ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Utility of Cleavable Isotope-Coded Affinity-Tagged Reagents for Quantification of Low-Copy Proteins Induced by Methylprednisolone Using Liquid Chromatography/Tandem Mass Spectrometry

View Author Information
The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York 14260-1200
Cite this: Anal. Chem. 2006, 78, 13, 4543–4552
Publication Date (Web):May 28, 2006
Copyright © 2006 American Chemical Society

    Article Views





    Other access options


    Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using 12C- (control) and 13C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.


    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. You can change your affiliated institution below.


     Corresponding author address:  The Department of Pharmaceutical Sciences, 539 Cooke Hall, University at Buffalo, State University of New York, Amherst, NY 14260-1200. Telephone:  (716) 645-2844, ext 243. Fax:  (716) 645-3693. E-mail:  [email protected].

    Cited By

    This article is cited by 31 publications.

    1. Qiong Wu, Baofeng Zhao, Yejing Weng, Yichu Shan, Xiao Li, Yechen Hu, Zhen Liang, Huiming Yuan, Lihua Zhang, Yukui Zhang. Site-Specific Quantification of Persulfidome by Combining an Isotope-Coded Affinity Tag with Strong Cation-Exchange-Based Fractionation. Analytical Chemistry 2019, 91 (23) , 14860-14864.
    2. Jun Qu, Rebeccah Young, Brian J. Page, Xiaomeng Shen, Nazneen Tata, Jun Li, Xiaotao Duan, James A. Fallavollita, and John M. Canty, Jr. . Reproducible Ion-Current-Based Approach for 24-Plex Comparison of the Tissue Proteomes of Hibernating versus Normal Myocardium in Swine Models. Journal of Proteome Research 2014, 13 (5) , 2571-2584.
    3. Eslam Nouri-Nigjeh, Ming Zhang, Tao Ji, Haoying Yu, Bo An, Xiaotao Duan, Joseph Balthasar, Robert W. Johnson, and Jun Qu . Effects of Calibration Approaches on the Accuracy for LC–MS Targeted Quantification of Therapeutic Protein. Analytical Chemistry 2014, 86 (7) , 3575-3584.
    4. Chengjian Tu, Jun Li, Rebeccah Young, Brian J. Page, Frank Engler, Marc S. Halfon, John M. Canty, Jr., and Jun Qu . Combinatorial Peptide Ligand Library Treatment Followed by a Dual-Enzyme, Dual-Activation Approach on a Nanoflow Liquid Chromatography/Orbitrap/Electron Transfer Dissociation System for Comprehensive Analysis of Swine Plasma Proteome. Analytical Chemistry 2011, 83 (12) , 4802-4813.
    5. De Lin, Jing Li, Robbert J. C. Slebos, and Daniel C. Liebler . Cysteinyl Peptide Capture for Shotgun Proteomics: Global Assessment of Chemoselective Fractionation. Journal of Proteome Research 2010, 9 (10) , 5461-5472.
    6. Jin Cao, Vanessa M. Covarrubias, Robert M. Straubinger, Hao Wang, Xiaotao Duan, Haoying Yu, Jun Qu and Javier G. Blanco . A Rapid, Reproducible, On-the-Fly Orthogonal Array Optimization Method for Targeted Protein Quantification by LC/MS and Its Application for Accurate and Sensitive Quantification of Carbonyl Reductases in Human Liver. Analytical Chemistry 2010, 82 (7) , 2680-2689.
    7. Xiaotao Duan, Rebeccah Young, Robert M. Straubinger, Brian Page, Jin Cao, Hao Wang, Haoying Yu, John M. Canty, Jr. and Jun Qu . A Straightforward and Highly Efficient Precipitation/On-Pellet Digestion Procedure Coupled with a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-Free Expression Profiling of the Swine Heart Mitochondrial Proteome. Journal of Proteome Research 2009, 8 (6) , 2838-2850.
    8. Hao Wang, Robert M. Straubinger, John M. Aletta, Jin Cao, Xiaotao Duan, Haoying Yu, Jun Qu. Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and drbitrap mass spectrometry. Journal of the American Society for Mass Spectrometry 2009, 20 (3) , 507-519.
    9. Tomoko Ichibangase,, Kyoji Moriya,, Kazuhiko Koike, and, Kazuhiro Imai. A Proteomics Method Revealing Disease-Related Proteins in Livers of Hepatitis-Infected Mouse Model. Journal of Proteome Research 2007, 6 (7) , 2841-2849.
    10. Chandrabose Selvaraj, Umesh Panwar, Karthik Raja Ramalingam, Rajendran Vijayakumar, Sanjeev Kumar Singh. Exploring the macromolecules for secretory pathway in cancer disease. 2023, 55-83.
    11. Vivaswath S. Ayyar, William J. Jusko, . Transitioning from Basic toward Systems Pharmacodynamic Models: Lessons from Corticosteroids. Pharmacological Reviews 2020, 72 (2) , 414-438.
    12. Jens Atzrodt, Volker Derdau, William J. Kerr, Marc Reid. Deuterium‐ und tritiummarkierte Verbindungen: Anwendungen in den modernen Biowissenschaften. Angewandte Chemie 2018, 130 (7) , 1774-1802.
    13. Jens Atzrodt, Volker Derdau, William J. Kerr, Marc Reid. Deuterium‐ and Tritium‐Labelled Compounds: Applications in the Life Sciences. Angewandte Chemie International Edition 2018, 57 (7) , 1758-1784.
    14. Gang Liu, Kai Cheng, Chi Y. Lo, Jun Li, Jun Qu, Sriram Neelamegham. A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis. Molecular & Cellular Proteomics 2017, 16 (11) , 2032-2047.
    15. Mingmei Yuan, Cong Feng, Shouyun Wang, Weiwei Zhang, Mo Chen, Hong Jiang, Xuesong Feng, . Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS. PLOS ONE 2017, 12 (9) , e0184152.
    16. Bo An, Ming Zhang, Jun Qu. LC/MS versus Immune‐Based Bioanalytical Methods in Quantitation of Therapeutic Biologics in Biological Matrices. 2015, 1-17.
    17. Osama Chahrour, Diego Cobice, John Malone. Stable isotope labelling methods in mass spectrometry-based quantitative proteomics. Journal of Pharmaceutical and Biomedical Analysis 2015, 113 , 2-20.
    18. Kubra Kamisoglu, Siddharth Sukumaran, Eslam Nouri-Nigjeh, Chengjian Tu, Jun Li, Xiaomeng Shen, Xiaotao Duan, Jun Qu, Richard R. Almon, Debra C. DuBois, William J. Jusko, Ioannis P. Androulakis. Tandem Analysis of Transcriptome and Proteome Changes after a Single Dose of Corticosteroid: A Systems Approach to Liver Function in Pharmacogenomics. OMICS: A Journal of Integrative Biology 2015, 19 (2) , 80-91.
    19. Gustaf Sandh, Margareta Ramström, Karin Stensjö. Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments. BMC Genomics 2014, 15 (1)
    20. Bo An, Ming Zhang, Jun Qu. Toward Sensitive and Accurate Analysis of Antibody Biotherapeutics by Liquid Chromatography Coupled with Mass Spectrometry. Drug Metabolism and Disposition 2014, 42 (11) , 1858-1866.
    21. Xiaomeng Shen, Rebeccah Young, John M. Canty, Jun Qu. Quantitative proteomics in cardiovascular research: Global and targeted strategies. PROTEOMICS – Clinical Applications 2014, 8 (7-8) , 488-505.
    22. Sapan Patel, Armand G. Ngounou Wetie, Costel C. Darie, Bayard D. Clarkson. Cancer Secretomes and Their Place in Supplementing Other Hallmarks of Cancer. 2014, 409-442.
    23. Cheng‐ye Guo, Hou‐yu Wang, Xiao‐Ping Liu, Liu‐yin Fan, Lei Zhang, Cheng‐xi Cao. Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis. ELECTROPHORESIS 2013, 34 (9-10) , 1343-1351.
    24. Jun Qu, Alan J Lesse, Aimee L Brauer, Jin Cao, Steven R Gill, Timothy F Murphy. Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses. BMC Microbiology 2010, 10 (1)
    25. Kaname Ohyama, Mari Tomonari, Tomoko Ichibangase, Hideto To, Naoya Kishikawa, Kenichiro Nakashima, Kazuhiro Imai, Naotaka Kuroda. A toxicoproteomic study on cardioprotective effects of pre-administration of docetaxel in a mouse model of adriamycin-induced cardiotoxicity. Biochemical Pharmacology 2010, 80 (4) , 540-547.
    26. Zuly Rivera-Monroy, Guenther K. Bonn, András Guttman. Fluorescent isotope-coded affinity tag (FCAT) I: Design and synthesis. Bioorganic Chemistry 2008, 36 (6) , 299-311.
    27. Vinzenz Lange, Johan A. Malmström, John Didion, Nichole L. King, Björn P. Johansson, Juliane Schäfer, Jonathan Rameseder, Chee-Hong Wong, Eric W. Deutsch, Mi-Youn Brusniak, Peter Buühlmann, Lars Björck, Bruno Domon, Ruedi Aebersold. Targeted Quantitative Analysis of Streptococcus pyogenes Virulence Factors by Multiple Reaction Monitoring. Molecular & Cellular Proteomics 2008, 7 (8) , 1489-1500.
    28. Petia Shipkova, Dieter M. Drexler, Robert Langish, James Smalley, Mary Ellen Salyan, Mark Sanders. Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides. Rapid Communications in Mass Spectrometry 2008, 22 (9) , 1359-1366.
    29. Andreas Prange, Daniel Pröfrock. Chemical labels and natural element tags for the quantitative analysis of bio-molecules. Journal of Analytical Atomic Spectrometry 2008, 23 (4) , 432.
    30. Xiaotao Duan, Xiaoyan Chen, Yiming Yang, Dafang Zhong. Precolumn derivatization of cysteine residues for quantitative analysis of five major cytochrome P450 isoenzymes by liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2007, 21 (20) , 3234-3244.
    31. . Current literature in mass spectrometry. Journal of Mass Spectrometry 2007, 407-418.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect