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• W2168049289 abstract "A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H2N-(His)6-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO2H, which consists of four functional elements: i) an affinity ligand (His6-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d4) or no (d0) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d4) or (d0) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d4 co-elute with their d0-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni2+ affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1–8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of 13C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities. A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H2N-(His)6-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO2H, which consists of four functional elements: i) an affinity ligand (His6-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d4) or no (d0) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d4) or (d0) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d4 co-elute with their d0-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni2+ affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1–8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of 13C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities. The capability of comparing levels of individual proteins between two or more biological samples is rapidly becoming essential for all mass spectrometry (MS) 1The abbreviations used are: MS, mass spectrometry; LC, liquid chromatography; MS/MS, tandem mass spectrometry; ICAT, isotope-coded affinity tag; SCX, strong cation exchange; DPDS, 2, 2′-dipyridyl disulfide; IMAC, immobilized metal affinity chromatography; Fmoc, 9-fluorenylmethoxycarbonyl; DTT, dithiothreitol; GB, gradient buffer; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; CID, collision-induced dissociation.1The abbreviations used are: MS, mass spectrometry; LC, liquid chromatography; MS/MS, tandem mass spectrometry; ICAT, isotope-coded affinity tag; SCX, strong cation exchange; DPDS, 2, 2′-dipyridyl disulfide; IMAC, immobilized metal affinity chromatography; Fmoc, 9-fluorenylmethoxycarbonyl; DTT, dithiothreitol; GB, gradient buffer; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; CID, collision-induced dissociation. -based proteomics (1Aebersold R. Mann M. Mass spectrometry-based proteomics..Nature. 2003; 422: 198-207Google Scholar). A direct comparison of mass spectra intensities recorded in two separate measurements of two samples usually cannot easily be used for estimation of relative protein ratios. However, as the ionization efficiency of various isotopic forms of chemically identical peptides are the same, the spectral intensities can be used as a measure their relative abundances. Therefore, directed labeling of proteins or peptides with stable isotopes has been frequently used in quantitative proteomics. Considering the way in which the stable isotope label is introduced into an investigated sample, the labeling methods can be divided into two types: the metabolic and the sample post-processing ones.Metabolic labeling of cultured cells is a classical approach already used half a century ago. For purposes of MS, either the whole media have been labeled with 15N (2Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Accurate quantitation of protein expression and site-specific phosphorylation..Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6591-6596Google Scholar, 3Conrads T.P. Alving K. Veenstra T.D. Belov M.E. Anderson G.A. Anderson D.J. Lipton M.S. Paša-Tolič L. Udseth H.R. Chrisler W.B. Thrall B.D. Smith R.D. Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling..Anal. Chem. 2001; 73: 2132-2139Google Scholar, 4Washburn M.P. Ulaszek R. Deciu C. Schieltz D.M. Yates J.R. 3rd.Analysis of quantitative proteomic data generated via multidimensional protein identification technology..Anal. Chem. 2002; 74: 1650-1657Google Scholar) or the media were supplemented with amino acids containing 2H or 13C isotopes, first for improving specificity in database searching (5Chen X. Smith L.M. Bradbury E.M. Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification..Anal. Chem. 1999; 72: 134-143Google Scholar, 6Veenstra T.D. Martinoviæ S. Anderson G.A. Paša-Tolič L. Smith R.D. Proteome analysis using selective incorporation of isotopically labeled amino acids..J. Am. Soc. Mass Spectrom. 2000; 11: 78-82Google Scholar, 7Martinoviæ S. Veenstra T.D. Anderson G.A. Paša-Tolič L. Smith R.D. Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level..J. Mass Spectrom. 2002; 37: 99-107Google Scholar, 8Pratt J.M. Robertson D.H. Gaskell S.J. Riba-Garcia I. Hubbard S.J. Sidhu K. Oliver S.G. Butler P. Hayes A. Petty J. Beynon R.J. Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting..Proteomics. 2002; 2: 157-163Google Scholar), and then for quantitative proteomics (9Ong S.E. Blagoev 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, 10Zhu H Pan S Gu S Bradbury EM Chen X Amino acid residue specific stable isotope labeling for quantitative proteomics..Rapid Commun. Mass Spectrom. 2002; 16: 2115-2123Google Scholar, 11Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling..Nat. Biotechnol. 2003; 21: 315-318Google Scholar). The sample post-processing approaches are mainly based on derivatization of the thiol-moiety of cysteines (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) or acylation (13Geng M. Ji J. Regnier F.E. Signature-peptide approach to detecting proteins in complex mixtures..J. Chromatogr. A. 2000; 870: 295-313Google Scholar,14Munchbach M. Quadroni M. Miotto G. James P. Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety..Anal. Chem. 2000; 72: 4047-4057Google Scholar) with reagents carrying isotope labels. Alternatively, up to two 18O atoms can be incorporated into carboxyl groups of peptides by digestion with trypsin or another endopeptidase in the presence of H218O (15Yao X. Freas A. Ramirez J. Demirev P.A. Fenselau C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus..Anal. Chem. 2001; 73: 2836-2842Google Scholar, 16Wang Y.K. Ma Z.X. Quinn D.F. Fu E.W. Inverse 18O labeling mass spectrometry for the rapid identification of marker/target proteins..Anal. Chem. 2001; 73: 3742-3750Google Scholar, 17Stewart. I.I. Thomson T. Figeys D. 18O labeling: A tool for proteomics..Rapid Commun. Mass Spectrom. 2001; 15: 2456-2465Google Scholar).In order to identify and quantify proteins in complex samples, isotope labeling in combination with affinity selection (18Gygi 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, 19Zhou H. Ranish J.A. Watts J.D. Aebersold R. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry..Nat. Biotechnol. 2002; 19: 512-515Google Scholar) has proven to be an especially successful tool. Isotope-coded affinity tags (ICATs) with a normal (H)- or stable heavy isotope-enriched (D)-labeled biotin reagent have been successfully applied for quantitative profiling of differentiation-induced microsomal proteins from human myeloid leukemia cells (20Han 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) and for quantitative analysis of Myc oncoprotein function (21Shiio Y. Donohoe S. Yi E.C. Goodlett D.R. Aebersold R. Eisenman R.N. Quantitative proteomic analysis of Myc oncoprotein function..EMBO J. 2002; 21: 5088-5096Google Scholar).In this study, we describe a novel tool for identification and quantification of cysteine-containing peptides in complex peptide mixtures: the HysTag reagent. This reagent is a decapeptide of the sequence (H)6ARAC that is activated with 2,2-dipyridiyl disulfide (DPDS), which selectively makes the HysTag reagent reactive toward cysteine side chains. In the “heavy” form of the reagent, Ala-9 contains four deuterium atoms. The amino-terminal histidines are the tag that enables selective isolation of tagged peptides by metal-affinity or cation-exchange chromatography. Subsequent digestion of tagged peptides with trypsin releases the H6AR portion of the HysTag from the peptide that is covalently bound via the disulfide bridge with the isotope-labeled dipeptide AC (see Fig. 1).To demonstrate its applicability for analysis of complex mixtures we used the HysTag for identification and relative quantification of proteins from two distinct areas of mouse brain. Fractions enriched in plasma membranes were prepared from fore- and hindbrain, and the proteins were labeled with the reagent direct on the membranes either with heavy or light reagents. The membranes from both brain compartments were mixed and treated with HysTag and digested with Lys-C. Following cation exchange chromatography and digestion with trypsin the released isotope-coded peptides were analyzed by liquid chromatography (LC)-microcapillary electrospray ionization tandem mass spectroscopy (MS/MS). A total of 355 unique proteins were identified, among which 281 could be quantified.EXPERIMENTAL PROCEDURESSolid-phase Synthesis of Isotope-labeled HysTag Peptides—The 10-mer HysTag peptides, light and heavy forms, were made by automated solid-phase peptide synthesis on a prototype Peptide Synthesizer (Intavis Bioanalytical Instruments, Cologne, Germany). Preloaded (Fmoc)-Cys(Trt)-NovaSyn® TFA-labile Wang-type amino resin was coupled with 9-fluorenylmethoxycarbonyl (Fmoc)-protected alanine (Novabiochem, Laufelfingen, Switzerland) using benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate/N-methylmorpholine/N,N-dimethylformamide activation. Fmoc-alanine-d4 (Euriso-top, Saint-Aubin, France) was used for synthesis of the heavy form of the HysTag peptide. All other residues were incorporated as Fmoc-amino acids with the same activation chemistry, using Pbf for Arg and Trt for His as the side-chain-protecting groups. After completion of the synthesis, the resin was washed in 3× 1 ml dichloromethane and dried over N2 for 15 min to remove all DMF. Simultaneous side-chain deprotection and cleavage was carried out using 2 ml of a solution of 5% triethylsilane/2.5% H2O/92.5% trifluoroacetic acid for 2 h. The trifluoroacetic acid solution was reduced under a gentle stream of N2, and the cleaved peptide was precipitated in 10 ml ice-cooled tert-butylmethylether as a white milky solution. The solution was centrifuged for 10 min (1000 rpm) and the ether-phase decanted to waste. The precipitated peptide was redissolved in water with 5% formic acid. Nano-electrospray MS and MS/MS spectra of the peptide were acquired as control of purity.Activation of the HysTag Peptide—The crude peptide solution was lyophilized and redissolved in 0.1 m Tris·HCl, pH 8.0. Dithiothreitol (DTT) was added to a final concentration of 10 mm, and the peptide solution was incubated for 30 min at room temperature to reduce potential disulfide bridges. The peptide solution was then reacted with 10 m excess of DPDS in 50%(v/v) acetonitrile for 1 h. Finally, the derivatized peptides were purified by reverse-phase chromatography on a C18 column and lyophilized.Preparation of Crude Cell Membranes—Adult mice were sacrificed by decapitation and brain material was dissected in less than 30 s. Fore- and hindbrain were separated and rinsed with PBS. Approximately 300 mg brain tissue (one forebrain or three hindbrains) was manually ground in 5 ml gradient buffer (GB) buffer containing 0.32 m sucrose, 10 mm HEPES·NaOH, 100 mm succinic acid, 1 mm EDTA, pH 7.4, 0.25 mm DTT, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 20 μm leupeptin hemisulfate, 150 μm aprotinin in a 15-ml glass potter on ice and the “initial homogenate” was centrifuged at 1000 × g at 4 °C for 10 min. The supernatant was discarded, and the pellet was homogenized in 4 ml GB buffer using an IKA Ultra Turbax blender (IKA-Ultra Turrax, Staufen, Germany) at maximum speed for 5–10 s (the “homogenate”). The suspension was centrifuged as above, and the supernatant was collected. The rehomogenization process was repeated twice, the supernatant pooled, and crude membranes collected at 50,000 × g at 4 °C for 30 min. The pellet (P2) was resuspended in 4 ml GB buffer with five strokes of the motorized Potter homogenizer (B. Braun Biotech, Melsungen, Germany). The suspension was sonicated twice on ice for 15 s using Soniprep 150 (Sanyo, Gallenkamp, UK).Preparation of Enriched Plasma Membranes by Density Gradient Centrifugation—In a 11.5-ml crimp tube (S/L, tube PA, 11.5 ml; Sorvall, Asheville, NC), the resuspended P2 fraction was mixed with 3.85 ml 100% Percoll (Amersham Biosciences, Piscataway, NJ) and 0.6875 ml 2 m sucrose. The tube was filled with GB buffer, closed, and centrifuged at 50,000 rpm in a fixed-angle rotor T 890 centrifuge (Sorvall) at 4 °C for 15 min. The gradient was fractionated from the top by the displacement method. Percoll was removed by centrifugation of the fractions in 1-ml PC tubes in Sorvall RC M150 GX using the S150AT rotor at 900,000 × g at 4 °C for 20 min.The composition of individual fractions was analyzed for γ-glutamyl transpeptidase (marker for plasma membranes) (22Orlowski M. Meister A. γ-Glutamyl-p-nitroanilide: A new convenient substrate for determination and study of l- and d-γ-glutamyltrasnpeptidase acticities..Biochim. Biophys. Acta. 1963; 73: 679-681Google Scholar), cytochrome c oxidase (marker for mitochondria) (23Price N.R. Some aspects of the inhibition of cytochrome c oxidase by phosphine in susceptible and resistant strains of.Rhysopertha dominicia. Insect Biochem. 1980; 19: 147-150Google Scholar), and NADPH-cytochrome c reductase (marker for endoplasmatic reticulum) (24Williams C.H. Kamin H. Microsomal triphosphoptridine nucleotide-cytochrome c reductase of liver..J. Biol. Chem. 1962; 237: 587-595Google Scholar) activities and total protein content using DC protein kit (Bio-Rad, Hercules, CA). The yield of plasma membranes was 2–3 mg protein/g brain.Labeling of Standard Proteins—Fifty micrograms of bovine serum albumin (BSA) and human Transferrin were reduced with 50 mm DTT at room temperature for 0.5 h. Following desalting of an HiTrap desalting 5-ml column (Amersham Biosciences), the proteins were incubated in the presence of 100-fold molar excess of either HysTag-d0 or HysTag-d4 in 150 μl of 4 m urea in 0.1 m Tris·HCl, pH 7.8, at room temperature for 4 h. The “light” and “heavy” samples were mixed and incubated with 1 μg of endoproteinase Lys-C at room temperature overnight.Labeling of Membrane Proteins—The method comprises repeated steps of membrane incubation and separation of the “solid phase” membranes from “liquid phase” by ultra centrifugation. During all incubations, the samples were gently mixed. The “phase” separations were achieved by centrifugation at 150,000 × rpm in 4 °C for 15 min using RC M150 GX centrifuge and the S150AT rotor. Fore- and hindbrain membrane fractions containing ∼1 mg total protein were pooled, and the membranes were collected by centrifugation at 150,000 × rpm in 4 °C for 15 min using RC M150 GX centrifuge and the S150AT. The pellets were resuspended in 400 μl of 0.2 m NaBr, 0.2 m KCl, 10 mm DTT, 50 mm Tris·HCl, pH 8.0. After 30 min incubation at room temperature the membranes were sedimented by centrifugation. Then the pellets were resuspended in 200 μl of 4 m urea in 0.1 m Tris·HCl, pH 8.0, and divided into two equal fractions. The fractions were mixed with 0.8 mg of either HysTag-d0 or HysTag-d4 and incubated at room temperature for 4 h. The membranes were collected by centrifugation (as above). “Heavy” and “light” membranes were mixed in a ratio 1:1 and digested with 5 μg of endoproteinase Lys-C at room temperature overnight. Following the next centrifugation, the supernatants were collected and the pellets discarded.Isolation of Tagged Peptides Using Cation Exchanger—Two hundred microliters Source 30S gel slurry (Amersham Biosciences) was pipetted into a 1-ml spin columns (type size) and centrifuged at 700 × g for 1 min. The columns were washed with 2× 500 μl elution buffer (25 mm 4-morpholinepropanesulfonic acid·NaOH, 1 m NaCl, pH 7.0) and equilibrated with 3× 500 μl binding buffer (8 m urea, 25 mm 4-morpholineethanesulfonic acid (MES), pH 5.5) The Lys-C digests were diluted 5-fold with the binding buffer to a final volume of 1.0 ml, and the pH was adjusted to 5.5 with 1 m HCl. The diluted samples were loaded into the spin columns, and incubated at room temperature for 1 h. After incubation, the spin columns were placed in 2 ml Eppendorf (Hamburg, Germany) tubes and centrifuged at 700 × g for 1 min, and the resin was washed twice with 500 μl 25 mm MES, 0.1 m NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), pH 5.5, followed by 2× wash with 25 mm MES, 0.1 m NaCl, pH 5.5. Finally, the bound peptides were eluted with 2× 100-μl elution buffer.Isolation of Tagged Peptides Using Immobilized Ni2+—Specific His6-Tag IMAC purification was performed using the nickel-chelated B-PER His6 spin column kit according to the manufacturer’s instructions (Pierce, Rockford, IL). Briefly, 1 ml of precharged nickel-chelated spin columns were washed and equilibrated with 2× 2 ml of B-PER Reagent. The Lys-C digests were diluted 10-fold with the B-PER Reagent to a final volume of 2 ml and applied to the spin column. After 1 h incubation at room temperature, the spin columns were placed in 2 ml Eppendorf tubes and centrifuged at 700 × g for 1 min and the resin was washed three times with 1 ml of B-PER washing buffer supplemented with 1% CHAPS to remove nonspecific-binding hydrophobic peptides. Finally, the bound peptides were eluted with 2 × 500 μl B-PER elution buffer (2% imidazole w/w solution).Tryptic Digestion of the Tagged Peptides—One microgram trypsin (Modified Sequence Grade; Promega, Madison, WI) was added to each peptide eluate and incubated overnight at 37 °C. The tryptic peptide mixtures were desalted and concentrated on Poros R2/Oligo R3 (1:1) resins (Perseptive Biosystems, Foster City, CA) packed in GELoader tips (Eppendorf) as described (25Neubauer G. Mann M. Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: Potentials and limitations..Anal. Chem. 1999; 71: 235-242Google Scholar) and eluted in 2× 2 μl (50% MeOH, 5% HCO2H) into 96-well plates, where they were diluted to a final volume of 20 μl (10% MeOH, 5% HCO2H).MS: Microcapillary LC-MS/MS Analysis—Microcapillary reverse-phase high-performance LC-MS/MS was performed using an Agilent 1100 capillary LC system with an μ-Autosampler (Agilent Technologies Inc., Palo Alto, CA) coupled to a QSTAR Pulsar hybrid quadrupole time-of-flight mass spectrometer (AB-MDS Sciex, Toronto, Canada) using a modified nano-electrospray ion source (Proxeon Biosystems, Odense, Denmark) interface.The tryptic peptide mixtures were auto-sampled at a flow rate of 5.0 μl/min onto a precolumn (150 μm id × 3 cm fused silica; Composite Metal Services, West Yorkshire, UK) in-house packed with C18 material (Zorbax C18 5-μm particles; Agilent Technologies) and then eluted with a linear gradient of H2O-MeCN in the presence of 0.4% acetic acid plus 0.005% heptafluorobutyric acid at a flow rate of 0.3 μl/min to 40% MeCN for 90 min. The precolumn eluate was separated on an analytical capillary C18 column (Zorbax C18 3.5-μm particles; Agilent Technologies) packed in a pulled fused silica capillary emitter (75 μm id × 8 cm; New Objectives, Cambridge, MA) mounted in the nano-electrospray ion source. A voltage of 2.0 kV was applied behind the emitter through a platin wire into one arm of the microcross T (Upchurch Scientific, Oak Harbor, WA) connecting the precolumn with the analytical column packed in the emitter.The mass spectrometer was operated in the information-dependent acquisition mode to automatically switch between MS and MS/MS acquisition controlled by the Analyst software. Survey MS spectra were acquired for 1 s with doubly, triply, and quadruply charged ions triggering the function switching (MS→MS/MS). The most intense ion was isolated and fragmented for 2 s by low-energy collision-induced dissociation (CID) MS/MS. The collision energy was automatically calculated and adjusted for each CID-MS/MS spectra individually. Former target ions were dynamic excluded for 180 s. Both MS and MS/MS spectra were acquired with the Q2-pulsing function switched on and optimized for optimal transmission of ions in the sequence tag mass region (m/z 400–1000).Database Searches: Peptide Identification—All MS/MS spectra files from each LC run were centroided and merged to a single file, which were searched using the Mascot Search Engine (Matrix Science, London, UK) against the mammalian NCBInr database with oxidized methionine (+15.99 Da) and HysTag-d0 and -d4 cysteine (+190.04 Da and +194.07 Da, respectively) as variable modifications. Searches were done with initial tolerance on mass measurement of 1.3 Da in MS mode and 0.13 Da in MS/MS mode. The rather large mass tolerance in MS mode was used to ensure identification of peptide ions selected and isolated by their 13C isotope instead of the 12C isotope via the Analyst software.RESULTS AND DISCUSSIONThe Reagent and Its PropertiesThe HysTag Labeling and Quantification Procedure—One of the major challenges of proteomics is quantification of the individual proteins in complex mixtures. This is a difficult task because of the complexity and dynamic range of proteomes. To address this problem, we have designed and tested a new protein identification and quantification tool, HysTag. The reagent is a derivatized decapeptide, H2N-(His)6-Ala-Arg-Ala-Cys(-2-pyridyl disulfide)-CO2H, which consists of four functional elements: i) an affinity ligand (His6-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) an Ala-9 residue that contains four (d4) or zero (d0) deuterium atoms, and iv) a thiol-reactive group (DPDS).The HysTag procedure consists of three main steps (Fig. 1): 1) covalent tagging of reduced and desalted protein samples with HysTag, followed by endoproteinase Lys-C digestion of the combined labeled protein samples; 2) isolation of tagged peptides by charge-dependent strong cation exchange (SCX), or 3) immobilized Ni2+ ion affinity chromatography (IMAC) followed by tryptic digest of the isolated peptides. Identification and quantification of the tagged tryptic peptides was determined by microcapillary LC-MS/MS. SCX isolation of the tagged peptides is based on the strong cationic properties of the HysTag. The imidazole ring of histidine side-chain has a pKa value of 6.2 (26Tanford C. The interpretation of hydrogen ion titration curves of proteins..Adv. Protein Chem. 1962; 17: 69-165Google Scholar), therefore at a pH below 6.2 the His-tag will have a net positive charge. This provides a sufficiently high density of positive charges to allow strong binding to a cation exchanger at 5.0 < pH < 6.0, whereas the majority of untagged peptides generated by Lys-C proteolysis have a net negative charge at pH > 5 (pI < 5). This therefore allows selective purification of His6-tagged peptides from undesired nontagged peptidesChromatographic and Mass Spectrometric Properties of Tagged Peptides—To analyze the chromatographic separation of HysTag-modified peptide pairs, human Transferrin and BSA were labeled with heavy and light HysTag reagent and processed as described above. The elution profiles from four pairs of the HysTag-labeled peptides originating from BSA and Transferrin pairs are shown in Fig. 2. Co-elution of the d0- and d4-labeled forms was observed for each peptide pair in all LC-MS runs. This property of the reagent simplifies the quantification procedure and increases the accuracy of quantification. The chromatographic behavior of the tagged peptides is unique, because usually deuterated peptides and the corresponding nondeuterated peptides do not co-elute (27Zhang R. Sioma C.S. Wang S. Regnier F.E. Fractionation of isotopically labeled peptides in quantitative proteomics..Anal. Chem. 2001; 73: 5142-5149Google Scholar). The expected chromatographic isotope effect is probably diminished due to the adjacent hydrophilic groups (28Zhang R. Sioma C.S. Thompson R.A. Xiong L. Regnier F.E. Controlling deuterium isotope effects in comparative proteomics..Anal. Chem. 2002; 74: 3662-3669Google Scholar).Fig. 2MS survey spectra and LC elution profiles (extracted ion current, XIC) of a BSA peptide (left) and a transferrin peptide (right) labeled with heavy and light HysTag reagent. The XIC for all peptide pairs demonstrates co-elution; this is an important feature of this HysTag reagent, because quantification of the pairs is readily obtained from their relative ratios in the MS survey spectra without the need for 13C reagents.View Large Image Figure ViewerDownload (PPT)It is a key feature of our method that a relatively long peptide with desired retention behavior is used for peptide isolation, whereas the relative small remaining mass added to the cysteine residue after tryptic digestion makes the CID-MS/MS spectra “clean” and easy to interpret. Removal of the His6-tag after affinity purification but before LC-MS generates MS/MS spectra that predominantly display b- and y-type fragment ions, whereas no abundant interfering losses from cleavage of the HysTag label are observed (Fig. 3). This makes the MS/MS spectra easy to identify via standard database search algorithms with cysteine specified as modified by the dipeptide.Fig. 3Microcapillary LC-MS/MS spectrum of HysTag-d0-derivatized tryptic human tran" @default.
• W2168049289 created "2016-06-24" @default.
• W2168049289 creator A5008902457 @default.
• W2168049289 creator A5009510298 @default.
• W2168049289 creator A5046276741 @default.
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• W2168049289 date "2004-01-01" @default.
• W2168049289 modified "2023-10-12" @default.
• W2168049289 title "HysTag—A Novel Proteomic Quantification Tool Applied to Differential Display Analysis of Membrane Proteins From Distinct Areas of Mouse Brain" @default.
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