Precolumn Isotope Dilution Analysis in nanoHPLC−ICPMS for

Feb 20, 2007 - The detection limit for sulfur was 45 μg L-1 which corresponded to 1−2 pmol of individual peptides. The method was validated by the ...
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Anal. Chem. 2007, 79, 2859-2868

Precolumn Isotope Dilution Analysis in nanoHPLC-ICPMS for Absolute Quantification of Sulfur-Containing Peptides Dirk Schaumlo 1 ffel,*,† Pierre Giusti,† Hugues Preud’Homme,† Joanna Szpunar,† and Ryszard Łobin´ski†,‡

Group of Bio-Inorganic Analytical Chemistry, CNRS UMR 5034, He´ lioparc, 2, Av. Pr. Angot, F-64053 Pau, France, and Department of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664-Warsaw, Poland

A novel generic approach based on precolumn isotope dilution nanoHPLC-ICPMS analysis was developed for the accurate absolute quantification of sulfur-containing peptides. A 34S-labeled, species-unspecific sulfur spike (sulfate), noninteracting with analyte peptides under the optimized HPLC condition, was added directly to the chromatographic eluents. Thus a generic sulfur standard permanently present during analysis was used for peptide quantification. Interference-free detection of the 32S and 34S isotopes in ICPMS was achieved by eliminating O + 2 ions in a collision cell using Xe gas at 130 µL min-1. The detection limit for sulfur was 45 µg L-1 which corresponded to 1-2 pmol of individual peptides. The method was validated by the analysis of a standard peptide solution showing high accuracy (recovery 103%) and good precision (RSD 2.1%). The combination of nanoHPLCICP IDMS with nanoHPLC-ESI MS/MS allowed the precise quantification and identification of sulfur-containing peptides in tryptic digests of human serum albumin and salt-induced yeast protein (SIP18) at the picomole level. The quantification of proteins is one of the major challenges in modern proteomics because many biological questions cannot be answered only by identifying proteins in a cell or tissue.1 The advancement of electrospray (ESI) and MALDI mass spectrometry in combination with multidimensional nanoHPLC for peptide separation has lead to an enormous progress in protein identification and spurred the development of MS-based techniques for protein quantification.2 These approaches are based on the labeling of proteins and peptides with stable isotopes (2H, 13C, 15N, or 18O) to be used as internal standards.2 The chemical synthesis of several labeled peptides has been reported.3 However, due to the impossibility to synthesize thousands of labeled protein and peptide standards the usual approach is to introduce a stable isotope in one of two samples.2 As result of the mass shift in MS * To whom correspondence should be addressed. Phone: +33-559-407760. Fax: +33-559-407781. E-mail: [email protected]. † Group of Bio-Inorganic Analytical Chemistry, CNRS UMR 5034. ‡ Warsaw University of Technology. (1) Kolker, E.; Higdon, R.; Hogan, J. M. Trends Microbiol. 2006, 14, 229-235. (2) MacCoss, M. J.; Matthews, D. L. Anal. Chem. 2005, 77, 295A-302A. (3) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci.U.S.A. 2003, 100, 6940-6945. 10.1021/ac061864r CCC: $37.00 Published on Web 02/20/2007

© 2007 American Chemical Society

caused by the introduced isotope the abundance ratio of each individual peptide can be measured, providing the relative amounts of individual proteins between the two samples. Hence, this approach allows only relative but not absolute protein quantification. Proteins and peptides can be labeled before of after enzymatic digestion. The isotopes can be introduced by digestion in 18Olabeled water4,5 or labeling with isotope-coded affinity tags (ICAT).6 Recent developments concern iTRAQ reagents,7 which contain isotope-coded isobaric tags that are released during MS/MS for quantification, or mammalian cells that are grown on cultures containing stable isotope-labeled amino acids (SILAC).8 The main advantage of these techniques is their applicability to highly complex protein mixtures. The ICAT approach uses an incorporated biotin tag for purification of peptide mixtures by affinity chromatography,6 while iTRAQ allows multiplex protein quantification.7 SILAC is compatible with virtually all cell culture conditions showing complete stable isotope incorporation which requires no chemical peptide labeling.8 Data on accuracy and precision of these techniques are rarely discussed. In the few comparative studies published the precision of the ICAT technique was reported to be between 4% and 28%9 and the precision of the iTRAQ approach in the range of 9-30%9 and 7-9%,10 respectively, depending strongly on the sample investigated. The main drawback of molecular MS-based approaches to quantitative proteomics is that the peptide ionization, and thus sensitivity, is compound dependent and, in addition, affected by the coeluting peptides and other sample components. Moreover, due to the lack of isotopically labeled peptide standards usually only relative quantification is possible.2 (4) Schno ¨lzer, M.; Jedrzejewski, P.; Lehmann, W. D. Electrophoresis 1996, 17, 945-953. (5) Yao, X. D.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842. (6) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (7) Ross, P. L.; Huang, Y. L. 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. (8) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376-386. (9) Wu, W. W.; Wang, G. H.; Baek, S. J.; Shen, R. F. J. Proteome Res. 2006, 5, 651-658. (10) Chong, P. K.; Gan, C. S.; Pham, T. K.; Wright, P. C. J. Proteome Res. 2006, 5, 1232-1240.

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An alternative can be elemental mass spectrometry using an inductively coupled plasma ionization source (ICPMS) to quantify specifically heteroatoms incorporated covalently in peptides and proteins, such as sulfur, selenium, iodine, or phosphorus.11 ICPMS is highly sensitive, element-specific, and capable of multielement/ multi-isotope detection, shows a large dynamic range, and the ionization is practically compound independent and not affected by coeluting species. These features make ICPMS the most powerful and versatile element-specific detector in chromatography. Other heteroelement detection methods such as chemiluminescent nitrogen detection (CLND) are less sensitive, have a limited dynamic range, and show no multielement or multi-isotope detection capabilities.12,13 The accuracy and precision of the quantitative ICPMS detection in chromatography can be considerably improved by the use of isotope dilution analysis (IDA) and isotope dilution mass spectrometry (IDMS), respectively, provided that the target element has at least two stable isotopes, which is the case of, e.g., selenium and sulfur.14 Isotope dilution is performed by the continuous addition of an isotopically labeled spike to the eluent, in principle after the chromatographic separation (postcolumn) in order to avoid isotope exchange between spike and different analyte molecules.15 The isotopically labeled spike is not required to have the same chemical form as the analyte, which make this method of particular interest for the quantification of heteroelements in biomolecules of unknown identity.16 Recently, postcolumn IDA quantification of selenopeptides issued from the digestion of selenomethionyl calmoduline at the low femtomole level was demonstrated.17 In view of the relatively rare occurrence of selenium in proteins, this approach cannot be extended to a wide range of applications. In contrast to selenium, however, sulfur being present in two fairly abundant (cumulative abundance of approximately 5% in natural peptides) amino acids, methionine and cysteine, can be an attractive internal elemental tag for quantitative proteomics. The determination of sulfur by elemental MS offers a potentially generic way for absolute quantification of peptides and proteins, provided that their primary structure and thus the number of sulfur-containing amino acids are known.18 Sulfur detection is hampered in ICPMS by diatomic oxygen ions such as 16O2+ and 16O18O+ which interfere with the 32S and 34S isotopes, respectively. A differentiation between 32S+ and 16O + 2 ions needs a spectral resolution of at least m/∆m 1800.19 High(11) Wind, M.; Lehmann, W. D. J. Anal. At. Spectrom. 2004, 19, 20-25. (12) Corens, D.; Carpentier, M.; Schroven, M.; Meerpoel, L. J. Chromatogr., A 2004, 1056, 67-75. (13) Lane, S.; Boughtflower, B.; Mutton, I.; Paterson, C.; Farrant, D.; Taylor, N.; Blaxill, Z.; Carmody, C.; Borman, P. Anal. Chem. 2005, 77, 4354-4365. (14) Heumann, K. G.; Gallus, S. M.; Radlinger, G.; Vogl, J. Spectrochim. Acta, Part B 1998, 53, 273-287. (15) Heumann, K. G.; Rottmann, L.; Vogl, J. J. Anal. At. Spectrom. 1994, 9, 13511355. (16) Schaumlo ¨ffel, D.; Lobinski, R. Int. J. Mass Spectrom. 2005, 242, 217-223. (17) Giusti, P.; Schaumlo¨ffel, D.; Encinar, J. R.; Szpunar, J. J. Anal. At. Spectrom. 2005, 20, 1101-1107. (18) Wind, M.; Wegener, A.; Eisenmenger, A.; Kellner, R.; Lehmann, W. D. Angew. Chem., Int. Ed. 2003, 42, 3425-3427. (19) Prohaska, T.; Latkoczy, C.; Stingeder, G. J. Anal. At. Spectrom. 1999, 14, 1501-1504. (20) Kru ¨ ger, R.; Ku ¨ bler, D.; Pallisse, R.; Burkovski, A.; Lehmann, W. D. Anal. Chem. 2006, 78, 1987-1994. (21) Becker, J. S.; Boulyga, S. F.; Pickhardt, C.; Damoc, E.; Przybylski, M. Int. J. Mass Spectrom. 2003, 228, 985-997.

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resolution ICP SFMS has been used to separate the oxygen interferences.20-22 The use of quadrupole ICPMS for this purpose is less straightforward, but some success with the use of reaction/ collision cells has recently been demonstrated. In dynamic reaction cells the addition of oxygen as a reaction gas generates SO+ ions which were detected with a mass shift of m/z +16 in the spectrum.23-25 An alternative way to eliminate O2+ interferences was the use of xenon as collision gas in ICP collision cell MS enabling one to detect the sulfur isotopes at their masses m/z 32 and m/z 34, respectively.26 No successful isotope dilution analysis of sulfur peptides using quadrupole ICPMS has to date been reported. The rare examples of postcolumn IDA for peptide quantification via the sulfur atom concerned capillary electrophoresis of metallothioneins and were carried out with a sectorfield ICPMS instrument.27,28 The objective of this study was to develop a generic method for accurate and precise absolute quantification of sulfur-containing peptides in protein tryptic digests by nanoHPLC-ICPMS with sulfur isotope dilution analysis. For this purpose a novel concept of precolumn addition of the isotopically labeled spike was developed, and the 32S/34S isotope ratio measurements using an ICP quadrupole mass spectrometer fitted with a xenon filled collision cell were optimized. Tryptic digests of human serum albumin and of SIP18 protein isolated from selenium-rich yeast served as model samples. EXPERIMENTAL SECTION Apparatus. Nanoflow Reversed-Phase HPLC System. The nanoHPLC pump (Agilent 1100, Waldbronn, Germany) used for this study was equipped with a nanoflow controller ensuring a stable flow rate of 300 nL min-1 during gradient analysis. nanoHPLC separations were performed using a 75 µm i.d. reversed-phase nanoHPLC column (C18 PepMap100, 75 µm × 15 cm, 3 µm, Dionex, Sunnyvale, CA). Injections were made using a model CN4 nanoinjection valve (Valco Instruments, Houston, TX) fitted with a rotor providing an internal injection volume of 11 nL. For online preconcentration the sample was introduced into a 1 µL loop of a model CN2 injection valve (Valco Instruments) and loaded on a preconcentration cartridge (C18 Acclaim 300, 300 µm × 5 cm, 5 µm, Dionex) by means of an Agilent 1100 micropump (capillary HPLC). The cartridge was mounted in the loop of another valve (CN2, Valco). All connections from the pump to the sample injection valve, from the valve to the column, and from the column to the nebulizer (or nanospray emitter) were made from fused silica capillaries (i.d. 20 µm, Polymicro Technologies, Phoenix, AZ) to keep the dispersion of the sample low. A (22) Van Lierde, V.; Chery, C. C.; Strijckmans, K.; Galleni, M.; Devreese, B.; Van Beeumen, J.; Moens, L.; Vanhaecke, F. J. Anal. At. Spectrom. 2004, 19, 888-893. (23) Bandura, D. R.; Baranov, V. I.; Tanner, S. D. Anal. Chem. 2002, 74, 14971502. (24) Hann, S.; Koellensperger, G.; Binger, C.; Furtmuller, P. G.; Stingeder, G. J. Anal. At. Spectrom. 2004, 19, 74-79. (25) Stu ¨ rup, S.; Bendahl, L.; Gammelgaard, B. J. Anal. At. Spectrom. 2006, 21, 201-203. (26) Pro ¨frock, D.; Leonhard, P.; Prange, A. Anal. Bioanal. Chem. 2003, 377, 132-139. (27) Schaumlo ¨ffel, D.; Prange, A.; Marx, G.; Heumann, K. G.; Bra¨tter, P. Anal. Bioanal. Chem. 2002, 372, 155-163. (28) Polec-Pawlak, K.; Schaumlo¨ffel, D.; Szpunar, J.; Prange, A.; Lobinski, R. J. Anal. At. Spectrom. 2002, 17, 908-912.

Figure 1. Schematic view of the instrumental setup for precolumn isotope dilution analysis by nanoHPLC-ICPMS. Table 1. Instrumental Settings of the ICPMS ICPMS (Agilent 7500ce) rf power sampling depth cones nebulizer gas flow cell gas flow extraction lens 1 extraction lens 2 octopole bias quadrupole bias

1500 W 8.5 mm nickel 1.15 L min-1 130-160 µL min-1 Xe 4V -130 V -43 V -19.5 V

schematic diagram of the nanoHPLC set up is represented in Figure 1. nanoHPLC-ICPMS Coupling. An ICPMS equipped with a collision cell (Agilent 7500ce, Tokyo, Japan) was used. Xenon was used as the collision gas in the octopole reaction cell in order to eliminate polyatomic ions interfering with 32S detection. The coupling of the nanoHPLC to the ICPMS was realized via an interface designed and described elsewhere.29 Briefly, a nanoflow total consumption nebulizer (nDS-200) was fitted with a low-deadvolume (3 cm3) drain-free spray chamber allowing 100% sample introduction into the ICP. The outlet capillary (20 µm i.d., 280 µm o.d.) of the nanoHPLC column was inserted into the nebulizer and connected without any dead volume to the nebulizer needle. ICPMS measurement conditions (nebulizer gas flow, rf power, and lens voltage) were optimized daily for the highest intensity of the 7Li signal. Therefore, 200 ng of Li µL-1 was added to all HPLC eluents. The instrumental parameters are summarized in Table 1. nanoHPLC-ESI TOF MS. An ESI-QTOFMS (Applied Biosystems QSTAR XL, Foster City, CA) instrument was used for nanoHPLC-ESI TOF MS/MS experiments. The nanoHPLC was coupled via a nanoelectrospray source (Applied Biosystems) to the mass spectrometer. The connection between the outlet capillary of the column and the nanospray needle (F360-50-15-N, New Objectives, Woburn, MA) was set with a special low-deadvolume union (Upchurch, Oak Harbor, WA). Mass spectra of sulfur and selenium-containing peptides were acquired in the range of m/z 300-2000 (pulsed mode). The needle voltage was (29) Giusti, P.; Lobinski, R.; Szpunar, J.; Schaumlo ¨ffel, D. Anal. Chem. 2006, 78, 965-971.

1800 V, and the entrance potential was 60 V. Nitrogen was used as curtain gas (1.38 bar) as well as collision gas, and the energy range was 20-90 eV. The recorded data were processed using Applied Biosystems/MDS-SCIEX Analyst QS software (Frankfurt, Germany). Reagents and Solutions. All reagents were of analytical grade and were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) unless stated otherwise. Water (18.2 MΩ cm) was obtained with a Milli-Q system (Millipore, Bedford, MA). The xenon (purity N 50) collision cell gas was from Air Liquide (Paris, France). A sulfur standard solution (1000 mg S L-1 as H2SO4 in water) was obtained from Spex CertiPrep (Metuchen, NJ). The isotopic spike solution enriched in the 34S isotope was prepared by dissolving 150 mg of elemental 34S (99.9%, Isoflex, San Francisco, CA) in 2 mL of a 30% (w/w) sodium hydroxide solution at 80 °C. In order to oxidize polysulfide to sulfate 3 mL of hydrogen peroxide solution (10% (v/v)) was added dropwise until the solution became clear. The solution was finally diluted to 50 mL with water resulting in a 34S concentration of 3 g L-1. Standard stock solutions of adrenocorticotropic hormone fragment 1-10 (90.2 mg S L-1) and fragment 4-10 (579 mg S L-1), neuropeptide Y fragment 1-24 (2.44 mg S L-1), oxytocin (534 mg S L-1), Met-Leu-Phe tripeptide (1.23 mg S L-1), methionine (100 mg S L-1), and sodium taurocholate (5.05 mg S L-1) were prepared by dissolving the corresponding compound in an aqueous solution of 0.2% (v/v) trifluoroacetic acid (TFA). If necessary, stock solutions were further diluted to the final concentration. Trypsin solution was prepared by dissolving 0.8 mg of TPCK-treated trypsin (from bovine pancreas; 13700 U mg-1 protein) in 1 mL of 0.1 mol L-1 TRIS buffer (pH 7.8). Procedures. Tryptic Digest Preparation. Human serum albumin (HSA; agarose gel electrophoresis grade, >99% purity, water content 3.3%) was digested following a protocol using tris(2carboxyethyl)phosphine hydrochloride (TCEP) as reductant in order to avoid sulfur-containing reagents. Briefly, 1.0 mg of HSA was dissolved in 0.1 mL of solution of 6 mol L-1 urea, 0.1 mol L-1 TRIS (pH 7.8). The sample was reduced by the addition of 5 µL of 0.1 mol L-1 TCEP at ambient temperature for 45 min, then alkylated with 10 µL of 0.2 mol L-1 iodoacetamide at ambient temperature for 60 min. An excess of iodoacetamide was avoided in order to minimize side reactions with methionine residues. Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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Afterward, 0.8 mL of water was added and tryptic digestion was performed by adding 20 µL of trypsine solution. The reaction was conducted for 16 h at 37 °C and then stopped by the addition of 10 µL of glacial acetic acid. The solution was filtered through a 10 kDa cutoff centrifuge filter (Ultrafree MC; Millipore, Bedford, MA) and concentrated by lyophilization. Before analysis, the lyophilizate was redissolved in 93.3 µL of water. Salt-induced protein (SIP 18) was isolated from selenium-rich yeast and digested as described elsewhere.30 The protocol was modified by replacing dithiothreitol by the sulfur-free reductant TCEP. The solution was filtered through a 10 kDa cutoff centrifuge filter (Ultrafree MC) and concentrated by lyophilization. Before analysis, the lyophilizate was redissolved in 76.4 µL of water. Chromatographic Conditions. The mobile phases A1 and B1 were 0.2% TFA in water and acetonitrile, respectively. A2 and B2 were the same eluents spiked with 10 µg mL-1 of the isotopically labeled 34S standard solution. When employing an on-line preconcentration step a 1 µL aliquot of the sample was injected and loaded on the preconcentration cartridge at 5 µL min-1 for 2 min. The eluents of the micropump for sample loading were the same solutions A1 and A2. Sulfur-containing peptides were separated on the nanoHPLC column by a linear gradient from 2% to 95% B in 25 min. The SIP18 protein digest was analyzed using a stepwise gradient: 0-10 min 2-12% B linear, 10-22 min 12% B isocratic, 2229 min 12-22% linear, 29-34 min 22-50% linear, 34-39 min 5095 linear, 39-42 min 95% isocratic. Total Sulfur Analysis in Protein Tryptic Digests. In order to determine the total sulfur content a 20 µL aliquot of the HSA tryptic digest solution was spiked with 3.75 µg of 34S and analyzed by nanovolume flow injection-ICPMS (nFI-ICPMS; injection volume, 5 µL; carrier, 0.25% (v/v) TFA in water at 6 µL min-1; three replicates) as described elsewhere.17 Quantification was made in the peak area mode by isotope dilution analysis on the basis of the 32S/34Se isotope ratio. The mass bias correction was performed by assuming the exponential model. Because only two sulfur isotopes, 32S and 34S, could be measured with sufficient sensitivity and as the natural isotopic abundances of sulfur isotopes vary in the range of (4% depending on their geological origin,31 titanium was used to determine the mass bias factor. Titanium, having five stable isotopes, could be measured interference-free with sufficient sensitivity. For this purpose three injections of 7.5 ng of Ti (IV) ((NH4)2TiF6, Spex CertiPrep) were made using the nFI-ICPMS system. A negative mass bias factor of 6.1% per mass unit was obtained. The masses of the titanium isotopes are close to the masses of the sulfur isotopes; thus, a similar mass discrimination was presumed for the Ti and S isotopes. By the same method the sulfur content was determined in the peptide standard solutions, in the sodium taurocholate solution, and in the SIP18 tryptic digest. In the latter the total selenium content was determined also. A 47 ns dead time correction was automatically applied for all the measurements. RESULTS AND DISCUSSION Analytical Strategy. The aim of this study was accurate absolute peptide quantification via sulfur IDA using the naturally present sulfur in methionine or cysteine residues as an internal heteroatom tag. In contrast to the existing species-unspecific HPLC-ICP IDMS methods in which the isotopic spike was added after the chromatographic separation of the analytes (postcolumn 2862

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isotope dilution), the nonspecific isotopically labeled sulfur spike (34S) in this work was directly added to the eluents used in HPLC (precolumn isotope dilution). This is only possible under the condition that the spiked isotope does not react with the analytes. A schematic view of the instrumental setup is shown in Figure 1. In contrast to postcolumn IDA systems, the precolumn IDA did not require any instrumental modifications of the nanoHPLCICPMS setup. A subsequent use of molecular mass spectrometry (ESI TOF MS) for structural characterization of the peptides serves to determine the number of sulfur atoms per peptide, which is a prerequisite for peptide quantification. The critical development steps are (i) the interference-free detection of both 32S and 34S using a quadrupole ICP mass spectrometer, (ii) the optimization of the spike addition to the mobile phase in the way that interactions between the spike and the analytes are avoided, and (iii) the accurate calibration of the sulfur spike mass flow in nanoHPLC. Sulfur Detection in ICP Collision Cell MS. In order to enable interference-free sulfur detection in quadrupole ICPMS the use of a collision cell with xenon as cell gas was optimized. Under the instrumental conditions used in this study (Table 1) the collision energy resulting from Xe-O2 collisions was calculated to be 37.8 eV as described elsewhere.26 This energy is theoretically sufficient to dissociate O2+ molecule ions requiring a dissociation energy of 6.66 eV.26 However, in order to have an efficient collision rate a high xenon flow would be necessary, which would decrease too much the overall transmission of the analyte ions in the cell. Therefore, in this study a low xenon flow rate of 130-160 µL min-1 (dependent on the daily optimization) was applied in combination with a very large energy discrimination voltage of 43.6 V (difference between octopole and quadrupole bias). An efficient removal of O2+ interferences was confirmed by a sulfur isotope ratios measurement in a standard solution containing 10 mg L-1 sulfur introduced into the ICPMS by a conventional PFA nebulizer with Scott spray chamber. After mass bias correction the 33S/32S and 34S/32S ratios were 0.00763 and 0.04329, respectively, which were close to the theoretical values (0.00788 and 0.04422). The detection limit for the 32S isotope was calculated to be 4 µg L-1 (3σ criterion). Sulfur Detection in nanoHPLC-ICP Collision Cell MS. The analytical performance of nanoHPLC-ICPMS for the detection of transient sulfur signals was investigated in terms of linearity, sample injection reproducibility, and detection limits. Analytical Performance of nanoHPLC-ICPMS. The linearity of the response was investigated by injection of methionine in the range between 2.5 and 265 mg L-1 (as S) under weak retention conditions (isocratic elution at 30% B). The regression coefficient (r2) of the calibration graph was 0.9999. The precision of 10 subsequent injections of methionine (100 mg S L-1) was 2.3%. On the basis of the signal-to-noise ratio (3σ criterion), the detection limit for transient 32S signal nanoHPLC-ICPMS was calculated to be 1 mg L-1 (11 pg). Analytical Performance of nanoHPLC-ICPMS with On-Line Preconcentration. In order to improve the detection limit for sulfur (30) Encinar, J. R.; Ouerdane, L.; Buchmann, W.; Tortajada, J.; Lobinski, R.; Szpunar, J. Anal. Chem. 2003, 75, 3765-3774. (31) Thode, H. G. In Stable Isotopes in the Assessment of Natural and Anthropogenic Sulphur in the Environment; Krouse, H. R., Grinenko, V. A., Eds.; John Wiley & Sons Ltd: Chichester, U.K, 1991; pp 1-26.

Figure 2. Effect of the acetonitrile concentration on the sulfur signal intensity in ICPMS. Intensities were normalized to those measured in pure aqueous solution.

a sample preconcentration step on a C18 reversed-phase cartridge was performed enabling the injection of 1 µL of sample. This was optimized with sodium taurocholate which is more hydrophobic than methionine and, hence, retained on the cartridge. The recovery was 97.2%, which made this compound suitable as calibration standard for sulfur in nanoHPLC-ICPMS. The precision of sample injection of was 3.5%, which was slightly higher than in nanoHPLC without preconcentration demonstrating the contribution of the preconcentration step to the measurement uncertainty. The detection limit for 32S was calculated to be 45 µg L-1 (45 pg) on the basis of the signal-to-noise ratio (3σ criterion), which was, as a result of the preconcentration step, a distinct improvement of the concentration sulfur detection limit by a factor of 22. Due to peak broadening, however, this was lower than the preconcentration factor of 90. Effect of Acetonitrile on the Intensity of the Sulfur Signal. The influence of acetonitrile typically used for gradient elution in reversed-phase HPLC on the sulfur signal intensity in ICPMS was investigated. In contrast to selenium,29 in this study the sulfur intensity decreased linearly (r2 ) 0.9921) with increasing acetonitrile concentration (0-90%) and was at background level at 100% acetonitrile (Figure 2). A similar effect was described by Wind et al. and explained by the sulfur oxide formation in the plasma.32 However, this intensity drift was compensated in our work by the isotopically labeled sulfur spike as internal standard. Precolumn Sulfur Isotope Dilution Analysis in NanoHPLC-ICP Collision Cell MS. The continuous addition of an isotopically labeled element in HPLC-ICPMS enables the accurate quantification of element species by on-line isotope dilution analysis (IDA).14 An important prerequisite in species-unspecific IDA is to avoid interactions such as isotope exchanges between analyte molecules and spike before a chromatographic separation.15 Therefore, the usual instrumental setup is a spike addition after the separation step (postcolumn).33 However, if no spike(32) Wind, M.; Eisenmenger, A.; Lehmann, W. D. J. Anal. At. Spectrom. 2002, 17, 21-26. (33) Rottmann, L.; Heumann, K. G. Fresenius’ J. Anal. Chem. 1994, 350, 221227.

analyte interactions occur a precolumn spike addition should be possible. Furthermore, analyte and the isotopically labeled spike have to be completely equilibrated prior to isotope measurement in ICPMS. Strictly speaking, in species-unspecific IDA complete equilibration between the isotopes of the regarded element in the analyte molecule and the spiked isotope is not achieved before but directly in the plasma, where all compounds are broken down into atoms, irrespective of their chemical forms. Thus, the point of spike addition (pre- or postcolumn) is irrelevant for isotope equilibration and isotope ratio measurement, in case no spikeanalyte interactions occur. Instrumental Setup. A clear advantage is the simplicity of the setup. For precolumn sulfur IDA the nanoHPLC-ICPMS system with on-line preconcentration was used without any instrumental modifications (Figure 1). Two solvent channels of the nanopump and one solvent channel of the micropump were used for eluents spiked with the sulfur isotope 34S; the other channels contained the same eluents without isotopic spike. Thus, the system could be easily switched from the conventional mode to the IDA mode. A flow controller on the nanoHPLC pump was indispensable to maintain a stable eluent flow of 300 ( 5 nL min-1 and thus a stable mass flow of the isotopically labeled 34S spike. Analyte-Spike Interactions. Because sulfur is covalently incorporated in methionine and cysteine an isotope exchange between the 34S spike (sulfate) and 32S in the amino acids seems highly unlikely. Nevertheless, it was verified by adding an excess of 34S (1.9 mg L-1) to a solution of standard peptides containing methionine and cysteine (200-350 µg S L-1). The adrenocorticotropic hormone fragments 1-10 and 4-10, the neuropeptide Y fragment 1-24 (all three peptides contain methionine), and oxytocin (containing cysteine) were analyzed by nanoHPLCICPMS before and after 34S addition. The excess of 34S, which was present at the eluent pH 2.8 as two inorganic species 34SO42and H34SO4-, was not retained on the preconcentration cartridge and thus washed out before injection on the separation column. The 34S/32S ratio measured on-line in the separated peptide did Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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Figure 3. Interaction of the 34SO42- spike with peptides containing basic amino acids. (a) Adduct formation of 34SO42- with adrenocorticotropic hormone fragment 4-10 (MEHFRWG). (b) Adduct formation of 34SO42- with tryptic peptides in HSA digest; peak assignments are given in Table 2. No adduct formation with adrenocorticotropic hormone fragment 4-10 (c) and HSA tryptic peptides (d) after increasing the TFA concentration to 0.2% and decreasing the 34S concentration to 10 mg L-1.

not change, which proved that neither isotope exchange nor substitutions or additions at the peptide side chain functional groups with the isotopically labeled spike occurred. However, when 34S spiked eluents were used for peptide separation additional peaks were observed for the two adrenocorticotropic hormone fragments 1-10 and 4-10 in the 34S chromatogram (Figure 3a). Both these peptides contain the two amino acids histidine and arginine which have positively charged basic functional groups in their side chain. Under the initial experimental conditions where the eluents contained 20 mg of 34S L-1 and 0.075% TFA (pH 2.8 in the aqueous eluent), 34SO 24 anions could form adducts with the basic functional groups of arginine and histidine in competition with TFA as the ion-pairing reagent. The same effect was observed when a tryptic digest of human serum albumin was analyzed under the same conditions showing several peaks between 16 and 21 min in the 34S trace (peak nos. 1-9 in Figure 3b). A subsequent ESI MS measurement revealed that all peptides forming sulfate adducts contained at least one but normally two or three basic amino acids, such as arginine, lysine, or histidine (Table 2). In order to avoid these undesirable adduct formations, the spike concentration in the eluents was decreased to 10 mg of 34S L-1 and the TFA concentration was increased to 0.2% resulting in a pH of 2.1 in the aqueous eluent. It was demonstrated that under these conditions no adduct formation occurred, neither with the standard peptides (Figure 3c) nor with the tryptic peptides from human serum albumin (Figure 3d). This can be explained by the fact that at pH 2.1 the 34SO 2-/H34SO - equilibrium is shifted to the H34SO - anions. 4 4 4 Furthermore, a higher TFA and lower spike concentration favored 2864 Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

Table 2. Assignment of Peaks in Figure 3ba peak no.

retention time min-1

1 2 3 4

16.7 17.1 17.6 18.0

5

18.4

6 7 8 9

18.6 19.1 19.2 20.1

peptide amino acid

sequenceb

ECCEKPLLEK VHTECCHGDLLECADDR VHTECCHGDLLECADDRADLAK KVPQVSTPTLVEVSR KYLYEIAR KQTALVELVK QTALVELVKHKPK SLHTLFGDK RPCFSALEVDETYVPK QNCELFEQLGEYK LVRPEVDVMCTAFHDNEETFLK

a Identified peptides in HSA 34SO 2-. b Basic amino acids: R 4

position in the HSA amino acid sequencec 277-286 241-257 241-262 mc 414-428 mc 137-144 mc 525-534 mc 526-538 mc 65-73 485-500 390-402 115-136

tryptic digest forming adducts with arginine, K lysine, H histidine. c mc:

miscleavage.

an ion pairing with the trifluoracetate ions instead of with the hydrogen sulfate ions. In conclusion, these results demonstrated that under optimized experimental conditions no analyte-spike interactions occurred. Thus, the prerequisite for precolumn sulfur isotope dilution analysis was fulfilled. Isotope Dilution Calculations. Isotope dilution analysis was performed in two steps: (i) calibration of the spike mass flow MFspike by reversed isotope dilution technique using a sulfur standard with natural isotopic abundances, and (ii) calculation of the sample mass flow MFsample and integration of the peaks in the mass flow chromatogram.

Figure 4. Sulfur mass flow chromatogram obtained from HSA tryptic digest by the precolumn isotope dilution technique. Peak assignments and quantitative data are given in Table 3. Table 3. Assignment of Peaks in Figure 4a peak no.

sulfur mass, pg

1 2 3 4 5 6

2427.2 1868.6 4382.0 1035.2 1397.1 5615.1

7

4929.2

8 9 10

2259.8 2571.8 3435.1

11 12 13 14 15 16 17 18 19 20 21 22 23 24

2150.3 1254.7 1305.1 999.6 772.0 1770.7 1126.0 1765.5 1737.6 1093.4 1436.1 2364.1 665.8 2134.8

peptide amino acid sequenceb

position in the HSA amino acid sequencec

peptide mass, ng

TCVADESAENCDK ETCFAEEGKK CCAAADPHECYAK CASLQKFGER ADDKETCFAEEGK AAFTECCQAADK YICENQDSISSK CCTESLVNR ETYGEMADCCAK QEPERNECFLQHK ECCEKPLLEK NECFLQHK LKECCEKPLLEK VHTECCHGDLLECADDR NECFLQHKDDNPNLPR AACLLPK LCTVATLR VHTECCHGDLLECADDRADLAK RPCFSALEVDETYVPK QNCELFEQLGEYK LVRPEVDVMCTAFHDNEETFLKK SLHTLFGDKLCTVATLR LVRPEVDVMCTAFHDNEETFLK EFNAETFTFHADICTLSEK AVMDDFAAFVEK SHCIAEVENDEMPADLPSLA ADFVESK TYETTLEKCCAAADPHECYAK MPCAEDYLSVVLNQLCVLHEK ALVLIAFAQYLQQCPFEDHV K

52-64 565-574 mc 360-372 200-209 mc 561-573 mc 163-174 263-274 476-484 82-93 94-106 mc 277-286 99-106 275-286 mc 241-257 99-114 mc 175-181 74-81 241-262 mc 485-500 390-402 115-137 mc 65-81 mc 115-136 501-519 546-557 287-313 352-372 mc 446-466 21-41

52.4 66.5 62.9 36.7 62.8

116.8 47.7 42.8 75.9 29.1 27.3 19.4 102.3 56.2 74.9 101.6 44.2 98.6 98.9 30.3

a Absolute quantification of sulfur-containing peptides in 1 mL of HSA tryptic digest via sulfur quantification. b Sulfur-containing amino acids: C cysteine, M methionine. c mc: miscleavage.

The mass flow calibration performed in the first step is a prerequisite to allow absolute quantification. Therefore, different sulfur compounds such as methionine, thiourea and its derivates, glutathione, and sodium taurocholate were tested as mass flow calibration standards. Among these compounds sodium taurocholate was the most suitable as it could be accurately weighed, was water soluble and thermodynamically stable, and showed no isotope exchange or other interactions with the 34S spike.

Furthermore this compound could be retained on the preconcentration cartridge and eluted with an acetonitrile gradient showing a similar retention time (∼20 min) as the sulfur-containing peptides. In normal-bore HPLC-ICP IDMS systems using postcolumn spike addition several milliliters of the calibration standard are usually injected by an additional valve fitted after the column resulting in a flat top peak with a steady-state signal of the Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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Table 4. Assignment of Peaks in Figure 5a peak no.

sulfur mass, pg

1 1′ 2 2′ 3 3′ 4 5 ) 4′

317.6

5′

420.2 291.9 704.5 1285.2

peptide amino acid sequenceb

peptide mass, ng 9.5

MGHDQSGTK XGHDGSGTK MNAGR XNAGR TYENMK TYENXK Ac-SNMMNK Ac-SNMXNK Ac-SNXMNK Ac-SNXXNK

7.2 7.1 8.4

a Identification of sulfur- and selenium-containing peptides SIP18 tryptic digest. Absolute quantification of sulfur-containing peptides in 1 mL of digest. b Sulfur- and selenium-containing amino acids: M methionine, X selenomethionine.

the spike mass flow calibration from a transient signal of the standard is mathematically more accurate than the method described in our former work for capillary electrophoresis-ICP IDMS.27 In the latter case simply an average value of Rcal in the transient signal was applied, while this new method respects the time dependence of Rcal. However, MFspike, determined by this procedure, represented strictly only the mass flow of the spike at the retention time of the calibration standard. Due to the constant pump flow it was deduced that MFspike was also constant during the entire IDMS measurement. A typical value for the spike mass flow was found to be 80.6 ( 2.5 pg s-1. MFspike was calibrated every 5 h and was stable during a working day (11 h). In the second step the mass flow of the sample MFsample was calculated as a function of time according to eq 4:

MFsample(t) ) MFspike measured isotopes.33 In order to avoid peak broadening in nanoHPLC caused by the dead volume of the additional valve, it was not installed, and the injection of the sulfur standard solution was performed by the sample injection valve. As a consequence, the measurement of the sulfur calibration standard resulted in a transient signal, and thus, the sulfur isotope ratio Rcal changed with the retention time. Rcal(t) is the on-line measured 32S/34S isotope ratio during chromatographic elution of the isotope-diluted standard. Therefore, the mass flow of the injected sulfur standard MFstd is a function of time too because it is a function of Rcal according to the equation given elsewhere:33 34 Mstd (h32 spike - hspikeRcal(t)) MFstd(t) ) MFspike Mspike (h34 R (t) - h32 ) std cal

(1)

std

Mstd denotes the atomic mass of sulfur in the standard solution, isotope Mspike is the atomic mass of sulfur in the spike solution, hspike is the isotope abundance of the respective isotope in the spike solution, and hisotope is that of the respective isotope in the std standard solution. From the injection volume and the concentration of the standard the injected sulfur mass mstd was calculated. It corresponds as well to the integral of the mass flow of the sulfur standard MFstd within the integration limits t1 and t2 of the transient signal:

mstd )

∫ MF t2

t1

std(t)

dt

(2)

By combination of eqs 1 and 2 a new equation was derived for the calculation of the mass flow of the spike MFspike:

Mspike MFspike ) mstd Mstd

∫

t2

t1

1 (h32 h34 spike spikeRcal(t)) 32 (h34 stdRcal(t) - hstd)

(3) dt

The value of the integral in eq 3 was obtained numerically by plotting this function against the retention time and integrating it within the limits t1 and t2 of the transient signal. This method for 2866

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34 Msample (h32 spike - hspikeRsample(t)) 32 Mspike (h34 R sample sample(t) - hsample) (4)

isotope Msample is the atomic mass of sulfur, and hsample is the isotope abundance of the respective isotope in the sample. Rsample(t) is the on-line measured isotope ratio during chromatographic separation of the isotope-diluted sample. The function MFsample(t) represents a mass flow chromatogram in which the integration of the peak area gives directly the mass of sulfur in an eluting sulfur-containing compound. In both steps the relevant isotope abundances were determined by ICPMS before the chromatographic run; thus, possible background values of the isotopes were taken into account. Mass bias correction is not required in on-line isotope dilution since the different isotopic terms in eqs 1, 3, and 4, influenced by mass discrimination effects, compensate each other.34 Validation of the Developed Method. The validation of the developed precolumn nanoHPLC-ICP IDMS method for the quantification of sulfur-containing peptides was performed by analyzing a solution of a standard peptide which was not used for the analyte-spike interaction experiments discussed above. After spike mass flow calibration three successive 1 µL injections of a Met-Leu-Phe tripeptide solution containing 1.23 mg of S L-1 were made. The average mass found was 1.268 ( 0.027 ng of sulfur, which corresponds to a recovery of 103% and demonstrates the accuracy of the developed method. The precision of the quantification was 2.1%, which was better than the sample injection reproducibility in nanoHPLC discussed above. This shows that on-line IDA compensates for instrumental drifts in ICPMS resulting in a more precise measurement. On the basis of the signalto-noise ratio (3σ criterion) in the mass flow chromatogram, the detection limit for sulfur by precolumn IDA in nanoHPLC-ICPMS was calculated to be 45 µg L-1 corresponding to 1.4 pmol peptide. Quantification of Sulfur-Containing Peptides in Protein Tryptic Digests. Analysis of Human Serum Albumin (HSA). The precolumn nanoHPLC-ICP IDMS method was applied to the quantification of sulfur-containing peptides in tryptic digest of human serum albumin (HSA). A 1 µL aliquot of the HSA digest was analyzed resulting in a mass flow chromatogram (Figure 4) with 24 peaks from sulfur-containing peptides. By integration of

(34) Heumann, K. G.; Gallus, S. M.; Radlinger, G.; Vogl, J. J. Anal. At. Spectrom. 1998, 13, 1001-1008.

Figure 5. Chromatogram of tryptic digest of SIP18 isolated from selenium-rich yeast. (a) Sulfur-containing peptides; (b) selenium-containing peptides. Peak assignments and quantitative data are given in Table 4.

the peak area the sulfur mass in each peak was obtained (Table 3). In order to calculate the peptide mass on the basis of the sulfur mass, the identity of the peptides is mandatory. In a subsequent nanoHPLC-ESI TOF MS/MS analysis of the sample the amino acid sequences of 29 sulfur-containing peptides were identified (Table 3). Tryptic digestion of HSA results theoretically in 25 sulfur-containing tryptic peptides. Among the total number of 29 peptides identified, 19 correctly cleaved peptides were found. Two further peptides (peak nos. 2 and 4) were identified having a miscleavage at position 205 and 573, respectively, of the HSA amino acid sequence. The remaining eight peptides concerned further miscleavages. At 27 peptides no additional modifications such as oxidation of methionine and alkylated cysteine residues and side reactions of iodoacetamide with methionine residues were detected. Only the peptides 8293 (peak no. 7) and 446-466 (peak no. 24) were oxidized at the methionine residues to about 30% and 25%, respectively. The first one showed also a side reaction (carboxymethylation) at methion-

ine (6.3%). The majority of the peptides identified contained only cysteine residues, five peptides contained cysteine and methionine, and one peptide (peak no. 22) contained a single methionine residue. The identified peptides were assigned to the mass flow chromatogram by retention times. The retention times of the peptide peaks were remarkably stable (0.2% RSD) due to the stability of the eluent flow rate and the gradient elution. Hence, the quantification of 20 peptides was possible (Table 3) because of their complete chromatographic separation. In four peaks coelution of several sulfur-containing peptides occurred, which hampered their quantification. This indicates that one-dimensional nanoHPLC used in this study is limited to separations of not very complex peptide mixtures. The application of the developed method to highly complex samples in modern proteomics will therefore require multidimensional separation approaches. The peptides were quantified with a precision of 4.3% (three replicates). This was distinctly better than in molecular MS-based peptide Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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quantification methods such as ICAT and iTRAQ showing a precision between 9% and 15% for bovine serum albumine.9 The mass balance of the method was evaluated by integrating the whole chromatogram and comparing the result to the total sulfur concentration in the sample determined by nFI-ICP IDMS. The resulting sulfur recovery was 103% demonstrating the accuracy of the peptide quantification. Analysis of Salt-Induced Protein 18 (SIP18). The SIP18 protein was isolated from a selenium-rich yeast sample as described elsewhere.30 Parallel sulfur and selenium detection in nanoHPLCICPMS demonstrated the presence of sulfur- and seleniumcontaining peptides in the tryptic digest of SIP18 (Figure 5). The well-separated peptides between 16 and 32 min were identified by ESI-TOF MS/MS revealing the analogy between methionineand selenomethionine-containing peptides. The sulfur-containing peptides were quantified by precolumn IDA. The results are presented in Table 4. The sulfur mass balance was 102%. The analysis of the total sulfur and selenium content in the SIP18 digest yielded a molar S/Se ratio of 70:30. This confirmed data of an earlier work where the random replacement of methionine by selenomethionine in selenium-containing proteins from seleniumrich yeast was estimated to be about 30%.35 CONCLUSIONS A method based on sulfur isotope dilution analysis in nanoHPLC-ICPMS was developed and successfully applied to precise and accurate absolute quantification of cysteine- and methioninecontaining peptides. Xenon collision cell technology in ICPMS in combination with on-line preconcentration and nanoHPLC allowed sensitive, compound-independent detection of sulfur-containing peptides at the low picomole range. Contrary to the commonly (35) McSheehy, S.; Kelly, J.; Tessier, L.; Mester, Z. Analyst 2005, 130, 35-37.

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applied postcolumn IDA technique in HPLC-ICPMS, a simplified instrumental setup was achieved by the novel concept of precolumn addition of an isotopically labeled, compound-unspecific spike. It compensated for instrumental drift and enabled determination of the sulfur content of peptides by on-line IDA. In order to correlate the sulfur determination with the peptide mass the parallel identification of the peptides by nanoHPLC-ESI TOFMS measurements is mandatory. High reproducibility of chromatographic retention times lead to unambiguous peak assignment in nanoHPLC-ICPMS chromatograms. In contrast to conventional quantification methods based on synthetic peptide labeling and molecular MS, the newly developed nanoHPLC-ICP IDMS method employing sulfur as a natural tag in peptides allows their absolute quantification and is more precise and accurate. Thus, it shows potential for a novel generic approach to protein quantification. ACKNOWLEDGMENT The study was carried out in the frame of the ACI CNRS (New Analytical Methods and Sensors) program. The Aquitaine Region is acknowledged for the support of instrumentation via CPER 20.6 and 21.6. The authors thank Dr. Mihaly Dernovics for preparing the HSA and SIP18 digests. The support of Agilent Technology for the ICPMS platform is greatly acknowledged. NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on February 20, 2007, with an error in the author byline and in Figure 3. The corrected version was posted on February 26, 2007. Received for review October 4, 2006. Accepted January 12, 2007. AC061864R