A Metal-coded Affinity Tag Approach to Quantitative Proteomics

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. 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). 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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 using ESI MS and ICP masses and of the DOTA complexes of the lanthanides used J.C. Benefiting from the unique properties of lanthanide ions.Acc. Chem. Res. 2006; 39: 53-61Crossref PubMed Scopus (900) Google Scholar, 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 mass DOTA in a new For the quantitative analysis of a protein MeCAT was combined with high resolution 2-D electrophoresis using dissolvable were and reaction at the cysteine with reagents the samples were and the mixtures were two which were using and 2-D After the of were for quantification by MS, or for identification using The is in Fig. The available peptides were with 2 at for a of reagent was The reaction was in for 2 at the reaction was using an of The proteins used in this study were bovine serum albumin and from Proteins were in 2 for at and using different reagents in a to cysteine The reaction was in a containing and for and and for and were used to of proteins. The reaction was for at and the reaction was using from a were on as previously J. C. A method for lens which the attached to the Res. PubMed Scopus Google Scholar). The proteins were in TCEP, and The of protein was determined by the A and sensitive method for the quantitation of of protein the of PubMed Scopus Google Scholar). and of lens protein A and were with for at The protein samples A and were with of or The reaction was in a containing and at and the reaction was using an of to affinity purification, of and of and were to of containing of peptide and of cell For affinity purification of peptides, a of et al. 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 magnetic After and of of magnetic with a magnetic the was and the were with of and followed by of and the were with of peptide as and for at with The was and the were with of and with of and three with of After of the peptides were with of and by for at and the containing the peptides was in a and at To labeling the labeling reaction proteins were to SDS-PAGE as by of structural proteins during the of the of PubMed Scopus Google Scholar) using gel and To the mass of metal-coded proteins, were stained with J. R. for in I. of PubMed Scopus Google Scholar) or a J. U. electrophoresis of an and for a analysis of the PubMed Scopus Google Scholar). purification or the samples were to 2-D electrophoresis proteins were by the high resolution 2-D electrophoresis technique to and J. U. electrophoresis of an and for a analysis of the PubMed Scopus Google Scholar). 2-D were from a gel and were For and for to of the combined lens samples were at the of the In the first was using in the of The was a SDS-PAGE as by of structural proteins during the of the of PubMed Scopus Google Scholar) using After the were stained with and were stained with For the protein were in gel or to the for MS For digestion were to and and The were with of with of and in a for of and 2 of were to the digestion was for at After the was and the were in of for and from the all were with of The were and samples were in acid to a concentration of For an was and enrichment were was using a with a flow of The A and acid; and acid; and For peptide the was the enrichment using a flow The separation was coupled to a a using a For MS and MS/MS the and were capillary High resolution MS and MS/MS were using a mass to a using the MS and MS/MS data were using the capillary lens Peptides were analyzed on a MALDI-TOF mass with acid as The samples were by the A of and was used for mass The of peptide mass was For analysis of lens proteins in samples from a combination of a and ICP mass was an between the and ICP MS the with a was used for and were using a standard containing and The analysis of standard proteins was on an in combination with a with a flow of for and were using a standard containing and To MeCAT as a to labeling techniques, to use in relative and to that it is a unique for absolute quantitative determination of proteins, it is to that the labeling of peptides and proteins is and To this the method was to peptides peptide and to single proteins serum albumin and and to the proteome of the lens it was to that the differently tagged peptides the in of were with one with and the with as metal were in a of and the mixtures were analyzed using LC/MS and The in Fig. two tagged The two peptides in were present at the of ESI We also used this to that the peptides be identified by a search data are not one the of the peptide is shown in Fig. In the MS/MS the and In the we to the reaction of all the available At the peptide level, samples of the were with different metal-coded reagents the reaction mixtures were combined in a of A mass of the peptide is in Fig. The were whereas no peptides were peptides are at with the sensitivity as Thus, the results that the labeling is To the of the with affinity purification, the of tagged peptides was with a E. cell Fig. the of the peptide peptides from the E. cell are After affinity purification, only the and of reagents were The of metals (Fig. For proteins, the reaction of was using and We the labeling reaction of different of proteins and For the were by the MeCAT changes the mass and the of the proteins the reaction be Thus, the the of whereas the protein should be in the first In Fig. the results of the separation of differently tagged are on the of MeCATBnz reagent and the of to all in the protein The were from to At a of no of the protein was To study the of the tagged proteins, a of was two and using different amounts of protein were by SDS-PAGE and this analysis, only one was (Fig. In Fig. the analysis of a of is To that both metals at the in the of the gel separation (Fig. the MS data of one are shown (Fig. The was and to MS for quantification (Fig. The two for and are In Fig. the data for the of the detection limit of ICP MS for protein quantification are To this different amounts of reaction and were using After the protein were and for MS data an has been (Fig. that is the of Detection limits of for and for were calculated using standard MS of and standard using the as used for proteins samples gel and metal shown are the calculated from the data of the metal In an to the detection of the and were tagged and ICP MS, of from were that all in were a of 110 was For of was to of In the the of the technique to an analysis of protein mixtures was Proteins from the eye lens were tagged and The proteins were under and and two samples were using the samples were and the proteins were using 2-D The were and with A. I. A. M. M. of peptides from proteins by tandem mass Biotechnol. PubMed Scopus Google Scholar) for MS/MS analysis and identification of analysis and quantitative analysis of S. scrofa eye lens proteins from 2-D A A A scrofa protein scrofa protein scrofa protein S. scrofa protein in a new To the proteins using ICP MS, the from a gel were and (Fig. the concentration of the metals was the samples were and analyzed using MS. In Table data with of proteins, and from 2-D gel are were identified as A were identified as and were identified as Table quantitative data of the The amounts of proteins from to for the and from to for To the detection one on this gel was analyzed with a resolution of The metal abundance was used to a of the entire (Fig. The data for the sensitivity of ICP MS that allows detection of low protein concentrations at the of the The data present that MeCAT is not only an to labeling techniques but a new for protein We showed that with reaction the labeling of proteins and peptides was In we of tagged peptides in and the for identification of peptides using LC/MSn data and a search as and proteins in one on SDS-PAGE and we are the of this for proteins. The affinity purification of peptides to be and we or of metals from the tag. The thermodynamic (see Table I) and the of the complexes is such that or with metal ions is J.M. Port M. I. M. S. C. and biological of by for magnetic a 2006; PubMed Scopus Google Scholar). Thus, the of peptides stable during the that the MeCAT is for quantitative proteomics. ICP MS for the quantitative detection of the metals was to the the and the precision of MeCAT the metal tag the molecular mass for ESI MS can be However, in MALDI MS for of the labeling reaction was and ESI MS was in generally for the proteins. In the of for proteins or peptides is of is as with chemical labeling techniques, this tagging technique changes the values of peptides and proteins. In this the values became Therefore, the 2-D gel of tagged proteins to of cysteine in proteins were the proteins to of the to the of the analysis; the information the of tags also The to the low lanthanide concentration and the detection of ICP MS the method with detection for the absolute quantification of proteins at 110 In the shown we have used the monoisotopic lanthanides to the in the ESI or MALDI In the metals also be used at the of of molecular isotopic and is at one be for metal that no with affinity preconcentration and purification should to the quantification and identification of low abundance proteins. However, the unique metal tag the for and We can on internal standards the only is a lanthanide metal Thus, in the analysis be that all can be for and A of this technique is that a of proteins in complex mixtures is access to the analysis of protein complexes and protein with unique for the quantitative protein analysis is the dynamic of concentration in biological at of R. M. Mass 2003; PubMed Scopus Google Scholar). only ICP MS a dynamic We have in the course of this a dynamic of of to the In for ICP MS to of for lanthanide samples has been shown High Scholar), that the dynamic is not by the analysis of one using MS is per and can be It is evident that MeCAT only be used for absolute quantification but also for relative quantification of or samples absolute determination is and different samples and can be this is unique of this The of such a dynamic and the sensitivity to the to MeCAT a to techniques available or that are under In the course of the research we that MeCAT can be used to proteins in an absolute and a relative fashion. The method was on model peptides and proteins and by analysis of an eye lens The be used in the to protein and protein in complex mixtures using ICP MS and ESI For the identification of proteins, strategies peptide and can be are in In 2-D LC/MS and capillary electrophoresis techniques are under development to the with the 2-D gel For the shown the in values of proteins has been in of the The capillary electrophoresis or techniques be to The to in the analysis based on internal standards should data that can be to the of ICP MS measurements.