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Cite this: Chem. Commun., 2014, 50, 1708 Received 17th October 2013, Accepted 6th December 2013
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Five-plex isotope dimethyl labeling for quantitative proteomics† Yue Wu, Fangjun Wang,* Zheyi Liu, Hongqiang Qin, Chunxia Song, Junfeng Huang, Yangyang Bian, Xiaoluan Wei, Jing Dong and Hanfa Zou*
DOI: 10.1039/c3cc47998f www.rsc.org/chemcomm
Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.
Mass spectrometry (MS)-based proteomics has become an indispensable tool for molecular and cellular biological studies at a systematic level.1 Quantitative proteomics is one of the most important techniques in proteomics for measuring protein expression levels of two or more physiological states of a biological system. In recent years, several MS-based quantitative techniques were developed to achieve robust, accurate, and high throughput proteome quantification. Most of these methods use stable isotope labeling to introduce differential mass tags to peptides or proteins. These isotopic mass tags can be incorporated metabolically by cell culture, such as stable isotope labeling by amino acids in cell culture (SILAC),2 or chemically, such as isobaric tags for relative and absolute quantitation (iTRAQ)3 and isotope dimethyl labeling.4 The chemical reaction of dimethylation at the N-terminus and lysine residues (a- and e-amino groups) was introduced to the field of quantitative proteomics in 2003.4 This reaction involves the formation of a Schiff base, followed by the reduction of cyanoborohydride to generate a dimethylamine. It exhibits high reaction efficiency and selectivity, and does not generate any significant by-products (except the rarely appearing N-terminal proline).5 Compared with other quantitative methods, dimethyl labeling is widely proven to be reliable, cost-effective, simple and applicable to any protein sample.6 The high efficient dimethyl labeling can be performed by in-solution, online, and on-column strategies to meet the requirements of different sample amounts.5 Efforts were also made towards the combination with specific enrichment methods, such as for Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. E-mail: [email protected], [email protected]; Fax: +86-411-84379620; Tel: +86-411-84379610 † Electronic supplementary information (ESI) available: Experimental details, LC-MS results. See DOI: 10.1039/c3cc47998f
1708 | Chem. Commun., 2014, 50, 1708--1710
phosphorylated7 or glycosylated8 peptides, and automation of the labeling procedures to facilitate sample handling and reduce sample loss.9 The dimethyl labeling strategy has been widely applied to different biological studies, such as stem cell, stimulation induced phosphorylation dynamics study and interaction proteomics.6 The conventional dimethyl labeling method can only quantify at most three protein samples in one experiment, which greatly limits its applications in high throughput analyses. Although multiplex proteome quantification can be easily achieved by isobaric isotope labeling or label free strategies, the interference of co-isolating ions or variations in different LC separation and MS detection will inevitably decrease the quantification accuracy of these strategies.10 Thus, we extended the conventional dual or triple dimethyl labeling to five-plex at the full MS scan (MS1) level without changing the handling procedures. Lys-C was used for protein digestion to generate at least two labeling sites for each peptide; thus the mass difference between the nearest labeled forms is at least 4 Da, which is important for reducing the overlapping of isotopic clusters in the MS1 spectra and increase the proteome quantification accuracy.11 The feasibility of this five-plex isotope labeling strategy was demonstrated in both proteome and phosphoproteome quantification of HeLa cell samples with high quantification accuracy. The five-plex isotope dimethyl labeling strategy was developed by simply combining different types of isotopic formaldehyde and cyanoborohydride (Fig. 1 and Table 1) using the in-solution labeling procedures.5 Briefly, for every 25 mg of LysC digested Hela protein sample (dissolved in 100 mM TEAB (pH 8) buffer), 4 mL of 4% (vol/vol) formaldehyde (CH2O, CD2O or 13CD2O) and 4 mL of 0.6 M cyanoborohydride (NaBH3CN or NaBD3CN) were added for the dimethylation reaction. After keeping the reaction solution in 37 1C for 1 h, 2 mL of 10% (vol/vol) ammonia and 5 mL of 10% (vol/vol) formic acid in water were successively added to quench the reaction (see ESI† for details about cell culture, protein extraction and other handling procedures). Lys-C was utilized for protein digestion to generate at least two active amino groups (lysine residue and N-termini) and thus obtain a mass difference of at least 4 Da, which was generally considered to be sufficient for differentiation of isotopomers.11
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Fig. 1
ChemComm
Schemes of the five-plex isotope dimethyl labeling method.
The MS1 resolution of the labeled peptides under different charge states was examined first. It is observed that the five-plex isotope clusters of +1, +2, +3 and +4 peptides (most frequently detected in shotgun proteomics) are clearly resolved with similar peak intensities (Fig. 2). On the other hand, if trypsin was utilized for protein digestion, a considerable portion of labeled clusters could not be resolved (Fig. S1, ESI†) due to the mass difference of only 2 Da derived from cleavage at arginine (without lysine residues in the digested peptides). Furthermore, we examined the labeling efficiency using a Mascot-based database search (ESI†); over 99% of the peptides were successfully labeled with dimethyl groups. Data analysis was accomplished by using MaxQuant software (http://maxquant.org/, version 1.3.0.3) against the IPI human database (v3.80) (ESI†). To validate the performance of this five-plex isotope dimethyl labeling strategy as well as the MaxQuant data processing, five identical Lys-C-digested HeLa protein samples (5 25 mg, 1 : 1 : 1 : 1 : 1) were differentially labeled and mixed for SCX-RP 2D LC-MS/MS analysis (ESI†). Finally, totally 5359 unique peptides and 1297 proteins were successfully identified at a false discovery rate (FDR) of 1%, among which 4530 unique peptides (85%) corresponding to 1190 proteins (92%) were successfully quantified (details listed in the ESI†). Among all of the quantified peptides and proteins for the five different protein samples, over 99% of the log2 ratios of both peptides and proteins fall in the range of [ 1, 1], within which no significant variation in proteome quantification is usually considered (Fig. 3a and b).12 The five proteins samples were also labeled and mixed in a ratio of 1 : 2 : 4 : 2 : 1. Finally, 4014 unique peptides (66% of the total 6078 identified peptides) corresponding to 1442 proteins (79% of the total 1833 identified proteins) were successfully quantified among all of the five protein samples (data shown in the ESI†). Though the quantification rates were slightly decreased due to the increased sample complexity, high quantification accuracy was still obtained Table 1
Fig. 2 Mass resolution of five-plex isotopically labeled peptides. MS spectra of Lys-C digested peptides with different charge states (+1, +2, +3 and +4) (1 : 1 : 1 : 1 : 1 ratio).
Fig. 3 Log2 ratio distributions of quantified peptides (a) and proteins (b) of the sample mixed with 1 : 1 : 1 : 1 : 1 ratio; log2 ratio distributions of quantified peptides (c) and proteins (d) of the sample mixed with 1 : 2 : 4 : 2 : 1 ratio and log2 ratio distributions of quantified phosphopeptides (e) and phosphorylation sites (f) of the sample mixed with 1 : 1 : 1 : 1 : 1 ratio.
as over 96% of the log2 ratios of both peptides and proteins are within one-fold change of the theoretical values (Fig. 3c and d). Protein phosphorylation is one of the most important protein PTMs, playing crucial roles in a wide variety of cellular functions, such as signal transduction, cell cycle regulation, apoptosis, and cell differentiation.13 High throughput phosphoproteome quantification
The combination of different isotopic reagents in the five-plex isotope dimethyl labeling method
Label
#1
#2
#3
#4
#5
Formaldehyde isotope Cyanoborohydride isotope DMass (Da, one active site) DMass (Da, two active sites)
H2CO NaBH3CN 28.0313 56.0626
H2CO NaBD3CN 30.0439 60.0878
D2CO NaBH3CN 32.0564 64.1128
D2CO NaBD3CN 34.0690 68.1380
D213CO NaBD3CN 36.0757 72.1514
This journal is © The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 1708--1710 | 1709
View Article Online
Published on 09 December 2013. Downloaded by UNIVERSITY OF ALABAMA AT BIRMINGHAM on 28/10/2014 03:32:50.
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strategies are also rapidly developing in recent years.14 In order to investigate the performance of this five-plex labeling strategy in phosphoproteome quantification, five identical protein samples were labeled and mixed together, followed by Ti4+-IMAC enrichment of the labeled phosphopeptides and SCX-RP 2D LC-MS/MS analysis (details shown in the ESI†). By using 5 100 mg starting materials, 1534 unique phosphopeptides (75% of the 2049 identified peptides) corresponding to 929 phosphoproteins (74% of the 1255 identified phosphoproteins) with 1976 phosphorylation sites (77% of the 2555 identified phosphorylation sites) were feasibly quantified among all of the five protein samples (data shown in the ESI†). Over 98% of all the log2 ratios of both phosphopeptides and phosphorylation sites fall in the range of [ 1, 1], demonstrating the high quantification accuracy of the method (Fig. 3e and f). All the above proteome and phosphoproteome experiments were performed twice and both the quantification accuracy and the number of quantified proteins and peptides was similar (Fig. S2 and Table S1, ESI†). In addition, we further spiked three standard proteins, ovalbumin, myoglobin and BSA, into five E. coli protein extraction samples (5 100 mg) to obtain standard/sample ratios (w/w) of 1 : 200, 2 : 200, 5 : 200, 10 : 200, 20 : 200 (BSA) and 20 : 200, 10 : 200, 5 : 200, 2 : 200, 1 : 200 (ovalbumin and myoglobin) (details shown in the ESI†). After digestion and labeling, the mixture of the five samples were analyzed by LC-MS. The results indicated that the E. coli proteome did not show significant change (over 99% log2 ratios of E. coli proteins fall in the range of [ 1, 1]), and measured ratios of all the three standard proteins were quite close to the theoretical ratios (Fig. 4) with RSDs o 0.16 in three parallel experiments. The quantified peptides of each protein standard were also examined, and most of the peptides showed good consistency with the theoretical ratios (Fig. S3, ESI†). Therefore, the above results demonstrated that high quantification accuracy can be feasibly obtained by using this five-plex isotope labeling method for comprehensive proteome and phosphoproteome quantification. The MS detection and data acquisition speed are greatly improved in recent years with the rapid development of new types of
Fig. 4 The quantification of three spiked-in protein standards. The standards were differentially mixed and then spiked into the E. coli samples to obtain standard/sample ratios (w/w) of 1 : 200, 2 : 200, 5 : 200, 10 : 200, 20 : 200 (BSA) and 20 : 200, 10 : 200, 5 : 200, 2 : 200, 1 : 200 (ovalbumin and myoglobin). Three quantification experiments were performed and the variations are shown as error bars.
1710 | Chem. Commun., 2014, 50, 1708--1710
Communication
mass spectrometers, which facilitates the application of the MS1 multiplex isotope labeling strategies to large-scale proteome and phosphoproteome quantification. Compared with our previous results of proteome or phosphoproteome quantification by using dual or triple dimethyl labeling,9,12 both the proteome coverage and the quantification accuracies are comparable (Fig. S4, ESI†). The quantification rates (quantified number/identified number) of conventional double or triple isotope dimethyl labeling (>90%) are higher than the five-plex dimethyl labeling (B78%). However, as the overlapping of two instrumental replicates is usually 60–80% in shotgun proteomics, it is still better to perform one five-plex quantification experiment than two or three conventional isotope labeling experiments separately. Therefore, we believe that the extension of the conventional double or triple dimethyl labeling to five channels has great potential in proteomic research that demands high throughput, high accuracy and simple operation. In this study, a new isotope dimethyl labeling method that enabled parallel analysis of five different protein samples in one experiment was developed. Both high throughput and high accuracy were obtained in comprehensive proteome and phosphoproteome quantification using this five-plex isotope labeling strategy. We believe that this multiplex isotope labeling strategy can be widely applied in high throughput proteome quantification of different types of biological samples, and will be beneficial in cellular dynamics investigations and biomarker discovery. Financial support from the China State Key Basic Research Program Grant (2013CB911203 and 2012CB910604), the NSFC (21021004, 81161120540 and 21235006), and the Analytical Method Innovation Program of MOST (2012IM030900) to H. Zou, and the financial support from NSFC (21305139) and ‘‘Hundred Talent Young Scientist Program’’ by DICP to F. Wang is gratefully acknowledged.
Notes and references 1 R. Aebersold and M. Mann, Nature, 2003, 422, 198–207. 2 S. E. Ong, B. Blagoev, I. Kratchmarova, D. B. Kristensen, H. Steen, A. Pandey and M. Mann, Mol. Cell. Proteomics, 2002, 1, 376–386. 3 P. L. Ross, Y. L. N. Huang, J. N. Marchese, B. Williamson, K. Parker, S. Hattan, N. Khainovski, S. Pillai, S. Dey, S. Daniels, S. Purkayastha, P. Juhasz, S. Martin, M. Bartlet-Jones, F. He, A. Jacobson and D. J. Pappin, Mol. Cell. Proteomics, 2004, 3, 1154–1169. 4 J. L. Hsu, S. Y. Huang, N. H. Chow and S. H. Chen, Anal. Chem., 2003, 75, 6843–6852. 5 P. J. Boersema, R. Raijmakers, S. Lemeer, S. Mohammed and A. J. R. Heck, Nat. Protoc., 2009, 4, 484–494. 6 D. Kovanich, S. Cappadona, R. Raijmakers, S. Mohammed, A. Scholten and A. J. Heck, Anal. Bioanal. Chem., 2012, 404, 991–1009. 7 S. Y. Huang, M. L. Tsai, C. J. Wu, J. L. Hsu, S. H. Ho and S. H. Chen, Proteomics, 2006, 6, 1722–1734. 8 Z. Sun, H. Qin, F. Wang, K. Cheng, M. Dong, M. Ye and H. Zou, Anal. Chem., 2012, 84, 8452–8456. 9 F. J. Wang, R. Chen, J. Zhu, D. G. Sun, C. X. Song, Y. F. Wu, M. L. Ye, L. M. Wang and H. F. Zou, Anal. Chem., 2010, 82, 3007–3015. 10 (a) L. Ting, R. Rad, S. P. Gygi and W. Haas, Nat. Methods, 2011, 8, 937–940; (b) L. V. DeSouza and K. W. Siu, Clin. Biochem., 2013, 46, 421–431. 11 M. Bantscheff, M. Schirle, G. Sweetman, J. Rick and B. Kuster, Anal. Bioanal. Chem., 2007, 389, 1017–1031. 12 C. Song, F. Wang, M. Ye, K. Cheng, R. Chen, J. Zhu, Y. Tan, H. Wang, D. Figeys and H. Zou, Anal. Chem., 2011, 83, 7755–7762. 13 T. Hunter, Cell, 2000, 100, 113–127. 14 (a) J. Villen, S. A. Beausoleil, S. A. Gerber and S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 1488–1493; (b) F. Wang, C. Song, K. Cheng, X. Jiang, M. Ye and H. Zou, Anal. Chem., 2011, 83, 8078–8085.
This journal is © The Royal Society of Chemistry 2014
ChemComm COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 1708 Received 17th October 2013, Accepted 6th December 2013
View Article Online View Journal | View Issue
Five-plex isotope dimethyl labeling for quantitative proteomics† Yue Wu, Fangjun Wang,* Zheyi Liu, Hongqiang Qin, Chunxia Song, Junfeng Huang, Yangyang Bian, Xiaoluan Wei, Jing Dong and Hanfa Zou*
DOI: 10.1039/c3cc47998f www.rsc.org/chemcomm
Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.
Mass spectrometry (MS)-based proteomics has become an indispensable tool for molecular and cellular biological studies at a systematic level.1 Quantitative proteomics is one of the most important techniques in proteomics for measuring protein expression levels of two or more physiological states of a biological system. In recent years, several MS-based quantitative techniques were developed to achieve robust, accurate, and high throughput proteome quantification. Most of these methods use stable isotope labeling to introduce differential mass tags to peptides or proteins. These isotopic mass tags can be incorporated metabolically by cell culture, such as stable isotope labeling by amino acids in cell culture (SILAC),2 or chemically, such as isobaric tags for relative and absolute quantitation (iTRAQ)3 and isotope dimethyl labeling.4 The chemical reaction of dimethylation at the N-terminus and lysine residues (a- and e-amino groups) was introduced to the field of quantitative proteomics in 2003.4 This reaction involves the formation of a Schiff base, followed by the reduction of cyanoborohydride to generate a dimethylamine. It exhibits high reaction efficiency and selectivity, and does not generate any significant by-products (except the rarely appearing N-terminal proline).5 Compared with other quantitative methods, dimethyl labeling is widely proven to be reliable, cost-effective, simple and applicable to any protein sample.6 The high efficient dimethyl labeling can be performed by in-solution, online, and on-column strategies to meet the requirements of different sample amounts.5 Efforts were also made towards the combination with specific enrichment methods, such as for Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. E-mail: [email protected], [email protected]; Fax: +86-411-84379620; Tel: +86-411-84379610 † Electronic supplementary information (ESI) available: Experimental details, LC-MS results. See DOI: 10.1039/c3cc47998f
1708 | Chem. Commun., 2014, 50, 1708--1710
phosphorylated7 or glycosylated8 peptides, and automation of the labeling procedures to facilitate sample handling and reduce sample loss.9 The dimethyl labeling strategy has been widely applied to different biological studies, such as stem cell, stimulation induced phosphorylation dynamics study and interaction proteomics.6 The conventional dimethyl labeling method can only quantify at most three protein samples in one experiment, which greatly limits its applications in high throughput analyses. Although multiplex proteome quantification can be easily achieved by isobaric isotope labeling or label free strategies, the interference of co-isolating ions or variations in different LC separation and MS detection will inevitably decrease the quantification accuracy of these strategies.10 Thus, we extended the conventional dual or triple dimethyl labeling to five-plex at the full MS scan (MS1) level without changing the handling procedures. Lys-C was used for protein digestion to generate at least two labeling sites for each peptide; thus the mass difference between the nearest labeled forms is at least 4 Da, which is important for reducing the overlapping of isotopic clusters in the MS1 spectra and increase the proteome quantification accuracy.11 The feasibility of this five-plex isotope labeling strategy was demonstrated in both proteome and phosphoproteome quantification of HeLa cell samples with high quantification accuracy. The five-plex isotope dimethyl labeling strategy was developed by simply combining different types of isotopic formaldehyde and cyanoborohydride (Fig. 1 and Table 1) using the in-solution labeling procedures.5 Briefly, for every 25 mg of LysC digested Hela protein sample (dissolved in 100 mM TEAB (pH 8) buffer), 4 mL of 4% (vol/vol) formaldehyde (CH2O, CD2O or 13CD2O) and 4 mL of 0.6 M cyanoborohydride (NaBH3CN or NaBD3CN) were added for the dimethylation reaction. After keeping the reaction solution in 37 1C for 1 h, 2 mL of 10% (vol/vol) ammonia and 5 mL of 10% (vol/vol) formic acid in water were successively added to quench the reaction (see ESI† for details about cell culture, protein extraction and other handling procedures). Lys-C was utilized for protein digestion to generate at least two active amino groups (lysine residue and N-termini) and thus obtain a mass difference of at least 4 Da, which was generally considered to be sufficient for differentiation of isotopomers.11
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View Article Online
Published on 09 December 2013. Downloaded by UNIVERSITY OF ALABAMA AT BIRMINGHAM on 28/10/2014 03:32:50.
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Fig. 1
ChemComm
Schemes of the five-plex isotope dimethyl labeling method.
The MS1 resolution of the labeled peptides under different charge states was examined first. It is observed that the five-plex isotope clusters of +1, +2, +3 and +4 peptides (most frequently detected in shotgun proteomics) are clearly resolved with similar peak intensities (Fig. 2). On the other hand, if trypsin was utilized for protein digestion, a considerable portion of labeled clusters could not be resolved (Fig. S1, ESI†) due to the mass difference of only 2 Da derived from cleavage at arginine (without lysine residues in the digested peptides). Furthermore, we examined the labeling efficiency using a Mascot-based database search (ESI†); over 99% of the peptides were successfully labeled with dimethyl groups. Data analysis was accomplished by using MaxQuant software (http://maxquant.org/, version 1.3.0.3) against the IPI human database (v3.80) (ESI†). To validate the performance of this five-plex isotope dimethyl labeling strategy as well as the MaxQuant data processing, five identical Lys-C-digested HeLa protein samples (5 25 mg, 1 : 1 : 1 : 1 : 1) were differentially labeled and mixed for SCX-RP 2D LC-MS/MS analysis (ESI†). Finally, totally 5359 unique peptides and 1297 proteins were successfully identified at a false discovery rate (FDR) of 1%, among which 4530 unique peptides (85%) corresponding to 1190 proteins (92%) were successfully quantified (details listed in the ESI†). Among all of the quantified peptides and proteins for the five different protein samples, over 99% of the log2 ratios of both peptides and proteins fall in the range of [ 1, 1], within which no significant variation in proteome quantification is usually considered (Fig. 3a and b).12 The five proteins samples were also labeled and mixed in a ratio of 1 : 2 : 4 : 2 : 1. Finally, 4014 unique peptides (66% of the total 6078 identified peptides) corresponding to 1442 proteins (79% of the total 1833 identified proteins) were successfully quantified among all of the five protein samples (data shown in the ESI†). Though the quantification rates were slightly decreased due to the increased sample complexity, high quantification accuracy was still obtained Table 1
Fig. 2 Mass resolution of five-plex isotopically labeled peptides. MS spectra of Lys-C digested peptides with different charge states (+1, +2, +3 and +4) (1 : 1 : 1 : 1 : 1 ratio).
Fig. 3 Log2 ratio distributions of quantified peptides (a) and proteins (b) of the sample mixed with 1 : 1 : 1 : 1 : 1 ratio; log2 ratio distributions of quantified peptides (c) and proteins (d) of the sample mixed with 1 : 2 : 4 : 2 : 1 ratio and log2 ratio distributions of quantified phosphopeptides (e) and phosphorylation sites (f) of the sample mixed with 1 : 1 : 1 : 1 : 1 ratio.
as over 96% of the log2 ratios of both peptides and proteins are within one-fold change of the theoretical values (Fig. 3c and d). Protein phosphorylation is one of the most important protein PTMs, playing crucial roles in a wide variety of cellular functions, such as signal transduction, cell cycle regulation, apoptosis, and cell differentiation.13 High throughput phosphoproteome quantification
The combination of different isotopic reagents in the five-plex isotope dimethyl labeling method
Label
#1
#2
#3
#4
#5
Formaldehyde isotope Cyanoborohydride isotope DMass (Da, one active site) DMass (Da, two active sites)
H2CO NaBH3CN 28.0313 56.0626
H2CO NaBD3CN 30.0439 60.0878
D2CO NaBH3CN 32.0564 64.1128
D2CO NaBD3CN 34.0690 68.1380
D213CO NaBD3CN 36.0757 72.1514
This journal is © The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 1708--1710 | 1709
View Article Online
Published on 09 December 2013. Downloaded by UNIVERSITY OF ALABAMA AT BIRMINGHAM on 28/10/2014 03:32:50.
ChemComm
strategies are also rapidly developing in recent years.14 In order to investigate the performance of this five-plex labeling strategy in phosphoproteome quantification, five identical protein samples were labeled and mixed together, followed by Ti4+-IMAC enrichment of the labeled phosphopeptides and SCX-RP 2D LC-MS/MS analysis (details shown in the ESI†). By using 5 100 mg starting materials, 1534 unique phosphopeptides (75% of the 2049 identified peptides) corresponding to 929 phosphoproteins (74% of the 1255 identified phosphoproteins) with 1976 phosphorylation sites (77% of the 2555 identified phosphorylation sites) were feasibly quantified among all of the five protein samples (data shown in the ESI†). Over 98% of all the log2 ratios of both phosphopeptides and phosphorylation sites fall in the range of [ 1, 1], demonstrating the high quantification accuracy of the method (Fig. 3e and f). All the above proteome and phosphoproteome experiments were performed twice and both the quantification accuracy and the number of quantified proteins and peptides was similar (Fig. S2 and Table S1, ESI†). In addition, we further spiked three standard proteins, ovalbumin, myoglobin and BSA, into five E. coli protein extraction samples (5 100 mg) to obtain standard/sample ratios (w/w) of 1 : 200, 2 : 200, 5 : 200, 10 : 200, 20 : 200 (BSA) and 20 : 200, 10 : 200, 5 : 200, 2 : 200, 1 : 200 (ovalbumin and myoglobin) (details shown in the ESI†). After digestion and labeling, the mixture of the five samples were analyzed by LC-MS. The results indicated that the E. coli proteome did not show significant change (over 99% log2 ratios of E. coli proteins fall in the range of [ 1, 1]), and measured ratios of all the three standard proteins were quite close to the theoretical ratios (Fig. 4) with RSDs o 0.16 in three parallel experiments. The quantified peptides of each protein standard were also examined, and most of the peptides showed good consistency with the theoretical ratios (Fig. S3, ESI†). Therefore, the above results demonstrated that high quantification accuracy can be feasibly obtained by using this five-plex isotope labeling method for comprehensive proteome and phosphoproteome quantification. The MS detection and data acquisition speed are greatly improved in recent years with the rapid development of new types of
Fig. 4 The quantification of three spiked-in protein standards. The standards were differentially mixed and then spiked into the E. coli samples to obtain standard/sample ratios (w/w) of 1 : 200, 2 : 200, 5 : 200, 10 : 200, 20 : 200 (BSA) and 20 : 200, 10 : 200, 5 : 200, 2 : 200, 1 : 200 (ovalbumin and myoglobin). Three quantification experiments were performed and the variations are shown as error bars.
1710 | Chem. Commun., 2014, 50, 1708--1710
Communication
mass spectrometers, which facilitates the application of the MS1 multiplex isotope labeling strategies to large-scale proteome and phosphoproteome quantification. Compared with our previous results of proteome or phosphoproteome quantification by using dual or triple dimethyl labeling,9,12 both the proteome coverage and the quantification accuracies are comparable (Fig. S4, ESI†). The quantification rates (quantified number/identified number) of conventional double or triple isotope dimethyl labeling (>90%) are higher than the five-plex dimethyl labeling (B78%). However, as the overlapping of two instrumental replicates is usually 60–80% in shotgun proteomics, it is still better to perform one five-plex quantification experiment than two or three conventional isotope labeling experiments separately. Therefore, we believe that the extension of the conventional double or triple dimethyl labeling to five channels has great potential in proteomic research that demands high throughput, high accuracy and simple operation. In this study, a new isotope dimethyl labeling method that enabled parallel analysis of five different protein samples in one experiment was developed. Both high throughput and high accuracy were obtained in comprehensive proteome and phosphoproteome quantification using this five-plex isotope labeling strategy. We believe that this multiplex isotope labeling strategy can be widely applied in high throughput proteome quantification of different types of biological samples, and will be beneficial in cellular dynamics investigations and biomarker discovery. Financial support from the China State Key Basic Research Program Grant (2013CB911203 and 2012CB910604), the NSFC (21021004, 81161120540 and 21235006), and the Analytical Method Innovation Program of MOST (2012IM030900) to H. Zou, and the financial support from NSFC (21305139) and ‘‘Hundred Talent Young Scientist Program’’ by DICP to F. Wang is gratefully acknowledged.
Notes and references 1 R. Aebersold and M. Mann, Nature, 2003, 422, 198–207. 2 S. E. Ong, B. Blagoev, I. Kratchmarova, D. B. Kristensen, H. Steen, A. Pandey and M. Mann, Mol. Cell. Proteomics, 2002, 1, 376–386. 3 P. L. Ross, Y. L. N. Huang, J. N. Marchese, B. Williamson, K. Parker, S. Hattan, N. Khainovski, S. Pillai, S. Dey, S. Daniels, S. Purkayastha, P. Juhasz, S. Martin, M. Bartlet-Jones, F. He, A. Jacobson and D. J. Pappin, Mol. Cell. Proteomics, 2004, 3, 1154–1169. 4 J. L. Hsu, S. Y. Huang, N. H. Chow and S. H. Chen, Anal. Chem., 2003, 75, 6843–6852. 5 P. J. Boersema, R. Raijmakers, S. Lemeer, S. Mohammed and A. J. R. Heck, Nat. Protoc., 2009, 4, 484–494. 6 D. Kovanich, S. Cappadona, R. Raijmakers, S. Mohammed, A. Scholten and A. J. Heck, Anal. Bioanal. Chem., 2012, 404, 991–1009. 7 S. Y. Huang, M. L. Tsai, C. J. Wu, J. L. Hsu, S. H. Ho and S. H. Chen, Proteomics, 2006, 6, 1722–1734. 8 Z. Sun, H. Qin, F. Wang, K. Cheng, M. Dong, M. Ye and H. Zou, Anal. Chem., 2012, 84, 8452–8456. 9 F. J. Wang, R. Chen, J. Zhu, D. G. Sun, C. X. Song, Y. F. Wu, M. L. Ye, L. M. Wang and H. F. Zou, Anal. Chem., 2010, 82, 3007–3015. 10 (a) L. Ting, R. Rad, S. P. Gygi and W. Haas, Nat. Methods, 2011, 8, 937–940; (b) L. V. DeSouza and K. W. Siu, Clin. Biochem., 2013, 46, 421–431. 11 M. Bantscheff, M. Schirle, G. Sweetman, J. Rick and B. Kuster, Anal. Bioanal. Chem., 2007, 389, 1017–1031. 12 C. Song, F. Wang, M. Ye, K. Cheng, R. Chen, J. Zhu, Y. Tan, H. Wang, D. Figeys and H. Zou, Anal. Chem., 2011, 83, 7755–7762. 13 T. Hunter, Cell, 2000, 100, 113–127. 14 (a) J. Villen, S. A. Beausoleil, S. A. Gerber and S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 1488–1493; (b) F. Wang, C. Song, K. Cheng, X. Jiang, M. Ye and H. Zou, Anal. Chem., 2011, 83, 8078–8085.
This journal is © The Royal Society of Chemistry 2014
