Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation

Edyta Marcon; Harshika Jain; Anandi Bhattacharya; Hongbo Guo; Sadhna Phanse; Shuye Pu; Gregory Byram; Ben C. Collins; Evan Dowdell; Maria Fenner; Xinghua Guo; Ashley Hutchinson; Jacob J. Kennedy; Bryan Krastins; Brett Larsen; Zhen-Yuan Lin; Mary F. Lopez; Peter Loppnau; Shane Miersch; Tin Nguyen; Jonathan B. Olsen; Marcin Paduch; Mani Ravichandran; Alma Seitova; Gouri Vadali; Maryann S. Vogelsang; Jeffrey R. Whiteaker; Guoqing Zhong; Nan Zhong; Lei Zhao; Ruedi Aebersold; Cheryl H. Arrowsmith; Andrew Emili; Lori Frappier; Anne-Claude Gingras; Matthias Gstaiger; Amanda G. Paulovich; Shohei Koide; Anthony A. Kossiakoff; Sachdev S. Sidhu; Shoshana J. Wodak; Susanne Gräslund; Jack F. Greenblatt; Aled M. Edwards

doi:10.1038/nmeth.3472

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation

Edyta Marcon, Harshika Jain, Anandi Bhattacharya, Hongbo Guo, Sadhna Phanse, Shuye Pu, Gregory Byram, Ben C. Collins, Evan Dowdell, Maria Fenner, Xinghua Guo, Ashley Hutchinson, Jacob J. Kennedy, Bryan Krastins, Brett Larsen, Zhen-Yuan Lin, Mary F. Lopez, Peter Loppnau, Shane Miersch, Tin NguyenJonathan B. Olsen, Marcin Paduch, Mani Ravichandran, Alma Seitova, Gouri Vadali, Maryann S. Vogelsang, Jeffrey R. Whiteaker, Guoqing Zhong, Nan Zhong, Lei Zhao, Ruedi Aebersold, Cheryl H. Arrowsmith, Andrew Emili, Lori Frappier, Anne-Claude Gingras, Matthias Gstaiger, Amanda G. Paulovich, Shohei Koide, Anthony A. Kossiakoff, Sachdev S. Sidhu, Shoshana J. Wodak, Susanne Gräslund, Jack F. Greenblatt, Aled M. Edwards

School of Biological Sciences

Research output: Contribution to journal › Article › peer-review

73 Citations (Scopus)

Abstract

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality—an absence that risks data integrity and reproducibility. We describe a mass spectrometry–based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Original language	English
Pages (from-to)	725-731
Number of pages	7
Journal	Nature Methods
Volume	12
Issue number	8
DOIs	https://doi.org/10.1038/nmeth.3472
Publication status	Published - 29 Jun 2015

Keywords

Proteomics
Proteomic analysis
Protein enrichment

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Marcon, E., Jain, H., Bhattacharya, A., Guo, H., Phanse, S., Pu, S., Byram, G., Collins, B. C., Dowdell, E., Fenner, M., Guo, X., Hutchinson, A., Kennedy, J. J., Krastins, B., Larsen, B., Lin, Z-Y., Lopez, M. F., Loppnau, P., Miersch, S., ... Edwards, A. M. (2015). Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation. Nature Methods, 12(8), 725-731. https://doi.org/10.1038/nmeth.3472

Marcon, Edyta ; Jain, Harshika ; Bhattacharya, Anandi ; Guo, Hongbo ; Phanse, Sadhna ; Pu, Shuye ; Byram, Gregory ; Collins, Ben C. ; Dowdell, Evan ; Fenner, Maria ; Guo, Xinghua ; Hutchinson, Ashley ; Kennedy, Jacob J. ; Krastins, Bryan ; Larsen, Brett ; Lin, Zhen-Yuan ; Lopez, Mary F. ; Loppnau, Peter ; Miersch, Shane ; Nguyen, Tin ; Olsen, Jonathan B. ; Paduch, Marcin ; Ravichandran, Mani ; Seitova, Alma ; Vadali, Gouri ; Vogelsang, Maryann S. ; Whiteaker, Jeffrey R. ; Zhong, Guoqing ; Zhong, Nan ; Zhao, Lei ; Aebersold, Ruedi ; Arrowsmith, Cheryl H. ; Emili, Andrew ; Frappier, Lori ; Gingras, Anne-Claude ; Gstaiger, Matthias ; Paulovich, Amanda G. ; Koide, Shohei ; Kossiakoff, Anthony A. ; Sidhu, Sachdev S. ; Wodak, Shoshana J. ; Gräslund, Susanne ; Greenblatt, Jack F. ; Edwards, Aled M. / Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation. In: Nature Methods. 2015 ; Vol. 12, No. 8. pp. 725-731.

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title = "Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation",

abstract = "Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality—an absence that risks data integrity and reproducibility. We describe a mass spectrometry–based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.",

keywords = "Proteomics, Proteomic analysis, Protein enrichment",

author = "Edyta Marcon and Harshika Jain and Anandi Bhattacharya and Hongbo Guo and Sadhna Phanse and Shuye Pu and Gregory Byram and Collins, {Ben C.} and Evan Dowdell and Maria Fenner and Xinghua Guo and Ashley Hutchinson and Kennedy, {Jacob J.} and Bryan Krastins and Brett Larsen and Zhen-Yuan Lin and Lopez, {Mary F.} and Peter Loppnau and Shane Miersch and Tin Nguyen and Olsen, {Jonathan B.} and Marcin Paduch and Mani Ravichandran and Alma Seitova and Gouri Vadali and Vogelsang, {Maryann S.} and Whiteaker, {Jeffrey R.} and Guoqing Zhong and Nan Zhong and Lei Zhao and Ruedi Aebersold and Arrowsmith, {Cheryl H.} and Andrew Emili and Lori Frappier and Anne-Claude Gingras and Matthias Gstaiger and Paulovich, {Amanda G.} and Shohei Koide and Kossiakoff, {Anthony A.} and Sidhu, {Sachdev S.} and Wodak, {Shoshana J.} and Susanne Gr{\"a}slund and Greenblatt, {Jack F.} and Edwards, {Aled M.}",

year = "2015",

month = jun,

day = "29",

doi = "10.1038/nmeth.3472",

language = "English",

volume = "12",

pages = "725--731",

journal = "Nature Methods",

issn = "1548-7091",

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Marcon, E, Jain, H, Bhattacharya, A, Guo, H, Phanse, S, Pu, S, Byram, G, Collins, BC, Dowdell, E, Fenner, M, Guo, X, Hutchinson, A, Kennedy, JJ, Krastins, B, Larsen, B, Lin, Z-Y, Lopez, MF, Loppnau, P, Miersch, S, Nguyen, T, Olsen, JB, Paduch, M, Ravichandran, M, Seitova, A, Vadali, G, Vogelsang, MS, Whiteaker, JR, Zhong, G, Zhong, N, Zhao, L, Aebersold, R, Arrowsmith, CH, Emili, A, Frappier, L, Gingras, A-C, Gstaiger, M, Paulovich, AG, Koide, S, Kossiakoff, AA, Sidhu, SS, Wodak, SJ, Gräslund, S, Greenblatt, JF & Edwards, AM 2015, 'Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation', Nature Methods, vol. 12, no. 8, pp. 725-731. https://doi.org/10.1038/nmeth.3472

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation. / Marcon, Edyta; Jain, Harshika; Bhattacharya, Anandi; Guo, Hongbo; Phanse, Sadhna; Pu, Shuye; Byram, Gregory; Collins, Ben C.; Dowdell, Evan; Fenner, Maria; Guo, Xinghua; Hutchinson, Ashley; Kennedy, Jacob J.; Krastins, Bryan; Larsen, Brett; Lin, Zhen-Yuan; Lopez, Mary F.; Loppnau, Peter; Miersch, Shane; Nguyen, Tin; Olsen, Jonathan B.; Paduch, Marcin; Ravichandran, Mani; Seitova, Alma; Vadali, Gouri; Vogelsang, Maryann S.; Whiteaker, Jeffrey R.; Zhong, Guoqing; Zhong, Nan; Zhao, Lei; Aebersold, Ruedi; Arrowsmith, Cheryl H.; Emili, Andrew; Frappier, Lori; Gingras, Anne-Claude; Gstaiger, Matthias; Paulovich, Amanda G.; Koide, Shohei; Kossiakoff, Anthony A.; Sidhu, Sachdev S.; Wodak, Shoshana J.; Gräslund, Susanne; Greenblatt, Jack F.; Edwards, Aled M.

In: Nature Methods, Vol. 12, No. 8, 29.06.2015, p. 725-731.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation

AU - Marcon, Edyta

AU - Jain, Harshika

AU - Bhattacharya, Anandi

AU - Guo, Hongbo

AU - Phanse, Sadhna

AU - Pu, Shuye

AU - Byram, Gregory

AU - Collins, Ben C.

AU - Dowdell, Evan

AU - Fenner, Maria

AU - Guo, Xinghua

AU - Hutchinson, Ashley

AU - Kennedy, Jacob J.

AU - Krastins, Bryan

AU - Larsen, Brett

AU - Lin, Zhen-Yuan

AU - Lopez, Mary F.

AU - Loppnau, Peter

AU - Miersch, Shane

AU - Nguyen, Tin

AU - Olsen, Jonathan B.

AU - Paduch, Marcin

AU - Ravichandran, Mani

AU - Seitova, Alma

AU - Vadali, Gouri

AU - Vogelsang, Maryann S.

AU - Whiteaker, Jeffrey R.

AU - Zhong, Guoqing

AU - Zhong, Nan

AU - Zhao, Lei

AU - Aebersold, Ruedi

AU - Arrowsmith, Cheryl H.

AU - Emili, Andrew

AU - Frappier, Lori

AU - Gingras, Anne-Claude

AU - Gstaiger, Matthias

AU - Paulovich, Amanda G.

AU - Koide, Shohei

AU - Kossiakoff, Anthony A.

AU - Sidhu, Sachdev S.

AU - Wodak, Shoshana J.

AU - Gräslund, Susanne

AU - Greenblatt, Jack F.

AU - Edwards, Aled M.

PY - 2015/6/29

Y1 - 2015/6/29

N2 - Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality—an absence that risks data integrity and reproducibility. We describe a mass spectrometry–based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

AB - Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality—an absence that risks data integrity and reproducibility. We describe a mass spectrometry–based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

KW - Proteomics

KW - Proteomic analysis

KW - Protein enrichment

U2 - 10.1038/nmeth.3472

DO - 10.1038/nmeth.3472

M3 - Article

VL - 12

SP - 725

EP - 731

JO - Nature Methods

JF - Nature Methods

SN - 1548-7091

IS - 8

ER -

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation

Abstract

Keywords

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