- Email: [email protected]
New ways in qualitative and quantitative protein analysis: Nano chromatography coupled to element mass spectrometry
ARTICLE IN PRESS
Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22 www.elsevier.de/jtemb
THIRD INTERNATIONAL FESTEM SYMPOSIUM
New ways in qualitative and quantitative protein analysis: Nano chromatography coupled to element mass spectrometry Dirk Schaumlo¨ffel Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, CNRS UMR 5254, He´lioparc, 2, Av du Pre´sident Angot, F-64053 Pau, France Received 30 June 2007; accepted 13 September 2007
Abstract The potential of inductively coupled plasma-mass spectrometry (ICP-MS), which allows element-specific detection of heteroelements (e.g. Se and S) incorporated in protein structures, is highlighted for sensitive qualitative and quantitative protein analysis. ICP-MS coupled to separation techniques such as size exclusion chromatography and gel electrophoresis (via laser ablation) can be employed at different steps in the proteomic workflow. Special emphasis is made on the couplings of capillary and nanoHPLC to ICP-MS that required the development of dedicated interfaces. Element-specific peptide mapping by nanoHPLC–ICP-MS has turned out to be a key technique in combination with peptide sequencing via nanoHPLC–electrospray MS. This could impressively be demonstrated for the identification of selenium-containing proteins in selenium-rich yeast. Furthermore the potential of sulfur isotope dilution analysis in nanoHPLC–ICP-MS is presented as generic tool for highly accurate, absolute protein quantification. r 2007 Elsevier GmbH. All rights reserved. Keywords: Nano chromatography; Element mass spectrometry
Introduction Proteomic research focuses to date on protein detection and identification, and on studying the protein function [1]. Generally, the proteins are separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) resulting in small protein-containing spots that are enzymatically digested [2]. Then the peptide mixture is separated by capillary or nanoHPLC [3]. Electrospray (ESI) or matrix-assisted-laser-desorptionionization (MALDI) mass spectrometry (MS) has turned out to be the most important technique for peptide sequencing and thus the identification of the proteins [4]. Analysis of the protein function has to Tel.: +33 559 407760; fax: +33 559 407781.
E-mail address: [email protected]. 0946-672X/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.09.007
include the quantification of individual proteins synthesized by a cell at a given moment and under specific conditions. Current MS-based approaches for protein quantification require isotopically (2H, 13C, 15N, or 18O) labeled derivates of the analytes as internal standards [5,6]. The isotopes are introduced, for example, via a tag (ICAT, iTRAQ) [7,8] or via amino acids (SILAC) [9]. The main drawback is that the peptide ionization is compound dependent and thus affected by the coeluting peptides and other sample components. Moreover, due to the lack of isotopically labeled peptide standards in most cases only relative quantification is possible [5]. Inductively coupled plasma mass spectrometry (ICPMS) allows highly sensitive multielement (multiisotope) determination and accurate quantification by isotope dilution analysis while the ionization process is practically
ARTICLE IN PRESS D. Schaumlo¨ffel / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22
compound and matrix independent [10]. These features make ICP-MS a valuable alternative for sensitive protein detection and quantification via the element signal under the condition that the protein structure contains detectable heteroelements such as phosphorus, sulfur, selenium or iodine (heteroatom proteomics). However, these heteroelements suffer from low ionization efficiency in the plasma of about 15–30% resulting in lower detection sensitivity than for most of the metals [11]. Moreover, detection of P, S, and Se is hampered by polyatomic interferences which requires the use of either a high resolution sector field ICP-MS [11] or quadrupole instruments equipped with reaction or collision cells [12,13]. For the analysis of heteroatom-containing proteins, ICP-MS can be employed at different steps in the bottom-up proteomics workflow for element-specific detection and quantification (Fig. 1). Size exclusion chromatography (SEC)–ICP-MS is useful for a first fractionation of the sample before subsequent separation steps. Examples are the fractionation of selenium-containing proteins extracted from selenized yeast [14] and metallothioneins in rat liver [15]. SEC–ICP-MS is preferably used for metalloprotein analysis due to its gentle separation mechanisms allowing to preserve the integrity of many metal–protein complexes [16]. Laser ablation coupled to ICP-MS (LA–ICP-MS) allows element-specific analysis of protein spots in gel
electrophoresis. Impressive progress was made by this technique for the determination of the phosphorylation degree of the human tau protein [17] and for the analysis of several metalloproteins in human brain [18]. Within this strategy, couplings of capillary and nanoHPLC to ICP-MS are key techniques that enable element-specific peptide mapping and absolute peptide quantification via the element signal. For example, phosphorus-to-sulfur ratio measurements by mLC-ICPMS allowed successfully the determination of phosphorylation degrees of model proteins [19] and of entire proteomes in bacteria and eukaryotes [20]. However, capillary/nanoHPLC–ICP-MS couplings are not straightforward and require the development of dedicated coupling interfaces. The objective of this paper is to give a short introduction to the emerging field of nanoHPLC–ICPMS with special emphasis to qualitative and quantitative peptide and protein analysis via the heteroelement signal. The few applications available to date concern mainly the analysis of selenium- and sulfur-containing peptides.
Capillary and nanoHPLC–ICP-MS couplings HPLC separations in small columns of 300 mm inner diameter or less have several advantages: high separation efficiency, low solvent consumption and small
celcell l oror tissue tissue
protein e extraction x tract ion protein fractionation fractiona t ion
LA - ICPMS • element - specific spot analysis • protein quantification
19
2D2D PAGE PAGE protein separation prote in separa tio n
SEC – ICP-MS • element - specific analysis of protein fractions
try tryptic p t ic ddigestion ig e s ti on
nanonanoHPLC-ESI-MS/MS HPL C -ES I -MS/M S separation pepeptide p t e sep ara tio n sequencing peppeptide tiide e seq sequencingd uencin g
databa database s e search sear ch
nano HPLC – ICP-MS • element - specific peptide mapping • absolute quantification
protein identity
Fig. 1. The use of ICP-MS in the bottom-up proteomics workflow for the analysis of heteroatom-containing proteins.
ARTICLE IN PRESS 20
D. Schaumlo¨ffel / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22
sample volumes [21]. Typically, capillary HPLC (300 mm i.d.) works at flow rates of around 4 mL min 1 and nanoHPLC (75 mm i.d.) at 0.3 mL min 1 and less. However, these flow rates are three orders of magnitude lower than those (ca. 700–1000 mL min 1) required by conventional nebulizers used for sample introduction in ICP-MS. Recently interfaces dedicated to couple capillary or nanoHPLC with ICP MS were developed [22,23]. They consist of a total consumption micronebulizer (flow rate 0.5–7 mL min 1 for capillary HPLC) and nanonebulizer (0.05–0.5 mL min 1 for nanoHPLC), respectively, combined with a single pass low volume spray chamber. These interfaces enable the complete transport of the low flow rates to the ICP-MS under preservation of the high chromatographic resolution due to reduced dead volumes.
Identification of selenium-containing proteins assisted by nanoHPLC–ICP-MS The trace element selenium is known to be either essential or toxic for living organisms depending on the concentration and the chemical form [24]. Selenium-rich yeast, a commercial product for selenium supplementation in human nutrition, is one of the most studied samples in selenium speciation analysis. Many of these studies propose selenomethionine as the major selenium compound present [25–27]. However, enzymatic digestion or acid hydrolysis usually applied during sample preparation liberates selenomethionine from the original protein amino acid sequences. Indeed, selenomethionine was suggested to replace methionine randomly in proteins [28]. To date only a few studies are dealing with the identification of selenium-containing proteins in selenium-rich yeast [14]. An analytical strategy was proposed for the identification of water-soluble selenium-containing proteins in yeast [29]. An aqueous extract from selenium-rich yeast was fractionated by SEC while ICP-MS enabled the detection of the selenium-containing protein fraction to be isolated and digested. NanoHPLC–ICP-MS with online preconcentration was applied for selenium-specific peptide mapping allowing the definition of targets for a subsequent nanoHPLC–ESI-MS analysis of the digest. This facilitated the detection and sequencing of the selenopeptides by ESI–MS/MS. Database search allowed the identification of two selenium-containing proteins, a 18 kDa salt-induced protein (SIP18) and 12 kDa heat-shock protein (HSP12). Recently, in a subsequent work, optimization of the protein extraction followed by 2D-PAGE protein separation, in-gel digestion and parallel nanoHPLC–ICP-MS and nanoHPLC–ESI-MS analysis enabled an additional identification of 16 selenium-containing proteins in selenium-rich yeast.
Quantification of sulfur-containing peptides by nanoHPLC–ICP-IDMS Incorporated in the amino acids cysteine (Cys) and methionine (Met), the element sulfur is almost omnipresent in all natural proteomes and plays therefore a key role in quantitative proteomics [30,31]. An interesting approach for protein quantification is the direct measurement of sulfur by ICP-MS. In combination with sulfur isotope dilution analysis it offers a potentially generic way for absolute peptide and protein quantification of high accuracy. The development of this approach was made possible due to improved sensitivity and accuracy of sulfur isotope ratio measurement by ICP-MS in recent years [32]. The unique feature of ICP-MS, compound-independent ionization, enables compound-unspecific sulfur IDA using a spike that is not required to have the same chemical form as the analyte [33]. This has the main advantage that only one generic sulfur standard (i.e. one isotopically labeled sulfur spike) is necessary to quantify accurately each peptide or protein in a sample provided that they are completely separated in chromatography and that their identities are known. The latter point requires usually complementary information by molecular MS analysis. Recently pre-column sulfur isotope dilution analysis in nanoHPLC–ICP-MS was introduced where the sulfur spike was added directly in the chromatographic eluents [34]. This approach was applied to sulfur-containing peptide quantification in the tryptic digest of human serum albumin. This protein contains 35 Cys and 6 Met residues and tryptic digestion results theoretically in 25 sulfur-containing peptides. Two sulfur isotopes were measured simultaneously in ICP-MS: 32S originated from the sample and 34S from the added spike. After calibration, the online measured 32S/34S isotope ratio was mathematically converted into a mass-flow chromatogram which has the advantage that an integration of the peaks resulted directly in the absolute mass of sulfur in the corresponding peptide. Hence, accurate absolute quantification of 20 sulfur-containing peptides at the low picomol level was possible. However, sulfur detection limits (45 mg L 1) in nanoHPLC–ICP-MS are still high compared to other elements. An interesting solution might be protein labeling with element tags, containing metals that are highly sensitive in ICP-MS, which promises a new, emerging research field in future.
Concluding remarks Element MS is a valuable alternative for sensitive protein detection and quantification via heteroelements incorporated in the protein structure such as selenium
ARTICLE IN PRESS D. Schaumlo¨ffel / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22
and sulfur. Hence, ICP-MS can be applied at different steps in the proteomics workflow for sensitive heteroelement-specific detection. NanoHPLC with ICP-MS and ESI MS/MS detection are key techniques in heteroatom proteomics, e.g. for the identification of selenium-containing proteins. Moreover, sulfur isotope dilution MS has the potential to become a generic tool for absolute protein quantification.
References [1] Patterson SD, Aebersold RH. Proteomics: the first decade and beyond. Nat Genet 2003;33:311–23. [2] Garfin DE. Two-dimensional gel electrophoresis: an overview. Trends Anal Chem 2003;22:263–72. [3] Ishihama Y. Proteomic LC-MS systems using nanoscale liquid chromatography with tandem mass spectrometry. J Chromatogr A 2005;1067:73–83. [4] Domon B, Aebersold R. Review-mass spectrometry and protein analysis. Science 2006;312:212–7. [5] MacCoss MJ, Matthews DE. Quantitative MS for proteomics: teaching a new dog old tricks. Anal Chem 2005;17:294A–302A. [6] Heck AJR, Krijgsveld J. Mass spectrometry-based quantitative proteomics. Expert Rev Proteom 2004;1: 317–26. [7] Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999;17:994–9. [8] Ross PL, Huang YLN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteom 2004;3: 1154–69. [9] Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteom 2002;1:376–86. [10] Montaser A. Inductively coupled plasma mass spectrometry. New York: Wiley; 1998. [11] Wildner H. Application of inductively coupled plasma sector field mass spectrometry for the fast and sensitive determination and isotope ratio measurement of nonmetals in high-purity process chemicals. J Anal At Spectrom 1998;13:573–8. [12] Bandura DR, Baranov VI, Tanner SD. Detection of ultratrace phosphorus and sulfur by quadrupole ICPMS with dynamic reaction cell. Anal Chem 2002;74: 1497–502. [13] Mason PRD, Kaspers K, Van Bergen MJ. Determination of sulfur isotope ratios and concentrations in water samples using ICP-MS incorporating hexapole ion optics. J Anal At Spectrom 1999;14:1067–74. [14] Ruiz Encinar J, Ouerdane L, Buchmann W, Tortajada J, Lobinski R, Szpunar J. Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24] [25]
[26]
[27]
21
MALDI-TOF and electrospray Q-TOF mass spectrometry. Anal Chem 2003;75:3765–74. Polec-Pawlak K, Schaumlo¨ffel D, Szpunar J, Prange A, Lobinski R. Analysis for metal complexes with metallothionein in rat liver by capillary zone electrophoresis using ICP double-focussing sector-field isotope dilution MS and electrospray MS detection. J Anal At Spectrom 2002;17:908–12. Hann S, Koellensperger G, Obinger C, Furtmueller PG, Stingeder G. SEC–ICP-DRCMS and SEC–ICP-SFMS for determination of metal-sulfur ratios in metalloproteins. J Anal At Spectrom 2004;19:74–9. Becker JS, Boulyga SF, Becker JS, Pickhardt C, Damoc E, Przybylski M. Structural identification and quantification of protein phosphorylations after gel electrophoretic separation using Fourier transform ion cyclotron resonance mass spectrometry and laser ablation inductively coupled plasma mass spectrometry. Int J Mass Spectrom 2003;228:985–97. Becker JS, Zoriy M, Przybylski M, Becker JS. Highresolution mass spectrometric brain proteomics by MALDI–FTICR–MS combined with determination of P, S, Cu, Zn and Fe by LA–ICP-MS. Int J Mass Spectrom 2007;261:68–73. Wind M, Wesch H, Lehmann WD. Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry. Anal Chem 2001;73:3006–10. Krueger R, Kuebler D, Pallisse R, Burkovski A, Lehmann WD. Protein and proteome phosphorylation stoichiometry analysis by element mass spectrometry. Anal Chem 2006;78:1987–94. Schaumlo¨ffel D. Capillary liquid separation techniques with ICP MS detection. Anal Bioanal Chem 2004;379:351–4. Schaumlo¨ffel D, Ruiz Encinar J, Lobinski R. Development of a sheathless interface between reversed-phase capillary HPLC and ICP MS via a micro-flow total consumption nebulizer for selenopeptide mapping. Anal. Chem. 2003;75:6837–42. Giusti P, Lobinski R, Szpunar J, Schaumlo¨ffel D. Development of a nebulizer for a sheathless interfacing of nanoHPLC and ICPMS. Anal Chem 2006;78:965–71. Alaejos MS, Romero CD. Analysis of selenium in body fluids—a review. Chem Rev 1995;95:227–57. Block E, Glass RS, Jacobsen NE, Johnson S, Kahakachchi C, Kaminski R, et al. Identification and synthesis of a novel selenium-sulfur amino acid found in selenized yeast: rapid indirect detection NMR methods for characterizing low-level organo selenium compounds in complex matrices. J Agric Food Chem 2004;52:3761–71. Dumont E, De Cremer K, Van Hulle M, Chery CC, Vanhaecke F, Cornelis R. Identification of the major selenium compound, Se-methionine, in three yeast (Saccharomyces cerevisiae) dietary supplements by on-line narrowbore liquid chromatography–electrospray tandem mass spectrometry. J Chromatogr A 2005;1071:191–6. Diaz Huerta V, Hinojosa Reyes L, Marchante-Gayon JM, Fernandez Sanchez ML, Sanz-Medel A. Total determination and quantitative speciation analysis of selenium in yeast and wheat flour by isotope dilution analysis ICP-MS. J Anal At Spectrom 2003:1243–7.
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[28] Whanger PD. Selenocompounds in plants and animals and their biological significance. J Am Commun Nutr 2002;21:223–32. [29] Giusti P, Schaumlo¨ffel D, Preud’homme H, Szpunar J, Lobinski R. Selenopeptide mapping in a selenium-yeast protein digest by parallel nanoHPLC–ICP-MS and nanoHPLC–electrospray-MS/MS after on-line preconcentration. J Anal At Spectrom 2006;21:26–32. [30] Wind M, Wegener A, Eisenmenger A, Kellner R, Lehmann WD. Sulfur as the key element for quantitative protein analysis by capillary liquid chromatography coupled to element mass spectrometry. Angew Chem 2003;42:3425–7. [31] Rappel C, Schaumlo¨ffel D. The role of sulfur and sulfur isotope dilution analysis in quantitative protein
analysis. Anal Bioanal Chem 2008; doi:10.1007/s00216007-1607-2. [32] Prohaska T, Latkoczy C, Stingeder G. Precise sulfur isotope ratio measurements in trace concentration of sulfur by inductively coupled plasma double focusing sector field mass spectrometry. J Anal At Spectrom 1999; 14:1501–4. [33] Heumann KG, Rottmann L, Vogl J. Elemental speciation with liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry. J Anal At Spectrom 1994;9:1351–5. [34] Schaumlo¨ffel D, Giusti P, Preud’Homme H, Szpunar J, Lobinski R. Precolumn isotope dilution analysis in nanoHPLC–ICPMS for absolute quantification of sulfur-containing peptides. Anal Chem 2007;79:2859–68.
Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22 www.elsevier.de/jtemb
THIRD INTERNATIONAL FESTEM SYMPOSIUM
New ways in qualitative and quantitative protein analysis: Nano chromatography coupled to element mass spectrometry Dirk Schaumlo¨ffel Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, CNRS UMR 5254, He´lioparc, 2, Av du Pre´sident Angot, F-64053 Pau, France Received 30 June 2007; accepted 13 September 2007
Abstract The potential of inductively coupled plasma-mass spectrometry (ICP-MS), which allows element-specific detection of heteroelements (e.g. Se and S) incorporated in protein structures, is highlighted for sensitive qualitative and quantitative protein analysis. ICP-MS coupled to separation techniques such as size exclusion chromatography and gel electrophoresis (via laser ablation) can be employed at different steps in the proteomic workflow. Special emphasis is made on the couplings of capillary and nanoHPLC to ICP-MS that required the development of dedicated interfaces. Element-specific peptide mapping by nanoHPLC–ICP-MS has turned out to be a key technique in combination with peptide sequencing via nanoHPLC–electrospray MS. This could impressively be demonstrated for the identification of selenium-containing proteins in selenium-rich yeast. Furthermore the potential of sulfur isotope dilution analysis in nanoHPLC–ICP-MS is presented as generic tool for highly accurate, absolute protein quantification. r 2007 Elsevier GmbH. All rights reserved. Keywords: Nano chromatography; Element mass spectrometry
Introduction Proteomic research focuses to date on protein detection and identification, and on studying the protein function [1]. Generally, the proteins are separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) resulting in small protein-containing spots that are enzymatically digested [2]. Then the peptide mixture is separated by capillary or nanoHPLC [3]. Electrospray (ESI) or matrix-assisted-laser-desorptionionization (MALDI) mass spectrometry (MS) has turned out to be the most important technique for peptide sequencing and thus the identification of the proteins [4]. Analysis of the protein function has to Tel.: +33 559 407760; fax: +33 559 407781.
E-mail address: [email protected]. 0946-672X/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.09.007
include the quantification of individual proteins synthesized by a cell at a given moment and under specific conditions. Current MS-based approaches for protein quantification require isotopically (2H, 13C, 15N, or 18O) labeled derivates of the analytes as internal standards [5,6]. The isotopes are introduced, for example, via a tag (ICAT, iTRAQ) [7,8] or via amino acids (SILAC) [9]. The main drawback is that the peptide ionization is compound dependent and thus affected by the coeluting peptides and other sample components. Moreover, due to the lack of isotopically labeled peptide standards in most cases only relative quantification is possible [5]. Inductively coupled plasma mass spectrometry (ICPMS) allows highly sensitive multielement (multiisotope) determination and accurate quantification by isotope dilution analysis while the ionization process is practically
ARTICLE IN PRESS D. Schaumlo¨ffel / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22
compound and matrix independent [10]. These features make ICP-MS a valuable alternative for sensitive protein detection and quantification via the element signal under the condition that the protein structure contains detectable heteroelements such as phosphorus, sulfur, selenium or iodine (heteroatom proteomics). However, these heteroelements suffer from low ionization efficiency in the plasma of about 15–30% resulting in lower detection sensitivity than for most of the metals [11]. Moreover, detection of P, S, and Se is hampered by polyatomic interferences which requires the use of either a high resolution sector field ICP-MS [11] or quadrupole instruments equipped with reaction or collision cells [12,13]. For the analysis of heteroatom-containing proteins, ICP-MS can be employed at different steps in the bottom-up proteomics workflow for element-specific detection and quantification (Fig. 1). Size exclusion chromatography (SEC)–ICP-MS is useful for a first fractionation of the sample before subsequent separation steps. Examples are the fractionation of selenium-containing proteins extracted from selenized yeast [14] and metallothioneins in rat liver [15]. SEC–ICP-MS is preferably used for metalloprotein analysis due to its gentle separation mechanisms allowing to preserve the integrity of many metal–protein complexes [16]. Laser ablation coupled to ICP-MS (LA–ICP-MS) allows element-specific analysis of protein spots in gel
electrophoresis. Impressive progress was made by this technique for the determination of the phosphorylation degree of the human tau protein [17] and for the analysis of several metalloproteins in human brain [18]. Within this strategy, couplings of capillary and nanoHPLC to ICP-MS are key techniques that enable element-specific peptide mapping and absolute peptide quantification via the element signal. For example, phosphorus-to-sulfur ratio measurements by mLC-ICPMS allowed successfully the determination of phosphorylation degrees of model proteins [19] and of entire proteomes in bacteria and eukaryotes [20]. However, capillary/nanoHPLC–ICP-MS couplings are not straightforward and require the development of dedicated coupling interfaces. The objective of this paper is to give a short introduction to the emerging field of nanoHPLC–ICPMS with special emphasis to qualitative and quantitative peptide and protein analysis via the heteroelement signal. The few applications available to date concern mainly the analysis of selenium- and sulfur-containing peptides.
Capillary and nanoHPLC–ICP-MS couplings HPLC separations in small columns of 300 mm inner diameter or less have several advantages: high separation efficiency, low solvent consumption and small
celcell l oror tissue tissue
protein e extraction x tract ion protein fractionation fractiona t ion
LA - ICPMS • element - specific spot analysis • protein quantification
19
2D2D PAGE PAGE protein separation prote in separa tio n
SEC – ICP-MS • element - specific analysis of protein fractions
try tryptic p t ic ddigestion ig e s ti on
nanonanoHPLC-ESI-MS/MS HPL C -ES I -MS/M S separation pepeptide p t e sep ara tio n sequencing peppeptide tiide e seq sequencingd uencin g
databa database s e search sear ch
nano HPLC – ICP-MS • element - specific peptide mapping • absolute quantification
protein identity
Fig. 1. The use of ICP-MS in the bottom-up proteomics workflow for the analysis of heteroatom-containing proteins.
ARTICLE IN PRESS 20
D. Schaumlo¨ffel / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22
sample volumes [21]. Typically, capillary HPLC (300 mm i.d.) works at flow rates of around 4 mL min 1 and nanoHPLC (75 mm i.d.) at 0.3 mL min 1 and less. However, these flow rates are three orders of magnitude lower than those (ca. 700–1000 mL min 1) required by conventional nebulizers used for sample introduction in ICP-MS. Recently interfaces dedicated to couple capillary or nanoHPLC with ICP MS were developed [22,23]. They consist of a total consumption micronebulizer (flow rate 0.5–7 mL min 1 for capillary HPLC) and nanonebulizer (0.05–0.5 mL min 1 for nanoHPLC), respectively, combined with a single pass low volume spray chamber. These interfaces enable the complete transport of the low flow rates to the ICP-MS under preservation of the high chromatographic resolution due to reduced dead volumes.
Identification of selenium-containing proteins assisted by nanoHPLC–ICP-MS The trace element selenium is known to be either essential or toxic for living organisms depending on the concentration and the chemical form [24]. Selenium-rich yeast, a commercial product for selenium supplementation in human nutrition, is one of the most studied samples in selenium speciation analysis. Many of these studies propose selenomethionine as the major selenium compound present [25–27]. However, enzymatic digestion or acid hydrolysis usually applied during sample preparation liberates selenomethionine from the original protein amino acid sequences. Indeed, selenomethionine was suggested to replace methionine randomly in proteins [28]. To date only a few studies are dealing with the identification of selenium-containing proteins in selenium-rich yeast [14]. An analytical strategy was proposed for the identification of water-soluble selenium-containing proteins in yeast [29]. An aqueous extract from selenium-rich yeast was fractionated by SEC while ICP-MS enabled the detection of the selenium-containing protein fraction to be isolated and digested. NanoHPLC–ICP-MS with online preconcentration was applied for selenium-specific peptide mapping allowing the definition of targets for a subsequent nanoHPLC–ESI-MS analysis of the digest. This facilitated the detection and sequencing of the selenopeptides by ESI–MS/MS. Database search allowed the identification of two selenium-containing proteins, a 18 kDa salt-induced protein (SIP18) and 12 kDa heat-shock protein (HSP12). Recently, in a subsequent work, optimization of the protein extraction followed by 2D-PAGE protein separation, in-gel digestion and parallel nanoHPLC–ICP-MS and nanoHPLC–ESI-MS analysis enabled an additional identification of 16 selenium-containing proteins in selenium-rich yeast.
Quantification of sulfur-containing peptides by nanoHPLC–ICP-IDMS Incorporated in the amino acids cysteine (Cys) and methionine (Met), the element sulfur is almost omnipresent in all natural proteomes and plays therefore a key role in quantitative proteomics [30,31]. An interesting approach for protein quantification is the direct measurement of sulfur by ICP-MS. In combination with sulfur isotope dilution analysis it offers a potentially generic way for absolute peptide and protein quantification of high accuracy. The development of this approach was made possible due to improved sensitivity and accuracy of sulfur isotope ratio measurement by ICP-MS in recent years [32]. The unique feature of ICP-MS, compound-independent ionization, enables compound-unspecific sulfur IDA using a spike that is not required to have the same chemical form as the analyte [33]. This has the main advantage that only one generic sulfur standard (i.e. one isotopically labeled sulfur spike) is necessary to quantify accurately each peptide or protein in a sample provided that they are completely separated in chromatography and that their identities are known. The latter point requires usually complementary information by molecular MS analysis. Recently pre-column sulfur isotope dilution analysis in nanoHPLC–ICP-MS was introduced where the sulfur spike was added directly in the chromatographic eluents [34]. This approach was applied to sulfur-containing peptide quantification in the tryptic digest of human serum albumin. This protein contains 35 Cys and 6 Met residues and tryptic digestion results theoretically in 25 sulfur-containing peptides. Two sulfur isotopes were measured simultaneously in ICP-MS: 32S originated from the sample and 34S from the added spike. After calibration, the online measured 32S/34S isotope ratio was mathematically converted into a mass-flow chromatogram which has the advantage that an integration of the peaks resulted directly in the absolute mass of sulfur in the corresponding peptide. Hence, accurate absolute quantification of 20 sulfur-containing peptides at the low picomol level was possible. However, sulfur detection limits (45 mg L 1) in nanoHPLC–ICP-MS are still high compared to other elements. An interesting solution might be protein labeling with element tags, containing metals that are highly sensitive in ICP-MS, which promises a new, emerging research field in future.
Concluding remarks Element MS is a valuable alternative for sensitive protein detection and quantification via heteroelements incorporated in the protein structure such as selenium
ARTICLE IN PRESS D. Schaumlo¨ffel / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 18–22
and sulfur. Hence, ICP-MS can be applied at different steps in the proteomics workflow for sensitive heteroelement-specific detection. NanoHPLC with ICP-MS and ESI MS/MS detection are key techniques in heteroatom proteomics, e.g. for the identification of selenium-containing proteins. Moreover, sulfur isotope dilution MS has the potential to become a generic tool for absolute protein quantification.
References [1] Patterson SD, Aebersold RH. Proteomics: the first decade and beyond. Nat Genet 2003;33:311–23. [2] Garfin DE. Two-dimensional gel electrophoresis: an overview. Trends Anal Chem 2003;22:263–72. [3] Ishihama Y. Proteomic LC-MS systems using nanoscale liquid chromatography with tandem mass spectrometry. J Chromatogr A 2005;1067:73–83. [4] Domon B, Aebersold R. Review-mass spectrometry and protein analysis. Science 2006;312:212–7. [5] MacCoss MJ, Matthews DE. Quantitative MS for proteomics: teaching a new dog old tricks. Anal Chem 2005;17:294A–302A. [6] Heck AJR, Krijgsveld J. Mass spectrometry-based quantitative proteomics. Expert Rev Proteom 2004;1: 317–26. [7] Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999;17:994–9. [8] Ross PL, Huang YLN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteom 2004;3: 1154–69. [9] Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteom 2002;1:376–86. [10] Montaser A. Inductively coupled plasma mass spectrometry. New York: Wiley; 1998. [11] Wildner H. Application of inductively coupled plasma sector field mass spectrometry for the fast and sensitive determination and isotope ratio measurement of nonmetals in high-purity process chemicals. J Anal At Spectrom 1998;13:573–8. [12] Bandura DR, Baranov VI, Tanner SD. Detection of ultratrace phosphorus and sulfur by quadrupole ICPMS with dynamic reaction cell. Anal Chem 2002;74: 1497–502. [13] Mason PRD, Kaspers K, Van Bergen MJ. Determination of sulfur isotope ratios and concentrations in water samples using ICP-MS incorporating hexapole ion optics. J Anal At Spectrom 1999;14:1067–74. [14] Ruiz Encinar J, Ouerdane L, Buchmann W, Tortajada J, Lobinski R, Szpunar J. Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24] [25]
[26]
[27]
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MALDI-TOF and electrospray Q-TOF mass spectrometry. Anal Chem 2003;75:3765–74. Polec-Pawlak K, Schaumlo¨ffel D, Szpunar J, Prange A, Lobinski R. Analysis for metal complexes with metallothionein in rat liver by capillary zone electrophoresis using ICP double-focussing sector-field isotope dilution MS and electrospray MS detection. J Anal At Spectrom 2002;17:908–12. Hann S, Koellensperger G, Obinger C, Furtmueller PG, Stingeder G. SEC–ICP-DRCMS and SEC–ICP-SFMS for determination of metal-sulfur ratios in metalloproteins. J Anal At Spectrom 2004;19:74–9. Becker JS, Boulyga SF, Becker JS, Pickhardt C, Damoc E, Przybylski M. Structural identification and quantification of protein phosphorylations after gel electrophoretic separation using Fourier transform ion cyclotron resonance mass spectrometry and laser ablation inductively coupled plasma mass spectrometry. Int J Mass Spectrom 2003;228:985–97. Becker JS, Zoriy M, Przybylski M, Becker JS. Highresolution mass spectrometric brain proteomics by MALDI–FTICR–MS combined with determination of P, S, Cu, Zn and Fe by LA–ICP-MS. Int J Mass Spectrom 2007;261:68–73. Wind M, Wesch H, Lehmann WD. Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry. Anal Chem 2001;73:3006–10. Krueger R, Kuebler D, Pallisse R, Burkovski A, Lehmann WD. Protein and proteome phosphorylation stoichiometry analysis by element mass spectrometry. Anal Chem 2006;78:1987–94. Schaumlo¨ffel D. Capillary liquid separation techniques with ICP MS detection. Anal Bioanal Chem 2004;379:351–4. Schaumlo¨ffel D, Ruiz Encinar J, Lobinski R. Development of a sheathless interface between reversed-phase capillary HPLC and ICP MS via a micro-flow total consumption nebulizer for selenopeptide mapping. Anal. Chem. 2003;75:6837–42. Giusti P, Lobinski R, Szpunar J, Schaumlo¨ffel D. Development of a nebulizer for a sheathless interfacing of nanoHPLC and ICPMS. Anal Chem 2006;78:965–71. Alaejos MS, Romero CD. Analysis of selenium in body fluids—a review. Chem Rev 1995;95:227–57. Block E, Glass RS, Jacobsen NE, Johnson S, Kahakachchi C, Kaminski R, et al. Identification and synthesis of a novel selenium-sulfur amino acid found in selenized yeast: rapid indirect detection NMR methods for characterizing low-level organo selenium compounds in complex matrices. J Agric Food Chem 2004;52:3761–71. Dumont E, De Cremer K, Van Hulle M, Chery CC, Vanhaecke F, Cornelis R. Identification of the major selenium compound, Se-methionine, in three yeast (Saccharomyces cerevisiae) dietary supplements by on-line narrowbore liquid chromatography–electrospray tandem mass spectrometry. J Chromatogr A 2005;1071:191–6. Diaz Huerta V, Hinojosa Reyes L, Marchante-Gayon JM, Fernandez Sanchez ML, Sanz-Medel A. Total determination and quantitative speciation analysis of selenium in yeast and wheat flour by isotope dilution analysis ICP-MS. J Anal At Spectrom 2003:1243–7.
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[28] Whanger PD. Selenocompounds in plants and animals and their biological significance. J Am Commun Nutr 2002;21:223–32. [29] Giusti P, Schaumlo¨ffel D, Preud’homme H, Szpunar J, Lobinski R. Selenopeptide mapping in a selenium-yeast protein digest by parallel nanoHPLC–ICP-MS and nanoHPLC–electrospray-MS/MS after on-line preconcentration. J Anal At Spectrom 2006;21:26–32. [30] Wind M, Wegener A, Eisenmenger A, Kellner R, Lehmann WD. Sulfur as the key element for quantitative protein analysis by capillary liquid chromatography coupled to element mass spectrometry. Angew Chem 2003;42:3425–7. [31] Rappel C, Schaumlo¨ffel D. The role of sulfur and sulfur isotope dilution analysis in quantitative protein
analysis. Anal Bioanal Chem 2008; doi:10.1007/s00216007-1607-2. [32] Prohaska T, Latkoczy C, Stingeder G. Precise sulfur isotope ratio measurements in trace concentration of sulfur by inductively coupled plasma double focusing sector field mass spectrometry. J Anal At Spectrom 1999; 14:1501–4. [33] Heumann KG, Rottmann L, Vogl J. Elemental speciation with liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry. J Anal At Spectrom 1994;9:1351–5. [34] Schaumlo¨ffel D, Giusti P, Preud’Homme H, Szpunar J, Lobinski R. Precolumn isotope dilution analysis in nanoHPLC–ICPMS for absolute quantification of sulfur-containing peptides. Anal Chem 2007;79:2859–68.