Benchmarking stable isotope labeling based quantitative proteomics ☆ A.F. Maarten Altelaar a, b, 1 , Christian K. Frese a, b, 1 , Christian Preisinger a, b , Marco L. Hennrich a, b , Andree W. Schram c , H.Th. Marc Timmers b, c , Albert J.R. Heck a, b, ⁎ , Shabaz Mohammed a, b, ⁎ a Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands b Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands c Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands ARTICLE INFO ABSTRACT Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or chemical isotope labeling with MS based quantification or chemical labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and chemical labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: EUPA 2012: NEW HORIZONS. © 2012 Elsevier B.V. All rights reserved. Keywords: Quantitation Dimethyl labeling SILAC Isobaric labeling 1. Introduction Robust and accurate quantification of (differential) protein expression levels is essential for deciphering the dynamics of proteomes. Mass spectrometry provides excellent means for quantitative proteomics [1] whereby the most common and accurate quantitative approaches utilize stable iso- topes [2–4]. In recent years several strategies have been JOURNAL OF PROTEOMICS XX (2012) XXX – XXX Abbreviations: MS/MS, Tandem Mass Spectrometry; SILAC, Stable Isotope Labeling by Amino acids in Cell culture; TMT, Tandem Mass Tag; iTRAQ, Isobaric Tag for Relative and Absolute Quantification; SCX, Strong Cation Exchange; FDR, False Discovery Rate; CID, Collision induced dissociation; HCD, Higher energy C-trap dissociation; AGC, Automatic Gain Control; NCE, Normalized Collision Energy; PSMs, Peptide to Spectrum Matches; AP-MS, Affinity Purification based Mass Spectrometry. ☆ This article is part of a Special Issue entitled: EUPA 2012: NEW HORIZONS. ⁎ Corresponding authors at: Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: +31 302539974; fax: + 31 302518219. E-mail addresses: a.j.r.heck@uu.nl (A.J.R. Heck), s.mohammed@uu.nl (S. Mohammed). 1 These authors contribute equally to this work. 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2012.10.009 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot JPROT-01198; No of Pages 13 Please cite this article as: Altelaar A.F.M, et al, Benchmarking stable isotope labeling based quantitative proteomics, J Prot (2012), http://dx.doi.org/10.1016/j.jprot.2012.10.009