Metal-Coded Affinity Tag Labeling: A Demonstration of Analytical

Feb 19, 2009 - MeCAT consists of the DOTA macrocycle for metal chelating (I) and ...... Neumann , B. Diploma Thesis, Technische Fachhochschule Berlin,...
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Anal. Chem. 2009, 81, 2176–2184

Metal-Coded Affinity Tag Labeling: A Demonstration of Analytical Robustness and Suitability for Biological Applications Robert Ahrends,† Stefan Pieper,† Boris Neumann,‡,§ Christian Scheler,‡ and Michael W. Linscheid*,† Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor Strasse 2, 12489 Berlin, Germany, Proteome Factory AG, Dorotheenstrasse 94, 10117 Berlin, Germany, and CCR/Institute of Pharmacology and Toxicology, Charite´ University Medicine, Hessische Strasse 3-4, 10115 Berlin, Germany Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanide-harboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a wellestablished technique for assessing concentrations down to low attomole ranges. This work investigates the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromatography/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MSn). Focus was given toward the separation behavior of labeled peptides and the dynamic range of detection and peptide charge distribution. Furthermore, the stability of MeCAT under harsh analytical conditions was investigated. With the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of changes in an Escherichia coli proteome, we successfully addressed the suitability to utilize MeCAT on biological samples. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%. Therefore, this technique is suitable to common proteomic workflows and enables relative as well as absolute differential peptide quantification.

quantification have been developed in the past decade.1 In general, these can be divided into two groups: peptide and protein quantification. The latter include mostly two-dimensional gel-based approaches in combination with fluorescence labeling (DIGE) or densitometry techniques.2 Unfortunately, two-dimensional gel electrophoresis has limitations, e.g., poor separation of alkaline proteins, and can hardly be automated.3 However, it is still the most commonly used method with the highest resolution on the protein level.4 On the peptide level, there are highly sensitive mass spectrometry (MS)-based relative quantification methods. Other methods like stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution MS also have the potential to be used in quantification of proteins but remain challenging due to missing separation at the protein level.5 However, relative quantification at the peptide level is the most frequently used approach to achieve relative protein quantification. In this approach peptides derived by proteolytic cleavage of proteins and complex mixtures are labeled with different isotopic tags, which can be introduced either in vivo (e.g., SILAC) or in vitro (e.g., 18O labeling, ICAT, ITRAQ).6-9 In order to overcome limitations such as matrix effects, small linear dynamic ranges, and difficult data handling of the established isotope labeling techniques, we have recently developed metal-coded affinity tags (MeCAT).10-12 Metal-harboring tags enable the use of inductively (1) (2) (3) (4) (5) (6) (7) (8)

Quantitative determination of peptides and proteins plays a key role in modern life sciences. Numerous methods for relative

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* To whom correspondence should be addressed. Phone: +493020937575. E-mail: [email protected]. † Humboldt-Universitaet zu Berlin. ‡ Proteome Factory AG. § Charite´ University Medicine.

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Linscheid, M. W. Anal. Bioanal. Chem. 2005, 381, 64–66. Miller, I.; Crawford, J.; Gianazza, E. Proteomics 2006, 6, 5385–5408. Ong, S. E.; Mann, M. Nat. Chem. Biol. 2005, 1, 252–262. Gorg, A.; Weiss, W.; Dunn, M. J. Proteomics 2004, 4, 3665–3685. Waanders, L. F.; Hanke, S.; Mann, M. J. Am. Soc. Mass Spectrom. 2007, 18, 2058–2064. Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994–999. Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591–6596. Pasaˇ-Tolic´, L.; Jensen, P. K.; Anderson, G. A.; Lipton, M. S.; Peden, K. K.; Martinovic´, S.; Tolic´, N.; Bruce, J. E.; Smith, R. D. J. Am. Chem. Soc. 1999, 121, 7949–7950. Ross, 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. Mol. Cell. Proteomics 2004, 3, 1154–1169. Krause, M.; Scheler, C.; Boettger, U.; Weisshoff, H.; Linscheid, M. W. Deutsches Patent und Markenamt; German Patent DE 102 27599, 2002. Linscheid, M.; Pieper, S.; Scheler, C.; Ahrends, R. World Intellectual Property Organization, Germany; International Patent WO 2007/118712 A1, 2007; p 49. 10.1021/ac802310c CCC: $40.75  2009 American Chemical Society Published on Web 02/19/2009

coupled plasma mass spectrometry12,13 (ICPMS) to obtain absolute quantification12 by using an external metal standard. Furthermore, commonly used relative quantification can also be achieved utilizing MeCAT in combination with standard MS techniques. Initially, Meares and colleagues introduced chemical tagging by using a metal chelate complex based on DOTA (1,4,7,10tetraazacyclododecane N,N′,N′′,N′′′-tetraacetic acid) and were able to demonstrate that the metal chelate complex itself is suited for an affinity enrichment of labeled peptides.14-16 Additionally, other metal-containing reagents like ferrocene complexes and p-hydroxymercuribenzoic acid can also be used for peptide and protein quantification.17,18 In the present study, DOTA was used to complex rare earth element ions. Besides the polydental macrocyclic DOTA, the noncyclic open form DTPA (diethylene triamine pentaacetic acid) was previously investigated.19 Both reagents, especially DOTA,20 generate extremely stable metal complexes with stability constants (log k) up to 25.4.20-23 Thus, those complexes tend to be well suited for many analytical and bioanalytical applications without the risk of metal loss or metal exchange. Liu et al. demonstrated the labeling of peptides using yttrium (Y) and terbium (Tb) DTPA complexes and observed no difference in elution time of the labeled peptides with different metals in liquid chromatography (LC). They identified differently tagged peptides using tandem MS.19 A difference in ionization efficiency of Tb- and Y-labeled peptides was observed but could be corrected statistically; thus, relative quantification of the tagged peptides was achieved. On the basis of DOTA-metal complexes a reagent that targets the thiol group of cysteine residues was developed (Figure 1).12 The different masses of the metal complexes allow the detection and relative quantification of differently labeled peptide species using nano liquid chromatography/electrospray ionization mass spectrometry (nano-LC/ESI-MS). In contrast to the method presented by Liu et al., MeCAT has the advantage of enhanced stability and the outstanding feature of absolute quantification applying ICPMS, with external calibration12 (Figure 2). Thus, the combination of nano-LC/ESI-MS and nano-LC/ICPMS24 will make MeCAT a valuable tool for quantification purposes in proteomics. (12) Ahrends, R.; Pieper, S.; Kuhn, A.; Weisshoff, H.; Hamester, M.; Lindemann, T.; Scheler, C.; Lehmann, K.; Taubner, K.; Linscheid, M. W. Mol. Cell. Proteomics 2007, 6, 1907–1916. (13) Patel, P.; Jones, P.; Handy, R.; Harrington, C.; Marshall, P.; Evans, E. H. Anal. Bioanal. Chem. 2008, 390, 61–65. (14) Whetstone, P. A.; Butlin, N. G.; Corneillie, T. M.; Meares, C. F. Bioconjugate Chem. 2004, 15, 3–6. (15) Lee, S.; Young, N. L.; Whetstone, P. A.; Cheal, S. M.; Benner, W. H.; Lebrilla, C. B.; Meares, C. F. J. Proteome Res. 2006, 5, 539–547. (16) Renn, O.; Meares, C. F. Bioconjugate Chem. 1992, 3, 563–569. (17) Seiwert, B.; Karst, U. Anal. Bioanal. Chem. 2008, 390, 181–200. (18) Kutscher, D. J.; Busto, M. E. D.; Zinn, N.; Sanz-Medel, A.; Bettmer, J. J. Anal. At. Spectrom. 2008, 23, 1359–1364. (19) Liu, H.; Zhang, Y.; Wang, J.; Wang, D.; Zhou, C.; Cai, Y.; Qian, X. Anal. Chem. 2006, 78, 6614–6621. (20) Port, M.; Idee, J. M.; Medina, C.; Dencausse, A.; Corot, C. Br. J. Radiol. 2008, 81, 258–259. (21) Byegard, J.; Skarnemark, G.; Skalberg, M. J. Radioanal. Nucl. Chem. 1999, 241, 281–290. (22) Moreau, J.; Guillon, E.; Pierrard, J. C.; Rimbault, J.; Port, M.; Aplincourt, M. Chemistry 2004, 10, 5218–5232. (23) Bunzli, J. C. Acc. Chem. Res. 2006, 39, 53–61. (24) Giusti, P.; Lobinski, R.; Szpunar, J.; Schaumloffel, D. Anal. Chem. 2006, 78, 965–971.

Figure 1. Structure of the MeCAT reagent used in this study for thiol group labeling. MeCAT exists in different metal variants depending on the incorporated lanthanide ion (M). MeCAT consists of the DOTA macrocycle for metal chelating (I) and affinity purification using DOTA antibodies, a spacer (II), which connects the macrocycle, and the maleimide group (III) for thiol-specific labeling.

The main objective of our study was to investigate the feasibility of relative quantification of differentially MeCAT-tagged peptides using nano-LC/ESI-MS. Focus was given on MeCAT stability, possible metal exchange between differentially tagged peptides, the elution behavior of tagged peptides, and the observed charge state. Furthermore, the dependency between the relative standard deviation of differently labeled peptide and the injected amount of sample and the linear dynamic range of quantification were investigated. Finally, the application of the technique to complex peptide mixtures was demonstrated. EXPERIMENTAL SECTION Protein Digestion and Labeling. An amount of 1 mg of bovine serum albumin (BSA) (Cohn V fraction, g96% Sigma) was dissolved in 2 mL of 50 mM ammonium hydrogen carbonate (pH 7.8) (Roth) containing 10% (v/v) acetonitrile (Roth). An amount of 10 µg of trypsin (Promega, sequencing grade) was added, and the sample was incubated for 6 h at 37 °C. A second portion of 10 µg of trypsin was added followed by another 6 h of incubation. Subsequently, the mixture was reduced with a 1-fold excess of tris(2-carboxyethyl) phosphine (TCEP, Sigma) for 1 h at 37 °C. Obtained peptides of BSA and model peptides were labeled in 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7. Peptides were diluted to 7.3 µM, divided into four aliquots, and tagged with a 10-fold molar excess of M-MeCAT reagent (M ) Tb(III), Ho(III), Tm(III), or Lu(III)) for 12 h at 37 °C (Proteome Factory AG, Berlin, Germany). Individual reactions were quenched using an excess of 20 mM DTT (1,4dithiothreitol) and subsequently combined. Peptide Analysis via Nano-LC/ESI-MSn. Labeled samples were diluted in 0.1% (v/v) formic acid to a final concentration of 0.73 or 0.073 µM, respectively. For LC/MS/MS, an 1100 nanoLC system (Agilent) was used. Separation was performed on a Zorbax 300 SB-C18 3.5 µm, 150 mm × 75 µm with a Zorbax 300 SB-C18 3.5 µm, 0.3 mm × 5 mm (Agilent) enrichment column and a binary mobile phase water/acetonitrile gradient with a maximum flow rate of 0.25 µL min-1. Samples were loaded onto the enrichment column at 20 µL min-1 and desalted with 98.5% deionized water, 1% acetonitrile, 0.5% formic acid (v/v/v). Peptides were separated with a 75 or 45 min gradient. The eluents used were as follows: As94.9% deionized water, 5% acetonitrile, 0.1% formic acid (v/v/v); Bs99.9% acetonitrile, 0.1% (v/v) formic acid. To determine reproducibility and dynamic range, each nano-LC/ESI-MS analysis was carried out at least three times. To investigate the dynamic range, different Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Figure 2. Current scheme of the MeCAT workflow. Cysteine residues of peptides or proteins from different samples are differentially labeled with metal-coded MeCAT reagents. After combination of the samples, the protein/peptide mixture can be separated and analyzed. Mass shift allows relative quantification and identification in ESI-MS, and the incorporation of a rare earth metal ion allows a sensitive detection and absolute quantification in ICPMS. The MeCAT tag is indicated by an asterisk.

volumes of a 36 fmol µL-1 dilution of the differently labeled (Ho/Lu) peptides were injected, ranging from 3.6 to 288 fmol. The nano-LC system was coupled to a Finnigan LTQ FT mass spectrometer (Thermo Fisher Scientific) using a Nanomate ESI interface (Advion). An electrospray voltage of 1.7 kV and a transfer capillary temperature of 200 °C were chosen. Collisioninduced dissociation (CID) experiments were performed and detected in the ion trap of the mass spectrometer. Elution experiments were carried out on a quadrupole timeof-flight (Q-TOF) system. For that purpose the separation column was coupled to a Q-STAR XL (Applied Biosystems). The instrument was used in positive ionization mode with a mass range of m/z 350-1600 for the survey scans and m/z 50-2500 for CID experiments. An ionization voltage of 1.7 kV and a nitrogen curtain gas flow of 15 L h-1 were applied. MeCAT-Metal Stability Experiments. pH Stability. A 7 µM Tm-MeCAT solution was treated with several dilutions (1 × 10-6 to 1 M) of hydrochloric acid for 30 min at 22 °C. Resulting solutions showed pHs over the range of pH 0 to pH 6. Subsequently, 1 µL of these solutions was injected. The peak intensity was determined by integration of the extracted ion chromatogram (EIC) of the Tm-MeCAT signal (m/z 827.19). NaCl Treatment. Identical conditions as for the pH stability experiments were used. Sodium chloride was added to final concentrations ranging from 1 × 10-5 to 1 M, and 1 µL aliquots were analyzed. Temperature Stability. Solutions of 14 µM Tm-MeCAT were heated to 22, 40, 60, 80, and 100 °C for 30 min and diluted with a 20 mM lanthanum(III) chloride (LaCl3) solutions to 7 µM Tm-MeCAT and incubated for further 30 min at 22 °C. The analyses were carried out as described before. Metal Exchange. The Tb-MeCAT- and Lu-MeCAT-labeled peptides, CRAEYSK (25 µM), were mixed in a ratio of 2:3 and treated with a 1-, 10-, or 100-fold molar excess of TbCl3 or LuCl3 for 2 h at 22 °C. Finally, 1 pmol of the mixtures was subjected to nano-LC/ESI-MS analyses. The peak intensity was determined by integration of the EIC of the Tb- and Lu-MeCATlabeled peptides. Labeling of an E.coli Lysate by MeCAT: Application on Biological Samples. To demonstrate the application of the metal labeling technique, a complex sample of Escherichia coli cell lysate 2178

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was utilized. Two different states of an E. coli K-12 TG125 culture were investigated. Samples were obtained from a cultivation experiment using minimal basal medium. Sample 1 was taken at a cultivation temperature of 30 °C. Subsequently, the growth temperature was raised to 40 °C and the second sample was taken 1 h after the temperature shift from the same culture. Amounts of 25 µg of E. coli protein mixtures were subjected to trypsin digestion overnight. Prior to labeling the peptides were reduced by incubation with 500 µM TCEP for 1 h. Then 35 pmol in 140 nmol of labeling reagents Lu-MeCAT or Ho-MeCAT were added to the peptide mixtures. The resulting mixture was analyzed by nano-LC/ESI-MSn on a Finnigan LTQ FT mass spectrometer, followed by a peptide fragment mass fingerprint analysis with OMSSA (version 2.1.1, E. coli K-12, target decoy database). The EICs of the identified differential peptides were integrated and used for differential quantification. RESULTS AND DISCUSSION Stability of the MeCAT-Metal Complex. Complex stability plays a critical role during separation and detection of labeled peptides. To assess whether the metal complex is stable during the process of tagging and subsequent analysis, Tm-MeCAT was treated with an increasing excess of salt and under acidic pH as well as increased temperature conditions. Additionally, the possible metal exchange of the complex was investigated. Figure 3A shows the signal intensity of the Tm-MeCAT complex (m/z 827.19) in dependence of different concentrations of hydrochloric acid for incubation, covering a range from pH 0 to pH 6 (1 M to 1 µM HCl). During these analyses, no change of intensity was observed, indicating that the metal chelate complex shows no dissociation or degradation under those harsh conditions. To combine MeCAT labeling with multidimensional chromatographic techniques (MudPIT), it is a prerequisite that the metal complex is stable under high salt conditions.26 For that purpose, a Tm-MeCAT complex was incubated with different concentrations of NaCl ranging from 10 µM to 1 M (Figure 3C). (25) Neumann, B. Diploma Thesis, Technische Fachhochschule Berlin, Germany, 2002. (26) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242–247.

Figure 3. Stability of the metal-MeCAT complex. (A) pH stability: A 7 µM solution of Tm-MeCAT was incubated with different amounts of HCl (1 µM to 1 M) to investigate the pH stability (pH 0-6). The integrated area of the EICs of Tm-MeCAT (m/z 827.19) is given in dependence of the HCl concentration used. (B) Temperature stability: A 7 µM Tm-MeCAT solution was heated for 30 min to 22, 40, 60, 80, and 100 °C and afterward incubated in 10 mM LaCl3. The peak area of the EICs (m/z 827.19) was plotted against the incubation temperature. (C) Salt stability: Tm-MeCAT was treated with increasing amounts of NaCl, ranging from 10 µM to 1 M, and incubation at 22 °C for 1 h. The sum of the intensities of the [M + H]+ and [M + Na]+ are plotted against the used NaCl concentration (for more information see the Supporting Information). (D) Complex stability toward metal exchange: A Tb/Lu-MeCAT-labeled peptide (ratio 2:3) was treated with increasing concentrations (25-2500 µM) of salt (LuCl3 or TbCl3). The peak areas of the EICs of Tb-MeCAT- and Lu-MeCAT-labeled peptide were plotted against the used salt excess. The ration was determined by dividing the Lu-MeCAT through Tb-MeCAT peak area.

High salt concentration induced the presence of sodium adducts as indicated by a characteristic mass shift of +22 m/z, but it did not effect the complexation of the trivalent coordinative bound lanthanide ion (Supporting Information). The complex was stable, and no metal loss was detected under these conditions (Figure 3C). To analyze if other lanthanides can effect or displace a DOTAcomplexed ion, two different experiments were performed. First, it was tested if a metal loss occurs during heating of the complex to up to 100 °C in an aqueous solution. To detect a metal loss the complex was incubated in the presence of an excess of another metal salt (LaCl3). No loss of signal intensity for the complex was observed under elevated temperature conditions (Figure 3B). Furthermore, no lanthanum metal complex was detected upon incubation with LaCl3. This demonstrates that the Tb-MeCAT complex is stable at high temperatures and is not prone to metal ion exchange. Second, a mixture (ratio of 2:3) of differentially Tb-MeCATand Lu-MeCAT-labeled peptide was incubated with different molar excesses of TbCl3 or LuCl3 (Figure 3D). The nano-LC/ESI-MS spectra and corresponding mass lists showed that the ratio of labeled peptides is unaffected from the treatment with free metal ions. All performed stability experiments gave no indication that the MeCAT metal complex is unstable under typical proteomics workflow conditions. Furthermore, the maleimide moiety also remained unaffected during the performed stability experiments. However, as previously reported the maleimide moiety is prone

to hydrolysis under alkaline conditions.27,28 The stability of the macrocycle and the maleimide moiety underlines that MeCAT metal complexes are well suited for a wide variety of separation techniques with even harder constraints like high salt concentrations or low pH. Efficiency of Labeling and Elution Behavior. The efficiency of labeling and the elution performance of modified peptides is important for quantification by ICPMS in combination with structure elucidation by ESI-MS. In general, maleimide derivatizations are carried out in micro- to millimolar concentration ranges. Therefore, a 2-50-fold excess of the maleimide derivative was previously used for derivatization of free thiol groups.29 It was also shown before that it is possible to label proteins in the low picomole to femtomole range over 3 orders of magnitude using maleimide derivatives.30 We investigated whether our labeling reaction of thiol groups is quantitative on the peptide level. No unmodified peptides were detected after MeCAT labeling with a variety of peptides using a 10-fold excess of labeling reagent (Figure 4). However, in case of very low copy numbers of proteins or peptides (attomole) the peptide or protein sample should be preconcentrated using tools like affinity purification or free flow electrophoresis. This is not only important for quantitative labeling, but also for quantitative detection, since in general uncertainty increases at very low (27) Wu, C. W.; Yarbrough, L. R.; Wu, F. Y. H. Biochemistry 1976, 15, 2863– 2868. (28) Ishii, Y.; Lehrer, S. S. Biophys. J. 1986, 50, 75–80. (29) Toedt, G. H.; Krishnan, R.; Friedhoff, P. Nucleic Acids Res. 2003, 31, 819– 825. (30) Pretzer, E.; Wiktorowicz, J. E. Anal. Biochem. 2008, 374, 250–262.

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Figure 4. Quantitative MeCAT labeling of thiol-containing peptides. Outer column left: EICs of four reduced thiol-containing peptides during a 75 min reversed-phase high-performance liquid chromatography (RP-HPLC) separation. Center column: EICs of the peptides after modification using a 10-fold molar excess of Tm-MeCAT (250 µM). Outer column right: EICs of thiol-containing peptides modified with Tm-MeCAT. For separations, 100 fmol of the peptide mixtures was injected into the HPLC system. The MeCAT tag is indicated by an asterisk.

Figure 5. Effect of MeCAT labeling on peptide retention time. The figure shows the EICs of unlabeled (blue) and Tb-MeCAT-labeled peptides (purple) on a Zorbax C18 RP capillary column using a 75 min, water/acetonitrile gradient. The MeCAT tag is indicated by an asterisk.

detection amounts. Furthermore, the use of such preconcentration steps in combination with prefractionation is also able to overcome the linear dynamic range problem (e.g., protein concentrations in the proteome varying between 1000-fold in bacteria up to 1012fold in mammalian plasma), which applies to all protein identification and quantification methods. In order to investigate the elution behavior of tagged peptides, nano-LC/ESI-MS experiments of labeled and unlabeled peptides 2180

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were performed (Figure 5). In these experiments, all tagged peptides showed a significantly increased retention time toward higher acetonitrile concentrations. The peak doubling of the EIC of m/z 886.3 (Figure 5D) arises from the two diastereomers being formed during the peptide labeling reaction with the maleimide reagent. This diastereomer formation is due to the maleimide double bond being opened in two different manners by the attaching thiol group. In most cases, the resulting diastereomers

Figure 6. Effect of different metals in the MeCAT complex. EICs of four differentially labeled peptides are shown. BSA peptides were labeled with Tb-, Ho-, Tm-, and Lu-MeCAT and combined for nano-LC/ESI-MS analysis. An amount of 200 fmol of labeled peptides was injected. The MeCAT tag is indicated by an asterisk.

coeluted during LC separation; however, in some cases both diastereomers were chromatographically separated. This peak splitting can result in a loss of intensity, and thus, this formation of diastereomers is currently under investigation in order to overcome the possible loss of intensity. To further investigate the elution behavior of differentially labeled peptides, four BSA digests were labeled using either Tb-, Ho-, Tm-, or Lu-MeCAT. The elution profiles of the MeCATlabeled peptides are shown by their EICs (Figure 6). Due to the fact that all labeled peptides species elute within less than 90 s peak width under the chosen conditions, a relative quantification of the differentially labeled BSA peptides from this mixture is achievable. However, in some cases of mainly doubly labeled peptides (e.g., 1ECCDKPLLEK10, Figure 6C) slightly different retention times were observed. Besides the elution behavior of MeCAT-labeled peptides on reversed-phase material, the retention behavior during strong cation-exchange separation (SCX) was investigated (data not shown). Under those conditions MeCAT-labeled peptides eluted at lower salt concentrations in comparison to unlabeled peptides. This observation can be attributed to the carboxylic acid groups reducing the retention on the cation-exchange resin. Charge State and Dynamic Range. During analyses of the differentially labeled BSA peptide mixture, signals of 64 MeCATlabeled peptides were observed and identified by the use of CID experiments. Of the investigated peptides, 60% were triply [M + 3H]3+ charged and 40% were doubly [M + 2H]2+ charged. Table 1 gives a summary of the charge states of labeled and unlabeled species, observed during LC/MS analyses.

With the use of search engines like MASCOT and OMSSA, it was possible to identify the sequences from all labeled peptides. Exemplarily, the fragmentation spectra of two Lu-MeCAT peptides are shown (Figure 7). With the use of the information extracted from the CID spectra with MASCOT and OMSSA, the two peptides 45GLVLIAFSQYLQQCPFDEHVK65 and 286YICDNQDTISSK299 were unambiguously identified with high scores. As apparent from the spectrum (Figure 7A, arrows), the mass difference between the ions y7 and y8 (∆m/z 832 + ∆m/z 103 for the cysteine residue, ∆m/z 935 in total) indicates that the thiol group of the peptide is labeled. Furthermore, the good signal-to-noise ratios in the CID spectra enabled an identification of peptides without an MS/MS spectrum clean up, which is usually applied with other quantification approaches such as ICAT. To investigate biological systems in a quantitative manner, it is important to gain knowledge on the dynamic range and the detection limit of the method. Therefore, a trypsin-digested BSA sample was split into four aliquots and labeled with Tb-, Ho-, Tm-, or Lu-MeCAT. After labeling, the Ho-MeCAT and Lu-MeCAT samples were combined using a molar ratio of 1:1 and diluted 100-fold. For quantitative analysis different amounts ranging from 3.6 to 288 fmol were subject to nanoLC/ESI-MS analysis. The signal intensity of each labeled peptide was determined by peak integration of the respective EIC. The average peak intensity of the three analyses with a differentially MeCAT (Ho/Lu) labeled BSA peptide was plotted against the injected sample amount (Figure 8A). A linear dynamic range of almost 2 orders of magnitude, with an average Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Table 1. Charge States of Tb-MeCAT-Labeled and Unlabeled Peptides Obtained from a BSA Trypsin Digesta unmodified peptide sequence

charge

m/z

charge

m/z

no. of cysteine residues

SLHTLFGDELCK100 139 LKPDPNTLCDEFK151 118 QEPERNECFLSHK130 123 NECFLSHK130 184 YNGVFQECCQAEDK197 286 YICDNQDTISSK299 198 GACLLPK204 223 CASIQK228 375 EYEATLEECCAK386 310 SHCIAEVEK318 387 DDPHACYSTVFDK399 413 QNCDQFEK420 499 CCTESLVNR507 508 RPCFSALTPDETYVPK523 581 CCAADDKEACFAVEGPK597 588 EACFAVEGPK597 300 ECCDKPLLEK309

3 3 n.d. n.d. 2 2 n.d. n.d. 2 n.d. 2 2 2 2 n.d. n.d. n.d.

454.9 507.3 n.d. n.d. 817.4 693.8 n.d. n.d. 694.3 n.d. 749.3 506.7 513.2 608.6 n.d. n.d. n.d.

3 3 3 2 3 3 2 2 3 2 3 2 3 3 4 2 3

727.0 779.3 811.6 897.3 1089.3 734.9 759.3 733.3 1007.6 916.3 771.9 914.3 886.3 880.6 1052.5 933.8 937.3

1 1 1 1 2 1 1 1 2 1 1 1 2 1 3 1 2

89

a

modified peptide

no. of MeCAT tags 1 1 1 1 2 1 1 1 2 1 1 1 2 1 3 1 2

n.d.: not detected.

Figure 7. ESI-CID mass spectra of Lu-MeCAT-labeled BSA peptides. The spectra show the y- and b-series of the triply charged (top) or doubly charged precursor ion (bottom). The MeCAT tag is indicated by an asterisk.

coefficient of determination of R2 ) 0.996, was achieved for the differently labeled peptides, with a run to run standard deviation below 15% for each measured point. 2182

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Additionally, the relative deviation between differentially MeCATlabeled samples has great impact on quantitative results and can be significantly high if different biological samples are compared.

Figure 8. Linear dynamic range and standard deviation of MeCAT-labeled peptides. (A) Linear dynamic range of the Ho- and Lu-MeCATlabeled BSA peptide 89SLHTLFGDELCK100 during reversed-phase separation. The intensities were calculated from EICs of the eluting peptides. The b and 9 symbols indicate intensities of the ion trap signal (IT), and the 2 and 1 symbols reflect the intensities of the labeled peptide in the FTICR (FT). The R2 values for both linear fits are 0.9995 (upper) and 0.99947 (lower). Panel B displays the fluctuation of the standard deviation against the average signal intensity of the Tb-, Ho-, Tm-, Lu-MeCAT-labeled peptide 483LCVLHEK489 over a molar range of more than 2 orders of magnitude; all intensities were normalized to the Tb-MeCAT-labeled peptide (100%).

Figure 9. Quantitative results of differentially labeled E. coli lysates. (A) Quantitative results obtained from nano-HPLC/ESI-MS experiments. The quotient of the peptides intensities of Ho-MeCAT- and Lu-MeCAT-labeled peptides is plotted against the identified proteins. (B) ESI-MS mass spectrum of unregulated Ho-, Lu-MeCAT-labeled peptide pair. (C) ESI-MS mass spectrum of regulated Ho-, Lu-MeCAT labeled peptide pair.

For this reason, the deviation of the applied technique must be minimized and should be independent from the sample amount. Therefore, the average intensities of an equimolar mixture of the Tm-, Ho-, Tm-, Lu-MeCAT-labeled BSA peptide 483LCVLHEK489 from the BSA digest was plotted against the injected amount. The standard deviation was lower than 10% in all cases between the differentially labeled peptides for over 2 orders of magnitude (Figure 8B). This indicates that the standard deviation of the signal of labeled peptides is stable over 2 orders of magnitude and independent from the amount of injected sample. Labeling of an E.coli Lysate. In order to investigate the suitability of MeCAT labeling for a complex protein mixture, two E. coli protein lysates, which were derived from two different

growth temperatures, were differentially labeled with Lu-MeCAT and Ho-MeCAT. A total of 561 unique E. coli proteins were identified from the obtained nano-LC/ESI-MS data. A set of 56 labeled peptides (min abs intensity >1 × 103; signal-to-noise >5) was analyzed to gain quantitative information on the abundance of the respective proteins. Five proteins showed a regulation factor of 2, respectively (Figure 9A). The regulated proteins were mainly heat shock proteins and other growth-related enzymes. Their abundances varied coherently with the induced temperature shift and fitted previous observations.25 Using MeCAT we were able to show that this labeling technique can be used to investigate differential expression of proteins from a complex mixture. However, the results revealed the need of high-resolution mass spectrometers for proper identification and quantification, Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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since commonly used isolation widths of ± m/z 1-2 for peptide MS/MS quantification experiments result in false positives caused by underlying peptide peaks (Figure 9C). CONCLUSION In the current work we demonstrated that the investigated MeCAT method fulfills all criteria to be used for quantitative analysis using nano-LC/ESI-MS. Remarkably, it was possible to achieve a linear dynamic range of 2 orders of magnitude, down to 3.6 fmol, with a run to run standard deviation below 15% for a differentially labeled BSA peptide, even at this low concentration. Furthermore, quantification of a mixture of differentially Tb-, Ho-, Tm-, and Lu-MeCAT-labeled peptides was achieved. The average standard deviation between the differently tagged BSA peptides was under 10% over a molar range of 2 orders of magnitude. Additionally, we showed that the MeCAT metal complexes are stable under a wide variety of conditions including high salt, low pH, and increased temperature. In comparison to the unlabeled peptides, it was observed that some modified peptides exhibit a higher charge state in ESI-MS (Table 1). This may be depending on the increased molecular mass upon labeling of the peptide. The higher charged state is deemed to be useful for electron-transfer dissociation (ETD) or electron capture dissociation (ECD) experiments to increase sequence information upon fragmentation. As expected, almost all MeCAT-tagged peptides exhibit a higher retention time compared to the unmodified peptides in reversed-phase chromatography, due to the increased size of the modified peptides. Representative of a complete proteomic workflow we demonstrated a labeling, identification, and quantification approach of complex E. coli cell lysates. Unfortunately, only 13% of the E. coli K-12 proteins contain a cysteine for labeling using the described maleimide MeCAT. In order to overcome this limitation, two strategies are currently being investigated. First, an alternative tagging strategy will be investigated that does not rely on modification of thiol groups and will allow us to generally label any peptide or protein, targeting amino and carboxyl groups during the labeling reaction. In future

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this will enable us to almost fully cover any proteome. Second, in order to separate labeled from unlabeled peptides, an enrichment method is currently under development based on the already describes antibody purification approach for the macrocycle.14 However, in case of different labeling strategies avoiding the use of maleimide such a separation might not be necessary. The aim of this work was to demonstrate the analytical robustness of the MeCAT labeling for proteomic workflows utilizing LC/ESI-MS. The investigations showed that MeCAT as a two-, three-, or fourplex metal label is well suited for relative quantification of proteins and peptides in conjunction with nanoLC and ESI-MS and provides a very useful alternative to other stable isotope tagging methods. Furthermore, MeCAT labeling also enables absolute quantification of labeled peptides since ICPMS is able to cover a huge dynamic range in quantitation.31 Currently, a work on the use of LC/ICPMS for absolute quantification of MeCAT-labeled peptides and proteins is in progress, and the combination of both techniques will enabled us to overcome common problems in protein quantification (e.g., matrix effects). ACKNOWLEDGMENT The excellent technical assistance of Florian Fricke is gratefully acknowledged. We thank the Stiftung Industrieforschung for generous financial support over the past 3 years. The authors thank Dr. Sebastian Beck and Professor Dr. Peter Friedhoff for critical comments and helpful discussions. We also want to thank Professor Dr. Popovic for providing the E. coli samples and Thermo Fisher Scientific for the friendly support. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 3, 2008. Accepted January 20, 2009. AC802310C (31) Thermo Finnigan. Technical Note 4; Thermo Fisher Scientific: Bremen, Germany, 2005.