Metabolic Labeling and Protein Linearization Technology Allow the Study of Proteins Secreted by Cultured Cells in Serum-Containing Media M. Colzani,* ,† P. Waridel, † J. Laurent, ‡ E. Faes, ‡ C. Ru ¨egg, ‡,§ and M. Quadroni* ,† Protein Analysis Facility, Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland, Division of Experimental Oncology, Multidisciplinary Oncology Center (CePO), Centre Hospitalier Universitaire Vaudois (CHUV), University of Lausanne, Lausanne, Switzerland, and National Center of Competence in Research (NCCR) Molecular Oncology, ISREC, School of Life Sciences, EPFL, Lausanne, Switzerland Received May 29, 2009 Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)- based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples. Keywords: stable isotope labeling • protein concentration linearization • secretome analysis • LC-MS/MS Introduction Proteins actively secreted by cells fulfill a number of biologi- cal functions, essential for the interactions with the external environment. Secreted proteins can affect the development (e.g., growth factors), the adhesion, and the migration (e.g., matrix proteins, chemokines) of neighboring cells. They can also influence the body’s metabolism (hormones) and mediate immune functions (e.g., antibodies, cytokines), through auto- crine, paracrine and endocrine signals. The study of the pool of secreted proteins (the secretome) can provide a better understanding of the molecular mechanisms underlying these processes in health and in disease conditions. 1 Besides actively secreted proteins, other cellular polypeptides can enter the bloodstream, either through passive release by leakage from damaged tissues or shedding from the cell surface. For all these reasons, numerous studies have focused on the serum and plasma proteomes to discover disease biomarkers and targets for therapeutics. 2-4 But the extreme complexity of the blood proteome and the very large dynamic range of protein con- centrations make the analysis of serum/plasma samples ex- tremely challenging. 5,6 Proteomic workflows based on sample fractionation and enrichment of low-abundance proteins have been applied to achieve a more comprehensive characterization of serum/plasma. 7-11 In spite of this, it is the common opinion that the analytical depth available for untargeted proteomics remains insufficient for comprehensively mapping serum/ plasma samples. Only a few studies have characterized the cellular secretome in vivo, 12,13 because of the inherent difficulties. As a proxy, a commonly used approach is the analysis of media conditioned by cells in culture (CM). 14-16 This model assumes that cells grown in vitro present a secretion phenotype similar to the one in vivo. The obvious advantage of cells in culture is the possibility to study variations of the secretome induced by specific events (e.g., treatment with growth factors, drugs or other stimuli). Despite the complexity reduction and the experimental advantages offered by in vitro model systems, even secretome profiling based on CM analysis presents serious challenges. The first one is the low concentration (as low as ng/mL) at which the proteins are secreted in the CM. 17 The second is that the actively secreted proteins are mixed with the ones passively released by cell death and lysis, which “contaminate” the sample. Proteins released by lysed cells can indeed represent an important proportion of the CM proteome and can be * To whom correspondence should be addressed. Manfredo Quadroni, phone 41-21-692-56-76, fax 41-21-692-57-05, e-mail manfredo.quadroni@ unil.ch. Mara Colzani, phone 41-21-692-56-77, fax 41-21-692-57-05, e-mail mara.colzani@unil.ch. † Center for Integrative Genomics, University of Lausanne. ‡ Centre Hospitalier Universitaire Vaudois (CHUV), University of Lausanne. § School of Life Sciences, EPFL. 10.1021/pr900476b CCC: $40.75  2009 American Chemical Society Journal of Proteome Research 2009, 8, 4779–4788 4779 Published on Web 08/27/2009