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Response to the comments on “Determining Allele-Specific Protein Expression (ASPE) Using a Novel QconCAT-Based Proteomics Method” Jian Shi, Xinwen Wang, Huaijun Zhu, Hui Jiang, Danxin Wang, Alexey Nesvizhskii, and Hao-Jie Zhu J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019
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Journal of Proteome Research
Response to the comments on “Determining Allele-Specific Protein Expression (ASPE) Using a Novel QconCAT-Based Proteomics Method” Jian Shi 1, Xinwen Wang 1, Huaijun Zhu 1, 2, Hui Jiang 3, Danxin Wang 4, Alexey Nesvizhskii 5, Hao-Jie Zhu 1* 1
Department of Clinical Pharmacy, University of Michigan, Ann Arbor, MI 48109
2
Department of Pharmacy, Drum Tower Hospital Affiliated to Medical School of Nanjing
University, Nanjing, Jiangsu, China 3 Department
4
of Biostatistics, University of Michigan, Ann Arbor, MI 48109
Department of Cancer Biology and Genetics, Center for Pharmacogenomics, School of
Medicine, Ohio State University, Columbus, OH 43210 5
Department of Pathology, Department of Computational Medicine and Bioinformatics,
University of Michigan, Ann Arbor, MI 48109 Corresponding author: Hao-Jie Zhu, Ph.D. Department of Clinical Pharmacy University of Michigan College of Pharmacy 428 Church Street, Room 3567 CCL Ann Arbor, MI 48109-1065 Tel: 734-763-8449, E-mail:
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Abstract Russell and colleagues deserve credit for being the first to use a QconCAT standard to simultaneously quantify both the wild-type and mutant peptides of a protein (i.e., CYP2B6) (J. Proteome Res. 2013,12 (12), 5934−5942, DOI: 10.1021/pr400279u). However, the rationale of their study was entirely different from ours (J. Proteome Res. 2018, 17 (10), 3606−3612, DOI: 10.1021/acs.jproteome.8b00620). Their study focused on the quantification of individual drugmetabolizing enzymes and transporters, whereas ours developed a targeted proteomics method to determine the allele-specific protein expression (ASPE) of a gene and advocated the use of the ASPE imbalance as the phenotype for identifying cis-regulatory genetic variants of the gene. More importantly, the digestion enzyme trypsin interacts with 3-4 amino acid residues around scissile bonds, and certain residues, such as negatively charged amino acids, can significantly affect the digestion efficiency. The QconCAT standard reported in our study differs from conventional QconCAT standards such as that used by Russell et al. in that at least 15 native flanking amino acids were included to ensure accurate measurement of ASPE ratios.
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Journal of Proteome Research
It was an oversight on our part to have missed the relevance of the referred publication1 during the literature search. Russell and colleagues deserve credit for being the first to use a QconCAT standard to simultaneously quantify both the wild-type and mutant peptides of a protein (i.e., CYP2B6). That said, the rationale of their study1 was entirely different from ours.2 Their study focused on the quantification of individual drug-metabolizing enzymes and transporters, whereas ours developed a targeted proteomics method to determine the allele-specific protein expression (ASPE) of a gene and advocated the use of the ASPE imbalance as the phenotype for identifying cis-regulatory genetic variants of the gene. More importantly, to accurately quantify the two allele expression ratios of a gene, the QconCAT standard should behave in the same way as the corresponding native protein during sample preparation (e.g., digestion and extraction) and MS analysis. The digestion enzyme trypsin interacts with 3-4 amino acid residues around scissile bonds, and certain residues, such as negatively charged amino acids, can significantly affect the digestion efficiency.3, 4 Cheung et al. assessed the effect of natural amino acid flanking sequence on the trypsin digestion efficiency of QconCAT proteins, and concluded that reliable quantification requires the inclusion of six or more amino acid flanking residues.5 Toward this end, different from conventional QconCAT standards such as that used by Russell et al., the QconCAT standard reported in our study included at least 15 native flanking amino acids to ensure accurate measurement of ASPE ratios.
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References 1.
Russell, M. R.; Achour, B.; McKenzie, E. A.; Lopez, R.; Harwood, M. D.; Rostami-
Hodjegan, A.; Barber, J., Alternative Fusion Protein Strategies to Express Recalcitrant QconCAT Proteins for Quantitative Proteomics of Human Drug Metabolizing Enzymes and Transporters. Journal of Proteome Research 2013, 12, (12), 5934-5942. 2.
Shi, J.; Wang, X.; Zhu, H.; Jiang, H.; Wang, D.; Nesvizhskii, A.; Zhu, H. J., Determining
Allele-Specific Protein Expression (ASPE) Using a Novel Quantitative Concatamer Based Proteomics Method. J Proteome Res 2018, 17, (10), 3606-3612. 3.
Schechter, I.; Berger, A., On the size of the active site in proteases. I. Papain. Biochem
Biophys Res Commun 1967, 27, (2), 157-62. 4.
Giansanti, P.; Tsiatsiani, L.; Low, T. Y.; Heck, A. J., Six alternative proteases for mass
spectrometry-based proteomics beyond trypsin. Nat Protoc 2016, 11, (5), 993-1006. 5.
Cheung, C. S. F.; Anderson, K. W.; Wang, M.; Turko, I. V., Natural Flanking Sequences
for Peptides Included in a Quantification Concatamer Internal Standard. Analytical Chemistry 2015, 87, (2), 1097-1102.
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