- Email: [email protected]
Peptide Labeling with Improved 18O Incorporation Method
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 38, Issue 1, January 2010 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2010, 38(1), 91–94.
RESEARCH PAPER
Peptide Labeling with Improved 18O Incorporation Method ZHAO Yan1,2, LU Zhuang1, JIA Wei1,2, YING Wan-Tao1,2, QIAN Xiao-Hong1,2,* 1 2
State Key Lab of Proteomics, Beijing Proteome Research Center, Beijing 102206, China Beijing Institute of Radiation Medicine, Beijing 100850, China
Abstract: To optimize the 18O-labeling method, two key aspects, peptide dispersion and trypsin deactivation, were discussed. The addition of RapiGestTM SF in H218O and microwave heating enhanced labeling efficiency of Į-casein digested peptides (18O/16O ratio > 99%). Chemical modification with tris (2-carboxyethyl) phosphine and iodoacetamide resulted in trypsin deactivation completely. No significant back-exchange from 18O to 16O was observed after labeled in 6 days. The experiment result with peptide mixture from thyroglobulin showed that the improved method could be effectively used to label protein and peptide. Key Words:
1
18
O stable isotope labeling; Peptide; Proteome
Introduction
Quantitative proteomics can provide important reference information to study pathogenic mechanism and clinical applications by large-scale screening and identify disease-related proteins based on the observation of normal and diseased cells or tissue in the protein expression profile. 18 O isotope labeling is one of the techniques commonly used in quantitative proteomics, which produces 4-Da mass differences by trypsin catalyzed 16O to 18O exchange[1–5]. The relative quantitative results were obtained by comparing the labeled peptide with unlabeled peptide peak area. Although the labeling reaction is a mild, nondiscriminatory, and universal method, its wide application in the laboratory was reduced because of difficulty in controlling the reaction conditions and the instability of labeled peptides. In this study, we have discussed and optimized the peptide dispersion and trypsin inactivation conditions, which are the two important factors of 18O isotope labeling. Compared with our previous research by Qian[6], the improved method can shorten the labeling time, enhance labeling efficiency, and inhibit 16O-18O back-exchange effectively. Experimental results show that even for protein digestion mixture, the improved method can get satisfied results (18O/16O peak area
ratio > 99%).
2 2.1
Experimental Instruments and reagents
Bovine thyroglobulin, Į-casein and Į-cyanohydroxy cinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sequencing-grade trypsin and iodoacetamide (IAA) were purchased from Promega (Madison, WI). H218O (97% 18O) was obtained from Shanghai Research Institute of Chemical Industry, China. Tris (2-carboxyethyl) phosphine (TCEP) was purchased from Pierce (USA). Acetonitrile was obtained from J. T. Baker (Phillipsburg, NJ). Trifluoroacetic acid was supplied by Acros (New Jersey, USA); RapiGestTM SF was purchased from Waters (USA). Mass spectrometric analysis was performed on 4800 matrix-assisted laser desorption/ionization-time of flight/time of flight (MALDITOF-TOF, Applied Biosystems, Framingham, MA, USA). 2.2
Protein digestion
After bovine thyroglobulin or Į-casein was dissolved in NH4HCO3 solution of 20 mM, RapiGestTM SF (final
Received 25 June 2009; accepted 16 September 2009 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20635010, 20735005) and the National Key Basic Research Development Project of China (No. 2007CB914104). Copyright © 2010, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(09)60017-7
ZHAO Yan et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 91–94
concentration was 0.1%) and TCEP (final concentration was 5 mM) were added and then incubated for 1 h at 56 ºC. Thereafter, IAA (final concentration was 5 mM) was added, and protein was alkylated at room temperature for 1 h in dark. Finally, protein was digested overnight with trypsin at an enzyme/substrate ratio of 1:50 at 37 ºC. 2.3
Peptides labeling and trypsin deactivation
The lyophilized protein digestion was redissolved in H218O and further incubated in microwave oven for 10 min. Trypsin remained in solution was reduced by adding TCEP to a final concentration of 100 mM, incubated for 1 h in 37 ºC water bath and further for 10 min in microwave oven, and subsequently alkylated by adding IAA to 100 mM and kept at room temperature for 1 h in dark. 2.4
Mass spectrometric analysis
Mass spectrometric analysis was performed on Explorer™ software-controlled 4800 MALDI-TOF-TOF (Applied Biosystems, Framingham, MA, USA) using Į-cyanohydroxy cinnamic acid as matrix and the GPS Explorer™ software 2.0 as data processing software. All MS data were acquired using the MS-1 kv reflective mode with accelerating voltage of 20 kV, scan range of 700–3500, laser energy of 4500, and each spectrum accumulated of 1500 times. Myoglobin trypsin digestion as an external standard calibrated mass accuracy with the error (d 0.1 Da) and the relative standard deviation (d 10 ppm).
3 3.1
Results and discussion Enhancement of labeling efficiency
Peptide 18O isotope labeling method is based on the mechanism of trypsin catalyzed two enzymes-peptide complex hydrolysis, which is a fast dynamic reversible reaction. Thus, there was often incomplete labeling efficiency, and 16O-18O back-exchange happened in this process. How to effectively control labeling efficiency and prevent from back-exchange is the key to the success of the entire labeling experiment[7,8]. In our study, optimization of peptide dispersion in the reaction solution and introduction of microwave-assisted heating as effective strategies were used to increase the reaction efficiency. When peptide dispersion was improved, the C-terminal of peptides obtains more opportunities to contact with trypsin and H218O. Thereby, the reaction in positive direction was promoted for 18O labeling. In this experiment, the addition of RapiGestTM SF (a surface-active agent often used to promote protein digestion) can enhance the solubility of proteins and peptides, which is beneficial for the exchange reaction of C-terminal carboxyl group. As
showed in the labeling results of Į-casein digestion, with the addition of RapiGestTM SF, the peptide labeling efficiency of m/z 1660, 1267 and 2316 increased significantly (Fig.1, a and d). Compared with the above results, three labeled peptides incompletely were observed and a part of unlabeled peptides still exists in the results obtained by traditional methods (Fig.1c). In traditional labeling process, 18O-labeling reaction overnight was performed in 37 ºC water bath to achieve high labeling efficiency. But even so, mild reaction conditions and longer reaction time were difficult to ensure labeling completely. The experimental process was lagged because of time-consuming digestion and labeling reaction. Furthermore, the reaction solution in the traditional method was placed in the H216O environment for a long time, which is risk factor for 18 O labeling. Isolated from the H216O, microwave-assisted heating was a more effective alternative method as the data shown in Fig.1 (d and e). The labeling efficiency of microwave-assisted heating in 10 min is superior to the labeling efficiency of incubation overnight in water bath. Compared with the traditional water-bath heating method, microwave heating provided a rapid and uniform heating method to achieve the desired labeling efficiency in a very short period of time 3.2
Prevention of 16O-18O back-exchange
16
O-18O back-exchange can be observed easily in experimental process because the labeling reaction is a reversible reaction. When 18O-labeled peptide placed in H216O, the isotopic label will be lost through the trypsin enzyme-facilitated mechanisms. To obtain quantitative accuracy, effective measures should be taken to prevent back-exchange. A variety of methods currently reported in the literature, including acid inhibition, ultrafiltration, and microwave heating, were used to trypsin inactivated with less effective[8]. In this experiment, trypsin remained in solution was inactivated completely by high concentration reduction reagents and alkylation reagents. The results demonstrated (Fig.2) that there was no apparent back-exchange phenomenon observed in mobile phase (2% acetonitrile: 98% water: 0.5% formic acid) in 6 days and the labeled peptides can be a follow-up analyzed in liquid chromatography. 3.3
Labeling results of complex peptides mixture
MALDI-TOF-TOF mass spectrometry can detect 24 peptides generated by digestion of bovine thyroid protein (molecular weight, 660 kDa). For evaluation of effectiveness and tolerance, the optimized method was applied to label bovine thyroid protein digestion. As shown in Fig.3, a satisfied labeling efficiency was obtained even for complex peptides mixture.
ZHAO Yan et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 91–94
Fig.1 MALDI-TOF-TOF mass spectrum of tryptic-digested peptides of Į-casein A, spectrum of unlabeled peptide mixture; B, spectrum of unlabeled peptides with m/z 1267, 1660 and 2316; C, spectrum of labeled peptides with m/z 1267, 1660 and 2316 without RapiGestTM SF at 37 ºC for 24 h; D, spectrum of labeled peptides with m/z 1267, 1660 and 2316 with RapiGestTM SF at 37 ºC for 24 h; E, spectrum of labeled peptides with m/z 1267, 1660 and 2316 with RapiGestTM SF and microwave heating for 10 min
Fig.2 Stability of labeled Į-casein peptides in 2% ACN: 98% water (0.5% FA) solution A, MALDI-TOF-TOF mass spectrum of labeled Į-casein; B, 0 day after labeling; C, 1 day after labeling; D, 3 days after labeling; E, 6 days after labeling. MALDI-TOF-TOF mass spectrum of labeled Į-casein peptides m/z 1267, 1660 and 2316
ZHAO Yan et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 91–94
trypsin to prevent back-exchange reaction. The optimized method with improved labeling efficiency, enhanced product stability, and less reaction time can label peptide quickly and efficiently to meet the needs of quantitative proteomics.
References [1] Fig.3
4
Labeling efficiency of thyroglobin peptides with 18O (18O incorporation efficiency was calculated by 18O/16O ratio of the first isotopic peak area)
8(8): 1645–1660 [2]
Jin S W, Peter G, Nathan E, Catherine F. J. Proteome Res.,
[3]
Catherine S L, Yu Q W, Richard B, William J G, Laurence H P.
[4]
Sui S H, Wang J L, Jia W, Lu Z, Lu J F, Song L N, Cai Y,
2007, 6(12): 4601–4607 Mol. Cell Proteomics, 2007, 6(6): 953–962
Conclusions 18
O-labeling method is one of the isotope-labeled methods used widely in quantitative proteomics. However, as the reaction is a dynamic reversible reaction, labeling efficiency and inhibition of back-exchange were difficult to be controlled, which lead to quantitative inaccuracy of 18O labeling. In recent years, although 18O labeling has been optimized in a variety of ways, the widespread use of this method in many laboratories has been limited because of poor stability and tolerance[9,10]. In this study, labeling efficiency and inhibition of back-exchange were optimized by the enhancement of peptide dispersion, the change of the auxiliary heating methods, and chemical modification of
Ching S E, Paul D V, Stuart G D, Eric C R. Proteomics, 2008,
Qian X H. Chinese J. Anal. Chem., 2008, 36(8): 1017–1023 [5]
Manfred H, Hassan M, Christoph M, Xu D Y. J. Am. Soc. Mass Spectrom., 2003, 14(7): 704–718
[6]
Qian L Y, Ying W T, Liu X, Lu Z, Cai Y, He J Y, Qian X H. Chinese J. Anal. Chem., 2007, 35(2): 161–165
[7]
Henricus F S, Robert V D H, Ubbo R T, Jan V D G. Rapid Commun. Mass Spectrom., 2006, 20(23): 3491–3497
[8]
Peggi M A, Ron O. Anal. Biochem., 2006, 359(1): 26–34
[9]
Shama P M, Andrew S G, Michael O. J. Proteome Res., 2008, 7(7): 3042–3048
[10]
Freije J R, Mulder P P M, Wendy W, Rienx L, Harm A G N, Verpoorte E, Bischoff R. J. Proteome Res., 2005, 4(5): 1805–1813
Cite this article as: Chin J Anal Chem, 2010, 38(1), 91–94.
RESEARCH PAPER
Peptide Labeling with Improved 18O Incorporation Method ZHAO Yan1,2, LU Zhuang1, JIA Wei1,2, YING Wan-Tao1,2, QIAN Xiao-Hong1,2,* 1 2
State Key Lab of Proteomics, Beijing Proteome Research Center, Beijing 102206, China Beijing Institute of Radiation Medicine, Beijing 100850, China
Abstract: To optimize the 18O-labeling method, two key aspects, peptide dispersion and trypsin deactivation, were discussed. The addition of RapiGestTM SF in H218O and microwave heating enhanced labeling efficiency of Į-casein digested peptides (18O/16O ratio > 99%). Chemical modification with tris (2-carboxyethyl) phosphine and iodoacetamide resulted in trypsin deactivation completely. No significant back-exchange from 18O to 16O was observed after labeled in 6 days. The experiment result with peptide mixture from thyroglobulin showed that the improved method could be effectively used to label protein and peptide. Key Words:
1
18
O stable isotope labeling; Peptide; Proteome
Introduction
Quantitative proteomics can provide important reference information to study pathogenic mechanism and clinical applications by large-scale screening and identify disease-related proteins based on the observation of normal and diseased cells or tissue in the protein expression profile. 18 O isotope labeling is one of the techniques commonly used in quantitative proteomics, which produces 4-Da mass differences by trypsin catalyzed 16O to 18O exchange[1–5]. The relative quantitative results were obtained by comparing the labeled peptide with unlabeled peptide peak area. Although the labeling reaction is a mild, nondiscriminatory, and universal method, its wide application in the laboratory was reduced because of difficulty in controlling the reaction conditions and the instability of labeled peptides. In this study, we have discussed and optimized the peptide dispersion and trypsin inactivation conditions, which are the two important factors of 18O isotope labeling. Compared with our previous research by Qian[6], the improved method can shorten the labeling time, enhance labeling efficiency, and inhibit 16O-18O back-exchange effectively. Experimental results show that even for protein digestion mixture, the improved method can get satisfied results (18O/16O peak area
ratio > 99%).
2 2.1
Experimental Instruments and reagents
Bovine thyroglobulin, Į-casein and Į-cyanohydroxy cinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sequencing-grade trypsin and iodoacetamide (IAA) were purchased from Promega (Madison, WI). H218O (97% 18O) was obtained from Shanghai Research Institute of Chemical Industry, China. Tris (2-carboxyethyl) phosphine (TCEP) was purchased from Pierce (USA). Acetonitrile was obtained from J. T. Baker (Phillipsburg, NJ). Trifluoroacetic acid was supplied by Acros (New Jersey, USA); RapiGestTM SF was purchased from Waters (USA). Mass spectrometric analysis was performed on 4800 matrix-assisted laser desorption/ionization-time of flight/time of flight (MALDITOF-TOF, Applied Biosystems, Framingham, MA, USA). 2.2
Protein digestion
After bovine thyroglobulin or Į-casein was dissolved in NH4HCO3 solution of 20 mM, RapiGestTM SF (final
Received 25 June 2009; accepted 16 September 2009 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20635010, 20735005) and the National Key Basic Research Development Project of China (No. 2007CB914104). Copyright © 2010, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(09)60017-7
ZHAO Yan et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 91–94
concentration was 0.1%) and TCEP (final concentration was 5 mM) were added and then incubated for 1 h at 56 ºC. Thereafter, IAA (final concentration was 5 mM) was added, and protein was alkylated at room temperature for 1 h in dark. Finally, protein was digested overnight with trypsin at an enzyme/substrate ratio of 1:50 at 37 ºC. 2.3
Peptides labeling and trypsin deactivation
The lyophilized protein digestion was redissolved in H218O and further incubated in microwave oven for 10 min. Trypsin remained in solution was reduced by adding TCEP to a final concentration of 100 mM, incubated for 1 h in 37 ºC water bath and further for 10 min in microwave oven, and subsequently alkylated by adding IAA to 100 mM and kept at room temperature for 1 h in dark. 2.4
Mass spectrometric analysis
Mass spectrometric analysis was performed on Explorer™ software-controlled 4800 MALDI-TOF-TOF (Applied Biosystems, Framingham, MA, USA) using Į-cyanohydroxy cinnamic acid as matrix and the GPS Explorer™ software 2.0 as data processing software. All MS data were acquired using the MS-1 kv reflective mode with accelerating voltage of 20 kV, scan range of 700–3500, laser energy of 4500, and each spectrum accumulated of 1500 times. Myoglobin trypsin digestion as an external standard calibrated mass accuracy with the error (d 0.1 Da) and the relative standard deviation (d 10 ppm).
3 3.1
Results and discussion Enhancement of labeling efficiency
Peptide 18O isotope labeling method is based on the mechanism of trypsin catalyzed two enzymes-peptide complex hydrolysis, which is a fast dynamic reversible reaction. Thus, there was often incomplete labeling efficiency, and 16O-18O back-exchange happened in this process. How to effectively control labeling efficiency and prevent from back-exchange is the key to the success of the entire labeling experiment[7,8]. In our study, optimization of peptide dispersion in the reaction solution and introduction of microwave-assisted heating as effective strategies were used to increase the reaction efficiency. When peptide dispersion was improved, the C-terminal of peptides obtains more opportunities to contact with trypsin and H218O. Thereby, the reaction in positive direction was promoted for 18O labeling. In this experiment, the addition of RapiGestTM SF (a surface-active agent often used to promote protein digestion) can enhance the solubility of proteins and peptides, which is beneficial for the exchange reaction of C-terminal carboxyl group. As
showed in the labeling results of Į-casein digestion, with the addition of RapiGestTM SF, the peptide labeling efficiency of m/z 1660, 1267 and 2316 increased significantly (Fig.1, a and d). Compared with the above results, three labeled peptides incompletely were observed and a part of unlabeled peptides still exists in the results obtained by traditional methods (Fig.1c). In traditional labeling process, 18O-labeling reaction overnight was performed in 37 ºC water bath to achieve high labeling efficiency. But even so, mild reaction conditions and longer reaction time were difficult to ensure labeling completely. The experimental process was lagged because of time-consuming digestion and labeling reaction. Furthermore, the reaction solution in the traditional method was placed in the H216O environment for a long time, which is risk factor for 18 O labeling. Isolated from the H216O, microwave-assisted heating was a more effective alternative method as the data shown in Fig.1 (d and e). The labeling efficiency of microwave-assisted heating in 10 min is superior to the labeling efficiency of incubation overnight in water bath. Compared with the traditional water-bath heating method, microwave heating provided a rapid and uniform heating method to achieve the desired labeling efficiency in a very short period of time 3.2
Prevention of 16O-18O back-exchange
16
O-18O back-exchange can be observed easily in experimental process because the labeling reaction is a reversible reaction. When 18O-labeled peptide placed in H216O, the isotopic label will be lost through the trypsin enzyme-facilitated mechanisms. To obtain quantitative accuracy, effective measures should be taken to prevent back-exchange. A variety of methods currently reported in the literature, including acid inhibition, ultrafiltration, and microwave heating, were used to trypsin inactivated with less effective[8]. In this experiment, trypsin remained in solution was inactivated completely by high concentration reduction reagents and alkylation reagents. The results demonstrated (Fig.2) that there was no apparent back-exchange phenomenon observed in mobile phase (2% acetonitrile: 98% water: 0.5% formic acid) in 6 days and the labeled peptides can be a follow-up analyzed in liquid chromatography. 3.3
Labeling results of complex peptides mixture
MALDI-TOF-TOF mass spectrometry can detect 24 peptides generated by digestion of bovine thyroid protein (molecular weight, 660 kDa). For evaluation of effectiveness and tolerance, the optimized method was applied to label bovine thyroid protein digestion. As shown in Fig.3, a satisfied labeling efficiency was obtained even for complex peptides mixture.
ZHAO Yan et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 91–94
Fig.1 MALDI-TOF-TOF mass spectrum of tryptic-digested peptides of Į-casein A, spectrum of unlabeled peptide mixture; B, spectrum of unlabeled peptides with m/z 1267, 1660 and 2316; C, spectrum of labeled peptides with m/z 1267, 1660 and 2316 without RapiGestTM SF at 37 ºC for 24 h; D, spectrum of labeled peptides with m/z 1267, 1660 and 2316 with RapiGestTM SF at 37 ºC for 24 h; E, spectrum of labeled peptides with m/z 1267, 1660 and 2316 with RapiGestTM SF and microwave heating for 10 min
Fig.2 Stability of labeled Į-casein peptides in 2% ACN: 98% water (0.5% FA) solution A, MALDI-TOF-TOF mass spectrum of labeled Į-casein; B, 0 day after labeling; C, 1 day after labeling; D, 3 days after labeling; E, 6 days after labeling. MALDI-TOF-TOF mass spectrum of labeled Į-casein peptides m/z 1267, 1660 and 2316
ZHAO Yan et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 91–94
trypsin to prevent back-exchange reaction. The optimized method with improved labeling efficiency, enhanced product stability, and less reaction time can label peptide quickly and efficiently to meet the needs of quantitative proteomics.
References [1] Fig.3
4
Labeling efficiency of thyroglobin peptides with 18O (18O incorporation efficiency was calculated by 18O/16O ratio of the first isotopic peak area)
8(8): 1645–1660 [2]
Jin S W, Peter G, Nathan E, Catherine F. J. Proteome Res.,
[3]
Catherine S L, Yu Q W, Richard B, William J G, Laurence H P.
[4]
Sui S H, Wang J L, Jia W, Lu Z, Lu J F, Song L N, Cai Y,
2007, 6(12): 4601–4607 Mol. Cell Proteomics, 2007, 6(6): 953–962
Conclusions 18
O-labeling method is one of the isotope-labeled methods used widely in quantitative proteomics. However, as the reaction is a dynamic reversible reaction, labeling efficiency and inhibition of back-exchange were difficult to be controlled, which lead to quantitative inaccuracy of 18O labeling. In recent years, although 18O labeling has been optimized in a variety of ways, the widespread use of this method in many laboratories has been limited because of poor stability and tolerance[9,10]. In this study, labeling efficiency and inhibition of back-exchange were optimized by the enhancement of peptide dispersion, the change of the auxiliary heating methods, and chemical modification of
Ching S E, Paul D V, Stuart G D, Eric C R. Proteomics, 2008,
Qian X H. Chinese J. Anal. Chem., 2008, 36(8): 1017–1023 [5]
Manfred H, Hassan M, Christoph M, Xu D Y. J. Am. Soc. Mass Spectrom., 2003, 14(7): 704–718
[6]
Qian L Y, Ying W T, Liu X, Lu Z, Cai Y, He J Y, Qian X H. Chinese J. Anal. Chem., 2007, 35(2): 161–165
[7]
Henricus F S, Robert V D H, Ubbo R T, Jan V D G. Rapid Commun. Mass Spectrom., 2006, 20(23): 3491–3497
[8]
Peggi M A, Ron O. Anal. Biochem., 2006, 359(1): 26–34
[9]
Shama P M, Andrew S G, Michael O. J. Proteome Res., 2008, 7(7): 3042–3048
[10]
Freije J R, Mulder P P M, Wendy W, Rienx L, Harm A G N, Verpoorte E, Bischoff R. J. Proteome Res., 2005, 4(5): 1805–1813