A Metal-coded Affinity Tag Approach to Quantitative Proteomics
2007; Elsevier BV; Volume: 6; Issue: 11 Linguagem: Inglês
10.1074/mcp.m700152-mcp200
ISSN1535-9484
AutoresRobert Ahrends, Stefan Pieper, Andreas Kühn, H. Weisshoff, M. Hamester, Torsten Lindemann, Christian Scheler, Karola Lehmann, Kerstin Taubner, Michael Linscheid,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures. The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures. Proteomics as a field of research is based on the characterization of an entire proteome of a biological system. A variety of approaches have been developed during the last decades to characterize such mixtures of proteins and peptides, and necessarily, all of them use separation techniques. At the protein level, separation has been achieved using 2-D 1The abbreviations used are: 2-D, two-dimensional; DPAGE, dissolvable PAGE; FIA, flow injection analysis; ICP, inductively coupled plasma; MeCAT, metal-coded affinity tag; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; M, lanthanide metal; HIV, human immunodeficiency virus. 1The abbreviations used are: 2-D, two-dimensional; DPAGE, dissolvable PAGE; FIA, flow injection analysis; ICP, inductively coupled plasma; MeCAT, metal-coded affinity tag; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; M, lanthanide metal; HIV, human immunodeficiency virus. gel electrophoresis (1O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) and densitometry of stained proteins or fluorescence detection (2Unlu M. 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Subsequently methods have been developed for the quantitative determination of proteins and peptides mainly based on chemical or metabolic isotopic labeling combined with LC/MSn detection (5Gygi 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: 994-999Crossref PubMed Scopus (4293) Google Scholar, 6Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3616) Google Scholar). Label-free LC/MS quantitative strategies are under development as well (7Wiener M.C. Sachs J.R. Deyanova E.G. Yates N.A. Differential mass spectrometry: a label-free LC-MS method for finding significant differences in complex peptide and protein mixtures.Anal. Chem. 2004; 76: 6085-6096Crossref PubMed Scopus (242) Google Scholar). Using such techniques, the investigation of changes of the proteome in biological systems has become possible. However, only relative changes can be monitored, and currently, with a few exceptions such as the absolute quantification (AQUA) method (8Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6940-6945Crossref PubMed Scopus (1523) Google Scholar) or QCAT (9Beynon R.J. Doherty M.K. Pratt J.M. Gaskell S.J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.Nat. Methods. 2005; 2: 587-589Crossref PubMed Scopus (388) Google Scholar), no labeling techniques are available to determine accurate amounts particularly of whole proteins. Here we describe a novel strategy for the relative and absolute quantification of peptides and proteins. The method is based on chemical labeling, but instead of isotopes, the different lanthanide ions in macrocyclic complexes are used (10Krause, M., Scheler, C., Boettger, U., Weisshoff, H., and Linscheid, M. (June 20,2002) Method and Reagent for Specifically Identifying and Quantifying one or More Proteins in a Sample. German Patent DE 102 27 599Google Scholar). The molecular structures of the metal labels used are shown in Fig. 1. If required, the structures of peptides can be determined using ESI or MALDI MS/MS data, but the quantitative information comes from inductively coupled plasma (ICP) MS measurements. ICP MS has been used already for the accurate and sensitive quantification of elements in many biological samples (11Beauchemin D. Kisilevsky R. A method based on ICP-MS for the analysis of Alzheimer's amyloid plaques.Anal. Chem. 1998; 70: 1026-1029Crossref PubMed Scopus (72) Google Scholar, 12Siethoff C. Feldmann I. Jakubowski N. Linscheid M. Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry.J. Mass Spectrom. 1999; 34: 421-426Crossref PubMed Scopus (86) Google Scholar, 13Wind M. Edler M. Jakubowski N. Linscheid M. Wesch H. Lehmann W.D. Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry.Anal. Chem. 2001; 73: 29-35Crossref PubMed Scopus (159) Google Scholar). Thus, it should be well suited for quantification of metal-labeled, low abundance proteins particularly in combination with enrichment and preconcentration procedures. The technique provides extraordinary detection capability, and it is unaffected by the nature of chemical structures (12Siethoff C. Feldmann I. Jakubowski N. Linscheid M. Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry.J. Mass Spectrom. 1999; 34: 421-426Crossref PubMed Scopus (86) Google Scholar), which allows the use of simple internal standards containing appropriate metals. Detection limits of 80 pg/g (parts per trillion) for 157Gd and 2 fg/g (parts per quadrillion (fg/ml)) for 175Lu have been reported by Zhang et al. (15Zhang A. Liu X. Zhang W. Determination of rare earth impurities in high purity europium oxide by inductively coupled plasma-mass spectrometry and evaluation of concentration values for europium oxide standard material.Eur. J. Mass Spectrom. (Chichester, Eng.). 2004; 10: 589-598Crossref PubMed Scopus (8) Google Scholar). The synthetic access to DOTA metal complexes has been shown already (16Whetstone P.A. Butlin N.G. Corneillie T.M. Meares C.F. Element-coded affinity tags for peptides and proteins.Bioconjug. Chem. 2004; 15: 3-6Crossref PubMed Scopus (109) Google Scholar). We used two different labeling reagents named MeCATBnz and MeCATBio (Fig. 1). They contain maleimide as the thiol-specific group and DOTA macrocycle, which has been used previously as an immunoglobulin recognition site for affinity purification (16Whetstone P.A. Butlin N.G. Corneillie T.M. Meares C.F. Element-coded affinity tags for peptides and proteins.Bioconjug. Chem. 2004; 15: 3-6Crossref PubMed Scopus (109) Google Scholar). The two reagents differ in the spacer region, which is short for MeCATBnz, whereas MeCATBio possesses a biotin-bearing spacer to allow affinity purification (17Ahrends R. Kosinski J. Kirsch D. Manelyte L. Giron-Monzon L. Hummerich L. Schulz O. Spengler B. Friedhoff P. Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.Nucleic Acids Res. 2006; 34: 3169-3180Crossref PubMed Scopus (38) Google Scholar, 18Girault S. Chassaing G. Blais J.C. Brunot A. Bolbach G. Coupling of MALDI-TOF mass analysis to the separation of biotinylated peptides by magnetic streptavidin beads.Anal. Chem. 1996; 68: 2122-2126Crossref PubMed Scopus (67) Google Scholar) The reaction of the DOTA macrocycle with lanthanide metal ions (M3+) results in stable metal chelate complexes (see Table I) (19Moreau J. Guillon E. Pierrard J.C. Rimbault J. Port M. Aplincourt M. Complexing mechanism of the lanthanide cations Eu3+, Gd3+, and Tb3+ with 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (dota)-characterization of three successive complexing phases: study of the thermodynamic and structural properties of the complexes by potentiometry, luminescence spectroscopy, and EXAFS.Chemistry. 2004; 10: 5218-5232Crossref PubMed Scopus (128) Google Scholar, 20Bunzli J.C. Benefiting from the unique properties of lanthanide ions.Acc. Chem. Res. 2006; 39: 53-61Crossref PubMed Scopus (900) Google Scholar). The different masses of the complexes (21Bohlke J.K. de Laeter J.R. De Bievre P. Hidaka H. Peiser H.S. Rosman K.J.R. Taylor P.D.P. Isotopic compositions of the elements, 2001.J. Phys. Chem. Ref. Data. 2005; 34: 57-67Crossref Scopus (231) Google Scholar) allow detection of differently labeled species using ESI MS and ICP MS.Table IExact masses and stability constants of the DOTA complexes of the lanthanides used (20Bunzli J.C. Benefiting from the unique properties of lanthanide ions.Acc. Chem. Res. 2006; 39: 53-61Crossref PubMed Scopus (900) Google Scholar, 21Bohlke J.K. de Laeter J.R. De Bievre P. Hidaka H. Peiser H.S. Rosman K.J.R. Taylor P.D.P. Isotopic compositions of the elements, 2001.J. Phys. Chem. Ref. Data. 2005; 34: 57-67Crossref Scopus (231) Google Scholar)159Tb165Ho169Tm175LuExact mass (g/mol)158.92535164.93032168.93421174.96700Complexing constants DOTA (log K)24.2224.5424.4125.41 Open table in a new tab For the quantitative analysis of a protein mixture, MeCAT was combined with high resolution 2-D electrophoresis using dissolvable gels (DPAGE, Proteome Factory AG). Samples were denatured and reduced before reaction at the cysteine residues with M-MeCAT reagents (M = lanthanides). Then the samples were mixed, and the mixtures were divided into two portions, which were separated using analytical and micropreparative 2-D electrophoresis, respectively. After staining, the spots of interest were picked, dissolved for quantification by FIA/ICP MS, or digested for identification using HPLC/MSn. The current workflow is displayed in Fig. 2. The commercially available peptides (Bachem, Weil am Rhein, Germany) were reduced with 2 mm TCEP at 37 °C for 30 min before a 10-fold excess of M-MeCATBio reagent (M = Lu(III), Ho(III), Tb(III), Tm(III)) was added. The reaction was carried out in 10 mm HEPES (pH 6.5) for 2 h at 37 °C. Finally the reaction was stopped using an excess of DTT. The proteins used in this study were bovine serum albumin (Bos taurus) and α-lactalbumin (B. taurus) from Sigma-Aldrich. Proteins (1.25 nmol) were reduced in 2 mm TCEP for 30 min at 37 °C and labeled using different M-MeCATBnz reagents (M = Lu(III), Ho(III), Tm(III)) in a 20-fold excess to each cysteine residue. The reaction was carried out in a solution containing 5 mm EDTA, 50 mm sodium acetate (NaOAc), and 5% acetone for α-lactalbumin and 5 mm EDTA, 25 mm NaOAc, and 10% acetone for BSA. Acetone and EDTA were used to achieve complete denaturation of proteins. The reaction mixture was incubated for 12 h at 37 °C, and the reaction was stopped using DTT. Fresh eyes lenses from a 1-year-old pig (Sus scrofa) were freshly prepared on ice as described previously (22Dewey J. Bartling C. Rae J.L. A non-enzymatic method for lens decapsulation which leaves the epithelial cells attached to the fibers.Curr. Eye Res. 1995; 14: 357-362Crossref PubMed Scopus (4) Google Scholar). The proteins were dissolved in 9 m urea, 70 mm TCEP, and 1% CHAPS. The amount of protein was determined by the Bradford assay (23Bradford 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: 248-254Crossref PubMed Scopus (211946) Google Scholar). Two hundred and fifty micrograms of lens protein (samples A and B) were reduced with 10 mm TCEP for 30 min at room temperature. The protein samples A and B were labeled with 800 μg of Lu(III)- or Tm(III)-MeCATBnz, respectively. The reaction was carried out in a solution containing 5 mm EDTA, 50 mm NaOAc, and 10% acetone at 37 °C, and the reaction was stopped after 12 h using an excess of DTT. Prior to affinity purification, 35 μl of water and 10 μl of 10-fold binding buffer (500 mm Tris-HCl, pH 7.4, and 10 mm DTT) were added to 55 μl of solution containing 20 pmol of M-MeCATBio (M = Ho(III), Lu(III), Tm(III), Tb(III))-labeled peptide (HIV integrase inhibitor) and 10 μg of tryptically digested Escherichia coli cell lysate. For affinity purification of MeCATBio-labeled peptides, a modified protocol of Girault et al. (18Girault S. Chassaing G. Blais J.C. Brunot A. Bolbach G. Coupling of MALDI-TOF mass analysis to the separation of biotinylated peptides by magnetic streptavidin beads.Anal. Chem. 1996; 68: 2122-2126Crossref PubMed Scopus (67) Google Scholar) was used using streptavidin-coated magnetic beads (M-280, Invitrogen). After incubation and immobilization of 10 μl of streptavidin-coated magnetic beads with a magnetic concentrator, the supernatant was removed, and the beads were washed twice with 100 μl of cleaning buffer (500 mm Tris-HCl, pH 7.4, 10 mm DTT, and 1 mg/ml BSA) followed by 100 μl of binding buffer (50 mm Tris-HCl, pH 7.4, and 1 mm DTT). Next the beads were mixed with 100 μl of peptide mixture, prepared as described above, and incubated for 60 min at 25 °C with gently shaking. The supernatant was removed, and the beads were washed four times with 100 μl of washing buffer (50 mm Tris-HCl, pH 7.4, and 0.01% (w/v) SDS), four times with 100 μl of 1 mm DTT, and finally three times with 100 μl of water. After removal of the supernatant, peptides were eluted with 50 μl of 0.1% (v/v) TFA and 40% (v/v) ethanol by incubation for 5–10 min at 60 °C, and the supernatant containing the eluted labeled peptides was dried in a SpeedVac and stored at −20 °C until analysis. To demonstrate labeling efficiency, following the labeling reaction MeCAT-coded proteins were subjected to SDS-PAGE as described by Laemmli (24Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) using 4% (w/v) stacking gel and 15% (w/v) separating gels. To visualize the mass shift of metal-coded proteins, gels were stained with silver (25Heukeshoven J. Dernick R. Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels.Electrophoresis. 1988; 9: 28-32Crossref PubMed Scopus (640) Google Scholar) or a colloidal Coomassie G-250 solution (26Klose J. Kobalz U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome.Electrophoresis. 1995; 16: 1034-1059Crossref PubMed Scopus (628) Google Scholar). Without further purification or fractionation, the M-MeCAT-labeled samples (M = Tm(III), Lu(III)) were subjected to 2-D electrophoresis analysis. Soluble proteins were separated by the high resolution 2-D electrophoresis technique according to Klose and Kobalz (26Klose J. Kobalz U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome.Electrophoresis. 1995; 16: 1034-1059Crossref PubMed Scopus (628) Google Scholar). Large (30 × 23-cm) soluble 2-D gels (DPAGE, Proteome Factory AG) were prepared from a ready-made gel solution. Analytical and preparative gels were 1 mm thick. For analytical runs 100 μg and for preparative runs up to 500 μg of the combined lens samples were applied at the anodic side of the gel. In the first dimension vertical IEF was performed using carrier ampholytes in the range of pH 2–11. The second dimension was a vertical SDS-PAGE as described by Laemmli (24Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) using 15% (w/v) DPAGE gels. After electrophoresis, the analytical gels were stained with silver nitrate, and preparative gels were stained with Coomassie Brilliant Blue G-250. For the subsequent analyses protein spots were digested either in gel or solubilized according to the DPAGE protocol for FIA/ICP MS analysis. For in-gel digestion pieces were cut out, transferred to vials, and destained (10% acetic acid, 30% ethanol, and 60% water (v/v/v)). The pieces were then rinsed with 100 μl of 50 mm ammonium hydrogen carbonate buffer, dehydrated with 100 μl of 100% acetonitrile four times, and dried in a SpeedVac for 15 min. Fifty microliters of 50 mm ammonium hydrogen carbonate buffer and 2 ng of trypsin were added to each vial; the digestion was carried out for 12 h at 37 °C. After centrifugation, the supernatant was carefully removed, and the pellets were reconstituted in 50 μl of 50 mm ammonium hydrogen carbonate buffer for 30 min, centrifuged, and freed from the supernatant. Finally all pellets were extracted with 50 μl of acetonitrile. The supernatants were pooled and analyzed. Digested samples were dissolved in 0.1% (v/v) formic acid to a final concentration of 500 fmol/μl. For LC/MS/MS, an 1100 nano-LC system (Agilent, Santa Clara, CA) was used. Zorbax 300SB-C18 3.5-μm, 150-mm × 75-μm and Zorbax 300SB-C18 3.5-μm, 0.3 × 5-mm (Agilent) enrichment columns were used. Separation was carried out using a binary mobile phase water/acetonitrile gradient with a maximum flow rate of 0.29 μl/min. The eluents were: A (isocratic pump): 98.5% deionized water, 1% acetonitrile, and 0.5% (v/v) formic acid; B (gradient pump): 94.9% deionized water, 5% acetonitrile, and 0.1% (v/v) formic acid; C (gradient pump): 99.9% acetonitrile and 0.1% (v/v) formic acid. For peptide separation, the sample was injected via autosampler onto the enrichment column using a 20-μl flow (A). The separation column was directly coupled to a Bruker Esquire 3000 plus (Bruker Daltonics, Bremen, Germany) via a nano-ESI source using a 10-μm-inner diameter PicoTip emitter (New Objectives, Woburn, MA). For MS and MS/MS experiments, the following mode and tuning parameters were used: scan range, 100–3000 m/z; polarity, positive; capillary voltage, 1550 V. High resolution MS and MS/MS experiments were carried out using a Finnigan LTQ FT mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) interfaced to a nano-HPLC system (Agilent 1100) using the conditions described above. MS and MS/MS data were recorded using the following parameters: scan range, 350–2000 m/z; polarity, positive; capillary voltage, 1600 V; tube lens voltage, 10 V. Peptides were analyzed on a Bruker Daltonics MALDI-TOF mass spectrometer (Reflex II) with α-cyano-4-hydroxycinnamic acid as matrix. The samples were prepared by the dried droplet method. A mixture of angiotensin I, angiotensin II, and substance P was used for external mass calibration. The accuracy of peptide mass measurement was about 10 ppm. For analysis of lens proteins in samples extracted from polyacrylamide gels, a combination of a Famos Ultimate II instrument (LC Packings, Sunnyvale, CA) and Elan 6000 ICP mass spectrometer (PerkinElmer Life Sciences) (plasma power, 1100 watts; nebulizer gas flow, 1.5 liters/min) was used. As an interface between the LC and ICP MS instruments the microconcentric nebulizer MCN-6000 with a membrane desolvation system was used (Cetac, Omaha, NE). Standard solutions for external calibration (18 fmol, 36 fmol, 72 fmol, 144 fmol, 288 fmol, and 2.88 pmol) were prepared using a multi-element standard mixture (Merck) containing holmium and lutetium. The analysis of labeled standard proteins was carried out on an Element XR (Thermo Fisher Scientific) (plasma power, 1450 watts; nebulizer gas flow, 0.87 liters/min; nebulizer, concentric PFA in combination with a Surveyor HPLC system (Thermo Fisher Scientific)) with a flow rate of 200 μl/min. Standard solutions for external calibration (2.5, 5, 10, 100, 1000, 5000, and 10,000 fmol) were prepared using a multi-element standard mixture containing holmium and lutetium. To demonstrate MeCAT as a viable alternative to other labeling techniques, to show its use in relative determinations, and to prove that it is a unique option for absolute quantitative determination of proteins, it is necessary to show that the labeling of peptides and proteins is complete, reproducible, and robust. To this end, the method described above was applied to peptides (HIV integrase inhibitor peptide and tryptic BSA digest), to single proteins (bovine serum albumin and α-lactalbumin), and to the proteome of the porcine lens (S. scrofa). Initially it was necessary to prove that the differently tagged peptides had the same retention time in HPLC separations. Two tryptic digests of BSA were labeled with M-MeCATBnz, one with holmium and the second with lutetium as metal core. Both digests were mixed in a ratio of 2:1, and the mixtures were analyzed using LC/MS and LC/MS/MS. The chromatogram in Fig. 3 shows two coeluting tagged peptides. The two peptides in each peak were present at the expected ratio of 2:1 (Ho(III):Lu(III); ESI MS). We also used this experiment to demonstrate that the labeled peptides could be identified by a common database search (Mascot data are not shown). As one example, the spectrum of the peptide LCVLHEK is shown in Fig. 4. In general, the CID MS/MS fragmentation pattern shows the typical y and b series. In the next step we had to prove the complete reaction of all the available cysteines. At the peptide level, four samples of the HIV integrase inhibitor (sequence, HCKFWW) were labeled with four different metal-coded M-MeCATBio reagents (M = Tb(III), Ho(III), Tm(III), Lu(III)). Then the resulting reaction mixtures were combined in a ratio of 2:1:2:1 (Tb(III):Ho(III):Tm(III):Lu(III)). A survey mass spectrum (MALDI-reflectron-TOF MS) of the labeled peptide mixture is given in Fig. 5A. The expected ratios were found, whereas no unlabeled peptides were detected. Generally unlabeled peptides are detectable at least with the same sensitivity as labeled peptides. Thus, the results indicate that the labeling is complete. To prove the quality of the recovery with affinity purification, the mixture of tagged peptides was spiked with a tryptically digested E. coli cell lysate. Fig. 5B shows the survey spectrum of the peptide mixture. Only peptides from the E. coli cell lysate are visible. After affinity purification, only the M-MeCAT-labeled HIV integrase inhibitor and traces of M-MeCAT reagents were detected. The ratio of metals remained unchanged (Fig. 5C). For proteins, the reaction behavior of M-MeCATBnz was tested using BSA and α-lactalbumin. We monitored the labeling reaction of different ratios of proteins and reagents. For comparison, the products were separated by 15% SDS-PAGE. Because the MeCAT label changes the mass and the pI of the proteins considerably, the reaction progress could be monitored. Thus, the slowest migrating band contains the highest number of tags, whereas the unlabeled protein should be found in the first band. In Fig. 6, the results of the separation of differently tagged α-lactalbumin are shown. Depending on the excess of MeCATBnz reagent (M = Ho(III)) and the amount of acetone added to improve denaturation, all eight cysteines in the protein reacted. The nine α-lactalbumin species were observed, ranging from unlabeled to fully labeled species. At a 20-fold excess of M-MeCAT/cysteine, no further shift of the protein band was apparent. To study the migration behavior of the tagged proteins, a reduced sample of BSA was divided into two aliquots and labeled using M-MeCATBnz (M = Tm(III), Lu(III)). Then different amounts of labeled protein were separated by SDS-PAGE and Coomassie-stained. Following this analysis, only one band was visible (Fig. 7). In Fig. 7 the analysis of a 1:1 (Tm(III):Lu(III)) mixture of M-MeCATBnz-tagged BSA is shown. To confirm that both metals appear at the expected ratio in the bands of the one-dimensional gel separation (Fig. 7A), the FIA/ICP MS data of one band are shown (Fig. 7B). The 1-μg BSA band was cut out, solubilized, and subjected to FIA/ICP MS for quantification (Fig. 7B). The two signals for thulium and lutetium are identical. In Fig. 8, the data for the calculation of the detection limit of ICP MS for protein quantification are shown. To this end, different amounts of labeled BSA reaction mixture (16, 31, 63, 125, 250, 500, and 1000 ng) were separated using SDS-PAGE. After silver staining, the protein bands were cut out, solubilized, and diluted for FIA/ICP MS analysis. From these data an external calibration curve has been constructed (Fig. 9) that is linear over the complete range covered here (4 orders of magnitude). Detection limits of 2.31 fmol for lutetium and 2.29 fmol for holmium were calculated using standard procedures.Fig. 9A, FIA/ICP MS of holmium and lutetium standard solutions made up using the same conditions as those used for proteins samples (dissolved gel matrix and metal salt). B, shown are the calibration curves calculated from the data of the measured metal standards. c, concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In an attempt to estimate the detection capabilities of the approach, BSA and lactalbumin were tagged and analyzed. With ICP MS, 3.95 fmol of
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