)69,564 (15.4) 900–4,000 Da (%)69,488 (10.3)47,072 (10.3)Lys-containing (%)11,565 (99.4)8,417 (99.7)153,576 (45.2)104,208 (45.5) 900–4,000 Da (%)92,404 (27.2)62,730 (27.4) Open table in a new tab The DIMAL-K method described in Fig. 1 was designed to measure changes in expression levels of individual proteins between two biological samples. The efficiency and accuracy of the DIMAL-K method was first investigated using standard proteins: myoglobin from horse skeletal muscle, albumin from chicken egg white, cytochrome c from horse heart, and lysozyme from chicken egg. 30 μg of each standard protein was dissolved in either 6 m urea, 6 m urea plus 1% SDS, or 6 m urea plus 4% CHAPS. The reaction was performed under mild pH conditions (pH 8.7) with either S-methyl thioacetimidate or S-methyl thiopropionimidate (30-fold molar excess of reagent over the total concentration of the amine groups). The gain in molecular weight between unlabeled and labeled standard proteins was assessed by ESI-Q-TOF mass analysis indicating that all ε-amino lysine groups were derivatized under these experimental conditions. For example, the labeling of myoglobin with S-methyl thioacetimidate resulted in a mass shift from 16,952 Da (unlabeled protein) to 17,774 Da (labeled protein), which corresponds to the expected 20 × 41 Da for the 19 lysine residues and the N-terminal amine (Fig. 2). Similar results were obtained from S-methyl thiopropionimidate. We detected an additional mass peak at 17,733 Da (of about 20% intensity compared with the mass peak of myoglobin with 20 labeled amine functions) corresponding to myoglobin bearing 19 labeled lysine residues. The pool of partially labeled myoglobin includes the unlabeled N-terminal residue because N-terminal amine shows lower reactivity with the reagent than the ε-amine of lysines. Similar results were obtained for cytochrome c and lysozyme (data not shown). The efficiency of derivatization of proteins was further confirmed by MALDI-TOF MS analysis of tryptic peptides. In MALDI-TOF MS analysis of the tryptic digest of unlabeled ovalbumin, among the four major signals detected, three of them came from cleavage after lysine: (K)HIATNAVLFFGR, (K)AFKDEDTQAMPFR, and (K)ISQAVHAAHAEINEAGR. In contrast, the MS analysis of the tryptic digestion of the labeled ovalbumin showed a different pattern in which only peptides resulting from cleavage after arginine were detected, including some peptides with labeled lysines in their sequence (Fig. 3). Moreover no peptide with unlabeled lysine was detected. These observations in addition to the Q-TOF mass analyses of labeled proteins prove that the reaction on the ε-position of lysine was total, leading to an Arg-C protease-like behavior of trypsin toward the derivatized proteins. In addition, we did not observe any ion signal corresponding to a potential side reaction. The same results were obtained for labeled myoglobin, cytochrome c, and lysozyme (data not shown). Ovalbumin labeled with either S-methyl thioacetimidate or S-methyl thiopropionimidate was identified by peptide mass fingerprint analysis with a high Mascot score of 170 and 24% sequence coverage (the statistical significance threshold was 62), indicating that the labeling reaction does not preclude identification. The specific parameters include Arg-C as the proteolytic enzyme and acetimidoyl or propionimidoyl as fixed lysine modifications. We detected two peptides with high intensities containing one (TQINKVVR) and two amidinated lysines (KIKVYLPR) in the peptide mass fingerprint of each labeled ovalbumin digest (Fig. 3). Identification could also be performed on mixtures of differentially labeled ovalbumin with a Mascot score of 136 and 24% sequence coverage. In this case, acetimidoyl and propionimidoyl labeling was set as variable lysine modifications leading to a scoring decrease. The accuracy of quantification by the DIMAL-K strategy is conditioned by the co-migration of the differentially labeled proteins on 2-D gels. Two ovalbumin samples were labeled with either S-methyl thioacetimidate or S-methyl thiopropionimidate and then mixed at various ratios ranging from 0.4 to 2 (Me/Et) to evaluate the sensitivity of the method for small differences in expression. 1 μg of unlabeled protein and each protein mixture were resolved by 2-D gel electrophoresis. Image analysis showed that protein labeling did not alter the profile of ovalbumin isoform separation by 2-D gel electrophoresis and preserved a good resolution (Fig. 4). Labeling only induced a slight increase in the apparent protein molecular weight. In contrast, 2-D analysis of mixtures of differentially labeled proteins showed no detectable Mr or pI shifts for proteins labeled with either of the two reagents, indicating that the proteins co-migrated, and thus that quantification was not biased by spot picking. This is consistent with the low difference in mass between differentially labeled proteins (14 mass units per lysine residue), which is below the resolution of SDS-PAGE. After excision, digestion, and MALDI-TOF mass analysis of 2-D spots of each isoform, quantitative analysis of differentially labeled ovalbumin mixtures was carried out from all spots detected on 2-D gels by measuring the ion signal intensities of two couples of tryptic peptides, which contain one (TQINKVVR, Mr = 998.6 and 1012.6) or two amidinated lysines (KIKVYLPR, Mr = 1,098.7 and 1,126.7) (Fig. 5a). Comparing the experimental ratio of the measured signal intensities of the monoisotopic peaks with the theoretical ratio indicated that the signal intensities were neither biased by differences in the reactivity of each reagent nor by differences in desorption-ionization potency between peptides derivatized by both reagents (Fig. 5b) because the average deviation from expected ratios was only 15%, yielding a linear dynamic range with a slope close to 0.93 and an R2 value of 0.992. To prove that the DIMAL-K method provides accurate and sensitive quantification, we also evaluated the differential quantification using a broader dynamic range up to a 120-fold quantitative difference over a protein amount range varying from 5 to 300 fmol. The observed ratios were linear across a 120-fold range of concentration ratios from 0.5 to 60 with R2 values of 0.993 for ovalbumin and 0.992 for myoglobin and with an error of less than 15% (Fig. 6).Fig. 6Observed versus expected ratios obtained from MALDI-TOF analyses of concentration ratios ranging from 0.5 to 60 over amounts ranging from 5 to 300 fmol of mixture of labeled Me-ovalbumin and Et-ovalbumin (a) and Me-myoglobin and Et-myoglobin (b). For each mixture of proteins, three experiments were performed for the entire range of relative concentrations.View Large Image Figure ViewerDownload (PPT) Previously we have identified the major proteins secreted by astrocytes, the major cell population in the mammalian central nervous system, using a proteomic approach combining the separation of proteins recovered from astrocyte culture-conditioned medium by 2-D electrophoresis and their identification by MALDI-TOF MS (24Lafon-Cazal M. Adjali O. Galéotti N. Poncet J. Jouin P. Homburger V. Bockaert J. Marin P. Proteomic analysis of astrocytic secretion in the mouse.J. Biol. Chem. 2003; 278: 24438-24448Google Scholar). We also showed that exposing cultured astrocytes to LPS or proinflammatory cytokines (interleukin 1β or tumor necrosis factor α) modified the profile of astrocytic protein secretion (24Lafon-Cazal M. Adjali O. Galéotti N. Poncet J. Jouin P. Homburger V. Bockaert J. Marin P. Proteomic analysis of astrocytic secretion in the mouse.J. Biol. Chem. 2003; 278: 24438-24448Google Scholar). Here we used the DIMAL-K strategy to quantify the variations in astrocytic protein secretion induced by treating cells with LPS (1 μg/ml, type 055:B5). We first evaluated the efficacy of the labeling reaction on astrocyte-conditioned medium containing 20 μg of total proteins using either S-methyl thioacetimidate or S-methyl thiopropionimidate. 2-D gel analysis of the derivatized proteins showed a profile (Fig. 7) similar to that found for unlabeled secreted proteins (24Lafon-Cazal M. Adjali O. Galéotti N. Poncet J. Jouin P. Homburger V. Bockaert J. Marin P. Proteomic analysis of astrocytic secretion in the mouse.J. Biol. Chem. 2003; 278: 24438-24448Google Scholar). Unambiguous identifications consistent with those previously obtained in unlabeled samples (24Lafon-Cazal M. Adjali O. Galéotti N. Poncet J. Jouin P. Homburger V. Bockaert J. Marin P. Proteomic analysis of astrocytic secretion in the mouse.J. Biol. Chem. 2003; 278: 24438-24448Google Scholar) were obtained from their peptide mass fingerprint with significant protein scores based on Arg" @default.
• W2099693553 created "2016-06-24" @default.
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• W2099693553 date "2005-08-01" @default.
• W2099693553 modified "2023-10-03" @default.
• W2099693553 title "Difference in Mass Analysis Using Labeled Lysines (DIMAL-K)" @default.
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