Development of a Nebulizer for a Sheathless Interfacing of NanoHPLC

Yuka Takasaki , Masaki Watanabe , Hiroshi Yukawa , Akhmad Sabarudin , Kazumi Inagaki , Noritada Kaji , Yukihiro Okamoto , Manabu Tokeshi , Yoshitaka ...
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Anal. Chem. 2006, 78, 965-971

Development of a Nebulizer for a Sheathless Interfacing of NanoHPLC and ICPMS Pierre Giusti, Ryszard Lobinski,† Joanna Szpunar, and Dirk Schaumlo 1 ffel*

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

A novel nebulizer (nDS-200) working at sample uptake rates of less than 500 nL min-1 was developed for a sheathless interfacing of nanoHPLC (75-µm column i.d.) with ICPMS. It was based on a hollow fused-silica needle of which the tip (i.d. 10 µm, o.d. 20 µm) centered in a 254-µm-i.d. sapphire orifice. The nebulizer, equipped with a 3-cm3 drain-free vaporization chamber, enabled a stable introduction into an ICP of aqueous mobile phases containing up to 95% acetonitrile at eluent flow rates between 50 and 450 nL min-1. The low dead volume of the interface resulted in a peak width of 1.3 s (at halfheight) and the entirely preserved chromatographic resolution. An example application of the coupling to the analysis of a tryptic digest of a SIP18 protein containing two to nine selenomethionine residues was described. The absolute detection limit was 25 fg (80Se), which allowed the detection of low-abundant selenopeptides at the femtomole level. In contrast to electrospray MS, the ICPMS detection in nanoHPLC is unaffected by the coeluting matrix and concomitant compounds and offers an elegant method for the detection and quantification of minor heteroelement-containing species prior to or in parallel with ESI MS analysis. Miniaturizing HPLC is an important challenge to bioanalytical chemistry when an amount of not more than several micrograms of a rare or toxic sample is available or when biological processes in single cells or in subcellular entities such as organelles need to be investigated.1 NanoHPLC using 75-µm-i.d. columns with eluent flow rates between 200 and 400 nL min-1 has become a key technique for high-efficiency separations of complex peptide mixtures. The nanoHPLC-electrospray MS/MS coupling is one of the principal techniques in today’s proteomics.2-4 A serious drawback of electrospray ionization is its being strongly compound dependent and vulnerable to matrix suppression. Total ion chromatograms in nanoHPLC-ES MS are mainly composed of signals of the major easily ionizable compounds while information on low-abundant peptides is often lost. If a minor species arrives at the ionization source accompanied by an easily * To whom correspondence should be addressed. Tel.: +33-559-407760. Fax: +33-559-407781. E-mail: [email protected]. † Warsaw University of Technology. (1) Nilsson, S.; Laurell, T. Anal. Bioanal. Chem. 2004, 378, 1676-1677. (2) Shen, Y.; Smith, R. D. Electrophoresis 2002, 23, 3106-3124. (3) Cutillas, P. R. Curr. Nanosci. 2005, 1, 65-71. (4) Ishihama, Y. J. Chromatogr., A 2005, 1067, 73-83. 10.1021/ac051656j CCC: $33.50 Published on Web 12/17/2005

© 2006 American Chemical Society

ionizable major one, the ionization of the former is likely to be suppressed leading to the absence of a relevant peak in the mass spectrum.5,6 Hence, there is a need for efficient purification protocols on the picomole scale and for analytical techniques allowing monitoring their optimization and efficiency. Many biomolecules contain in their structure a heteroatom, such as, for example, sulfur, phosphorus, and selenium, and are therefore amenable to detection by inductively coupled plasma mass spectrometry (ICPMS). Its numerous advantages including element specificity, high sensitivity and response linearity within a wide dynamic range, and high tolerance to matrix make it complementary to electrospray MS.7-9 However, the loss of any structural information is the major obstacle in ICP ionization. A wider exploration of the ICPMS and ES MS/MS tandem detection in speciation analysis and heteroatom-tagged proteomics is hampered by the lack of interfaces allowing ICPMS detection in nanoHPLC in parallel to electrospray MS/MS. Indeed, whereas the coupling of normal-bore (4.6-mm i.d.) and microbore (1-mm i.d.) LC to ICPMS is easy to realize by simply connecting the column exit to a conventional nebulizer whose sample uptake rate is compatible with the eluent flow rate,10 the use of capillary columns with an i.d. of 0.3 mm or less requires specific micronebulizers working at uptake rates at the lowmicroliter level.11 The few application examples of capillary HPLC-ICPMS have included the analysis of selenoamino acids,12 and sulfur-containing,7 phosphorylated,13,14 and selenium-containing peptides.15 Recent microflow nebulizers used for a sheathless coupling of capillary HPLC to ICPMS provide an optimal nebulization at flow rates of 3-7 µL min-1. This range could be extended down (5) Stewart, I. I. Spectrochim. Acta B 1999, 54B, 1649-1695. (6) Tomlinson, A. J.; Chicz, R. M. Rapid Commun. Mass Spectrom. 2003, 17, 909-916. (7) Wind, M.; Wegener, A.; Eisenmenger, A.; Kellner, R.; Lehmann, W. D. Angew. Chem., Int. Ed. 2003, 42, 3425-3427. (8) Szpunar, J. Analyst 2005, 130, 442-465. (9) Lobinski, R.; Schaumlo ¨ffel, D.; Szpunar, J. Mass Spectrom. Rev., in press. (10) Szpunar, J.; Lobinski, R.; Prange, A. Appl. Spectrosc. 2003, 57, 102A-111A. (11) Schaumlo ¨ffel, D. Anal. Bioanal. Chem. 2004, 379, 351-354. (12) Ruiz Encinar, J.; Schaumlo ¨ffel, D.; Ogra, Y.; Lobinski, R. Anal. Chem. 2004, 76, 6635-6642. (13) Wind, M.; Edler, M.; Jakubowski, N.; Linscheid, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 29-35. (14) Wind, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 3006-3010. (15) Schaumlo ¨ffel, D.; Ruiz Encinar, J.; Lobinski, R. Anal. Chem. 2003, 75, 6837-6842.

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Figure 1. (a) Instrumental setup of the nanoHPLC-ICPMS coupling: (1) nanoflow HPLC pump; (2) nanoliter-volume injection valve; (3) nanoHPLC column; (4) nanoflow nebulizer nDS-200; (5) spray chamber; (6) ICP torch. (b) Enlargement of the zero-dead-volume connection in the nebulizer: (7) nanocolumn outlet (fused-silica capillary 20-µm i.d.); (8) zero-dead-volume connection of the nanocolumn outlet to the nebulizer needle in a through-hole (i.d. 300 µm); (9) fused-silica hollow needle. (c) Enlargement of the nebulizer tip: (9) fused-silica hollow needle; (10) sapphire orifice.

to 0.5 µL min-1, but that resulted in increasing signal instability.15,16 In a recent study, a dedicated capHPLC-ICPMS interface was tested at 0.5 µL min-1 for a nanoHPLC (100-µm i.d.) coupling, which turned out to be unsuccessful because it resulted in a distinct loss of chromatographic resolution and sensitivity.16 However, this nebulizer sample uptake rate is nevertheless still too high for a direct, sheathless coupling of nanoHPLC working at 0.2-0.4 µL min-1. The only to date nanoHPLC-ICPMS coupling was based on the use of the commercially available (CETAC, Omaha, NE) DS-5 microflow (5 µL min-1) nebulizer and the addition of a sheath flow.17 The main problem was analyte dispersion by the makeup flow, which affected negatively chromatographic resolution and detection limits. A further disadvantage was the rather complex instrumental setup including two independent HPLC pumps. The development of a first dedicated nanoflow nebulizer allowing a robust sheathless coupling of nano (75-µm i.d.) HPLC to ICPMS was the objective of this work. The interface to be developed should enable high-resolution separations and sensitive heteroatom-specific detection in sample volumes of several nanoliters only. The performance of nanoHPLC-ICPMS as a truly complementary technique to the common nanoHPLC-ES MS coupling was demonstrated by the sensitive detection of lowabundant selenopeptides at the subpicomole level in a protein tryptic digest. EXPERIMENTAL SECTION Apparatus. Nanoflow Reversed-Phase HPLC System. NanoHPLC separations were performed using a nanoflow HPLC pump (Ultimate, LC Packings, Amsterdam, The Netherlands) providing a stable flow rate of less than 500 nL min-1 demanded by nanoHPLC columns (i.d. 25-100 µm). In this work, a 75-µm-i.d. reversed-phase nanoHPLC column (C18 PepMap100, 75 µm i.d. × 15 cm, 3 µm, LC Packings) with a flow rate of 300 nL min-1 was used. Injections were made using a model CN4 nanoinjection (16) Pro¨frock, D.; Leonhard, P.; Ruck, W.; Prange, A. Anal. Bioanal. Chem. 2005, 381, 194-204. (17) Giusti, P.; Schaumlo ¨ffel, D.; Ruiz Encinar, J.; Szpunar, J. J. Anal. At. Spectrom. 2005, 20, 1124-1130.

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valve (Valco Instruments, Houston, TX) fitted with a rotor providing an internal injection volume of 11 nL. All connections from the pump to the sample injection valve, from the valve to the column, and from the column to the nebulizer were made from fused-silica capillaries (i.d. 20 µm, Polymicro Technologies, Phoenix, AZ) to keep the dispersion of the sample low. Nanoflow Nebulizer. A novel nanoflow nebulizer (nDS-200) was designed employing a hollow fused-silica capillary needle (New Objective, Woborn, MA) as nebulizer capillary.18 The small needle tip (i.d. 10 µm, o.d. 20 µm) was centered in a 254-µm-i.d. sapphire orifice allowing a stable and continuous nebulization of nanoliter flow rates of less than 500 nL min-1, which could be optimized by adjusting the position of the needle tip in the nebulizer orifice. The nebulizer was fitted with a low-dead-volume (3 cm3) spray chamber without drain. This arrangement allowed a complete introduction of nanoliter sample volumes into the plasma. NanoHPLC-ICPMS. The instrumental setup of the nanoHPLC-ICPMS coupling with enlargements of technical details of the nebulizer is shown in Figure 1. An ICPMS equipped with a collision cell (Agilent 7500ce, Yokogawa Analytical Systems, Tokyo, Japan) was used. The outlet capillary (20-µm i.d., 280-µm o.d.) of the nanoHPLC column was fitted into the nebulizer via a zero-dead-volume connection to the nebulizer needle in order to minimize the broadening of the narrow chromatographic peaks. The zero-dead-volume connection is realized in a small throughhole (i.d. 300 µm), which allowed a precise abutting of the capillaries (Figure 1b). NanoHPLC-ESI TOF MS. An ESI Q TOF MS/MS (Applied Biosystems QSTAR XL, Foster City, CA) was used for parallel nanoHPLC-ESI TOF 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 was set with a special low-dead-volume union (Upchurch, Oak Harbor, WA), with an internal webbed through-hole that minimizes breaking of fused silica at the junction point while adding only 13-nL swept volume. (18) Schaumlo¨ffel, D.; Giusti, P.; Szpunar, J.; Lobinski, R. Patent Application No. 05 05884 France, 2005.

Table 1. Instrumental Parameter of the 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 6-8 mm nickel 1.1 L min-1 4.2 mL min-1 H2 2.9 V, -170 V -18 V -16 V

The use of a conductive perfluoroelastomer ferrule (Upchurch) for 360-µm-o.d. needles allows the voltage to translate through the metallic low-dead-volume union to the flow path. Reagents, Solutions, and Materials. Analytical reagent grade chemicals purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) were used throughout unless stated otherwise. Water (18.2 MΩ cm) was obtained with a Milli-Q system (Millipore, Bedford, MA). A selenium standard solution (1000 mg of Se L-1 as H2SeO3 in 2% HNO3) was obtained from Merck Eurolab (Fontenay-sous-Bois, France). The hydrogen (purity N 55) collision cell gas was from Air Liquide (Paris, France). A tryptic digest of a high molecular weight fraction isolated from a water extract of selenized yeast was prepared as described elsewhere19 and used as example sample. Among other proteins, this fraction contained a selenium-containing salt-stress-induced SIP 18 protein (Mr 8874), in which two to nine methionine residues are replaced by selenomethionine. NanoHPLC-ICPMS Conditions. The mobile phases A and B were 0.05% trifluoroacetic acid in water and in acetonitrile, respectively. The solvents were degassed by purging with helium. Selenomethionine was eluted isocratically with 30% B for the determination of the detection limit as well as for the calibration of the system. An 11-nL aliquot of the tryptic digest solution containing ∼0.5 pmol of each selenopeptide was injected. These peptides were separated using a stepwise gradient: 0-10 min 2-50% B linear; 10-12 min 50-75% B linear; 12-14.5 min 7595% B linear. ICPMS measurement conditions (nebulizer gas flow, rf power, and lens voltage) were optimized daily for the highest intensity of the 7Li, 89Y, 140Ce, and 205Tl signals as well as for low oxide ion (CeO+/Ce+) and low doubly charged ion (Ce2+/Ce+) rate (Table 1). For this purpose and in order to monitor the nebulization and sample introduction process, solutions of Li+, Y3+, Ce3+, and Tl+ (200 µg L-1 each) were added to the mobile phases A and B. Furthermore, the continuous signal of 89Y served as internal standard to correct selenium intensities for instrumental drifts and eluent composition changes during gradient elution allowing a semiquantitative selenium analysis. The selenium isotopes 78Se and 80Se were monitored on-line by ICPMS after nanoHPLC separation in order to detect selenomethionine- and seleniumcontaining peptides. To remove Ar2+ interferences for sensitive selenium detection, the H2 flow rate in the collision cell was optimized to 4.2 mL min-1. The value for the octopole bias was set to -18 V and that for the quadrupole bias to -16 V. The (19) Ruiz Encinar, J.; Ouerdane, L.; Buchmann, W.; Tortajada, J.; Lobinski, R.; Szpunar, J. Anal. Chem. 2003, 75, 3765-3774.

optimal setting of these values was controlled by measuring the 82Se/80Se ratio. Being close to the natural isotope ratio demonstrated a complete removal of the Ar2+ interference. The nebulization process was optimized by adjusting the position of the fused-silica needle tip in the nebulizer orifice while monitoring the 7Li, 89Y, 140Ce, and 205Tl signals by ICPMS. The signal intensity, precision, and stability over time were considered. The peak shape was monitored by injection of an 11-nL sample aliquot containing 250 µg L-1 selenium (as selenomethionine) under weak retention conditions (30% B). The effect of acetonitrile on the signal intensity was studied by introducing solutions of different acetonitrile concentrations (0-100%) containing selenomethionine (1.5 mg L-1 Se) and Y3+, Ce3+, and Tl+ (200 µg L-1 each) with a Harvard apparatus model 22 syringe pump. ESI TOF MS Conditions. Mass spectra of selenium-containing peptides were acquired in the range m/z 420-2100. The needle voltage was 2150 V and the entrance potential 60 V. Nitrogen was used as curtain gas (1.38 bar). RESULTS AND DISCUSSION Performance of the Nanoflow Nebulizer and the NanoHPLC-ICPMS Interface. The performance of the nanoflow nebulizer was characterized in terms of flow rate working range, ICPMS signal intensity and precision, and formation rates of oxide and doubly charged ions. In contrast to microflow nebulizers working in the range of 0.5-11 µL min-1, which were employed for capillary HPLC15,16 or CE-ICPMS20 couplings, in this study the novel nanoflow nebulizer (nDS-200) was custom designed for liquid flow rates of less than 0.5 µL min-1 used in nanoHPLC. A key point of the construction was the design of the nebulizer tip (Figure 1c). The taper shape of the fused-silica hollow needle led to a very small wall thickness of ∼5 µm and an i.d. of ∼10 µm at the tip. The small inner diameter resulted in a high velocity of the liquid flow (∼4.2 cm s-1 at 200 nL min-1) in the tip, which is similar to that in the commercial DS-5 nebulizer operated at 5 µL min-1. Moreover, the small cross-sectional area and wall thickness of the tip facilitated the formation of small droplets, which were nebulized by the Venturi effect due to the argon expansion after the narrow orifice. Therefore, unlike the above-mentioned micronebulizers, which have flat ended nebulizer capillaries, this new design allowed the generation of a fine and stable aerosol at nanoliter flow rates. The nebulizer was fitted in a low-dead-volume single-pass spray chamber to ensure complete sample introduction and minimized peak broadening. In this spray chamber, the aerosol was immediately evaporated resulting in a gas-phase introduction of the analytes into the plasma. Uptake Rate, Sensitivity, and Precision. The nebulizer was tested for liquid sample introduction in ICPMS by introducing a solution containing Li, Y, Ce, and Tl in 30% B at flow rates between 50 and 450 nL min-1. The choice of these elements assured vast elemental mass range coverage. The signal intensity for all four elements increased linearly as function of the flow rate demonstrating the total-consumption characteristics of the interface (Figure 2 shows 89Y as example). The linearity range attained its limit at ∼2 µL min-1 as shown in the inset in Figure 2. The use of higher flow (20) Schaumlo ¨ffel, D.; Prange, A. Fresenius' J. Anal. Chem. 1999, 364, 452456.

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Figure 2. Intensity and relative standard deviation of the

89Y

signal as function of the nebulizer flow rate.

rates was restricted by the high back pressure of the fused-silica needle. It provoked a nonlinear increase in the signal intensity at flow rates above 2 µL min-1 followed by a destruction of the needle above 3.5 µL min-1. The nebulizer developed is, to our knowledge, the first one for ICP sample introduction working below 500 nL min-1. It provided a stable signal at nebulizer gas flow rates between 0.9 and 1.2 L min-1 with an intensity maximum between 1.05 and 1.15 L min-1. The relative standard deviation (RSD) of the signal intensity was obtained from steady-state transient signals of elements in the introduced sample solution at the corresponding nanoflow rate. The measurement was performed during 10 min with a short detector dwell time of 0.4 s. The measurement precision decreased with increasing flow rate and was lower than 5% at flow rates higher than 200 nL min-1 (Figure 2). Formation of Oxide Ions and Doubly Charged Ions. The formation of oxide ions and doubly charged ions in the plasma is a critical issue in ICPMS because they can lead to possible interferences in the mass spectrometer. Former studies have shown that in particular the formation of oxide ions is critically dependent on the nebulizer performance for aerosol generation.21 The reason is that aerosol droplets create cold regions in the plasma where oxide ions cannot decompose completely.22 In this study, the oxide formation rate CeO+/Ce+ was investigated as a function of the nebulizer uptake rate. It remained stable at ∼0.2% at flow rates between 50 and 450 nL min-1. This was in contrast to a previously described microflow DS-5 nebulizer that showed an increase of the oxide formation rate with the increasing liquid flow rate.15 At nanoflow rates, the nebulizer performance has virtually no influence on the oxide formation. This result suggests that the nDS-200 developed assured a complete volatilization of the liquid in the spray chamber resulting in a gas-phase sample introduction into the ICP. Former studies of micronebulizers (21) Liu, H.; Clifford, R. H.; Dolan, S. P.; Montaser, A. Spectrochim. Acta, Part B: At. Spectrosc. 1996, 51B, 27-40. (22) Tanner, S. D. J. Anal. At. Spectrom. 1993, 8, 891-897.

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combined with desolvatization systems indicate a 50-100 times decrease of the oxide formation rate in comparison to standard sample introduction systems (oxide ion formation rate ∼5% at the sample uptake rate of ∼1 mL min-1) due to gas-phase sample introduction and thus the absence of water droplets in the plasma.23,24 In the present study, the formation of doubly charged ions was stable at ∼1.5%, which was observed by measuring the Ce2+/Ce+ ratio at flow rates between 50 and 450 nL min-1 (data not shown). Effect of Acetonitrile on the Signal Intensity. The influence of acetonitrile typically used as organic modifier in reversed-phase HPLC was investigated on the ionization of Se as well as Y, Ce, and Tl in the plasma. In contrast to normal-bore LC-ICPMS, where the use of high organic solvent flow rates has a detrimental effect on the ICP stability,25,26 in capillary LC, organic solvent introduction at microliter flow rates led to an enhancement of the signal intensity.15 This effect can be explained by a charge-transfer reaction from ionized carbon in the plasma to incompletely ionized analytes.27 In this study, at the flow rate of 300 nL min-1, the signal intensities for 80Se, 89Y, 140Ce, and 205Tl increased 2-2.5 times, reaching a maximum at 60-70% acetonitrile in comparison with the measurement in pure aqueous solution (Figure 3). Note that for the first time the signal enhancement effect by carbon ions in the plasma could be observed for changes of the solvent composition at nanoliter flow rates. NanoHPLC-ICPMS Coupling. Peak Shape. The development of a dedicated nanoflow nebulizer allowed a sheathless coupling between nanoHPLC and ICPMS. To investigate the (23) Field, M. P.; Sherrell, R. M. Anal. Chem. 1998, 70, 4480-4486. (24) Fragniere, C.; Haldimann, M.; Eastgate, A.; Kraehenbuehl, U. J. Anal. At. Spectrom. 2005, 20, 626-630. (25) Ferrarello, C. N.; Ruiz Encinar, J.; Centineo, G.; Garcia Alonso, J. I.; Fernandez de la Campa, M. R.; Sanz-Medel, A. J. Anal. At. Spectrom. 2002, 17, 1024-1029. (26) Kahen, K.; Strubinger, A.; Chirinos, J. R.; Montaser, A. Spectrochim. Acta, Part B: At. Spectrosc. 2003, 58B, 397-413. (27) Larsen, E. H.; Stuerup, S. J. Anal. At. Spectrom. 1994, 9, 1099-1105.

Figure 3. Influence of the acetonitrile concentration on the signal intensity of 80Se, 89Y, 140Ce, and 205Tl in ICPMS. Intensities were normalized to those measured in pure aqueous solution.

Figure 4. Transient signals in nanoHPLC-ICPMS. Comparison of the peak shapes obtained after an injection of 250 µg L-1 Se (as selenomethionine) under weak retention conditions (isocratic elution at 30% B) in (1) sheathless nanoHPLC-ICPMS (C18 column 75 µm i.d. × 150 mm) (this work), (2) sheath flow nanoHPLC-ICPMS (C18 column 75 µm i.d. × 150 mm),17 and (3) sheathless capillary HPLC-ICPMS (C18 column 300 µm i.d. × 150 mm).15 Chromatograms were set off and normalized to the intensity maximum for the sake of the clarity of presentation. Background intensity of 80Se and the signals of two 80-fg Se injections, isocratic elution (30% B).

dispersion of the analyte in the nanoHPLC-ICPMS system, an 11-nL sample aliquot containing 250 µg L-1 Se (as selenomethionine) was injected under weak retention conditions (isocratic elution at 30% B) on a C18 nanocolumn. The peak produced had a width of 1.3 s measured at half-height and 3.7 s at the base. The peaks produced by a sheathless nanoHPLC-ICPMS coupling were therefore twice as narrow as those in nanoHPLC-ICPMS using the sheath flow interface (2.6 s at half-height)17 and more than four times in capillary HPLC-ICPMS using a sheathless interface (5.6 s at half-height).15 Figure 4a compares selenomethionine peaks produced under the same chromatographic conditions by these three different couplings. Sample Injection Reproducibility. To evaluate the reproducibility of the nanoliter-volume sample injection, six subsequent 11-nL injections of selenomethionine (100 µg Se L-1) were performed.

The precision of the peak area was 4.3%, which was similar to the precision of a steady-state signal obtained by nebulization at a 300 nL min-1 flow rate. This high reproducibility of the injection demonstrated that the contribution of the sample injection uncertainty to the overall measurement uncertainty budget was negligible. Linearity of the Response and Detection Limits. The linearity of the response in nanoHPLC-ICPMS was investigated by injection of selenomethionine amounts in the range between 50 and 1200 µg L-1 (0.55 and 13.08 pg). The regression coefficient (r2) of the calibration graph was 0.9994. The detection limit was determined by two subsequent injections of 80 fg of Se (7.3 µg L-1) in 30% (v/v) acetonitrile solution. The corresponding transient signals are shown in Figure 4b. On the basis of the signal-to-noise ratio (3σ criterion), the detection limit for 80Se was calculated to be 25 Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

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Figure 5. NanoHPLC analysis of the tryptic digest of a high molecular weight fraction isolated from an aqueous extract of selenized yeast: (a) 80Se chromatogram obtained from nanoHPLC-ICPMS for element-specific selenopeptide detection. (b) Total ion chromatogram from nanoHPLC-ES MS. (c) Extracted ion chromatograms from nanoHPLC-ES MS of five identified selenopeptides (cf. Figure 6).

fg (2.3 µg L-1). That is, to our knowledge, the lowest absolute detection limit ever reported for Se in HPLC-ICPMS. It was distinctly lower that the value reported in our earlier work for a sheath flow nanoHPLC-ICPMS coupling (40 fg)17 and a sheathless capillary HPLC-ICPMS coupling (75 fg).12 Parallel NanoHPLC-ICPMS and NanoHPLC-ESI MS for Selenopeptide Identification. The developed nanoHPLCICPMS coupling was applied to selenopeptide detection in a tryptic digest of a high molecular weight fraction isolated from an aqueous extract of selenized yeast.19 Selenium detection allowed the optimization of the elution gradient for the selenopeptide separation regardless of other compounds present. The nanoHPLC-ICPMS chromatogram shows five well-separated selenopetides between 3 and 11 min and larger fragments at 13-15 min 970

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(Figure 5a). Even under optimized conditions, a baseline separation of the latter was not possible because of the presence of a large number of very similar selenopeptides. Repeated sample injection (n ) 3) demonstrated that the retention times of the first five peptides were stable with a precision of 8% RSD. Stable retention times are mandatory for a complementary use of nanoHPLC-ICPMS and nanoHPLC-ESI Q TOF MS on-line couplings as a new approach for improved selenopeptide identification. The newly developed nanoHPLC-ICPMS has the unique feature to detect specifically and sensitively low-abundant selenopeptides in the presence of other compounds in excess where electrospray ionization fails. Indeed, the total ion chromatogram of the tryptic digest analyzed by nanoHPLC-ESI MS showed a

Figure 6. MS spectra with selenium-characteristic isotopic pattern found in nanoHPLC-ES MS at retention times of the selenopeptide peaks previously detected by nanoHPLC-ICPMS (cf. Figure 5). Comparison with the exact molecular mass (M + H+) of selenopeptides described elsewhere19 confirmed their amino acid sequence (X denotes selenomethionine). (a) Doubly charged molecule ion; (b)-(e) monocharged molecule ions, signals at (m + 16)/z correspond to oxidized forms of the molecule ions.

number of unresolved peaks corresponding to countless peptides that were superposing the selenopeptide signals (Figure 5b). The stability of the selenopeptide retention times (RSD 8%) in nanoHPLC-ICPMS allowed us to define elution zones of interest, which assisted us in finding the mass spectra of low-abundant selenopetides in nanoHPLC-ESI MS. They were recognized by the typical selenium isotopic pattern in MS. The ESI MS spectra of the five selenopeptides are shown in Figure 6. Their molecular masses correspond to selenopeptides that were previously identified as tryptic peptides of a selenium-containing salt-stress-induced SIP 18 protein (Mr 8874).19 From the molecular masses obtained from the mass spectra, extracted ion chromatograms were reconstructed for each selenopeptide (Figure 5c). The retention times of the peptide peaks matched perfectly with those of their corresponding peaks in the nanoHPLC-ICPMS chromatogram. Furthermore, the selenium signal in ICPMS, which is virtually compound-independent, enabled a semiquantitative analysis of the selenopeptides by external calibration. The amounts of the first five peptides in the chromatogram (cf. Figure 5a) were in the range of 403-714 fmol (37-65 nmol g-1) measured with a precision of ∼15% (three replicates). CONCLUSIONS The nebulizer developed is the first device to allow a stable introduction of liquids at flow rates in the nanoliter per minute range into an ICP and enables a robust sheathless coupling of

nanoHPLC and ICPMS. In this way, an eluate from nanoHPLC can be screened for the presence of heteroatom-containing species, which, on one hand, allows the identification of elution zones of interest and, on the other hand, facilitates the ES MS data interpretation or the optimization of purification protocols to acquire ES MS data at all. Especially for the detection of lowabundant peptides that contain a heteroatom, nanoHPLC-ICPMS has turned out to be a valuable complement to nanoHPLC-ES MS in proteomics research. Future work will focus on adaptation of the 2D proteomics protocols for sample introduction and exploration of the potential of nanoHPLC-ICPMS for quantitative analysis. ACKNOWLEDGMENT The work was funded by the CNRS ACI “New analytical methods and sensors” and Nuclear Toxicology national research programs. The support of the Aquitaine Region (CPER 20.6) and Agilent Technologies with the acquisition of instrumentation is acknowledged. The authors thank Dr. H. Preud’homme for his help in electrospray data acquisition and interpretation and Dr. M. Dernovics for his help in sample preparation.

Received for review September 16, 2005. Accepted November 21, 2005. AC051656J

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