A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes Freek Bouwman, Johan Renes and Edwin Mariman Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands Differential gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a commonly used protein profiling method. However, observed changes can be explained in multiple ways, one of which is by the protein turnover rate. In order to easily and rapidly obtain information on both the identity and turnover of individual proteins, we applied a combination of protein labeling with L-(ring-2,3,4,5,6 2 H 5 ) phenylalanine and MALDI-TOF MS. While the spectrum reveals the identity of a protein, mass isotopomer analysis provides information about the rate of protein labeling as a measure of synthesis or turnover. Using this approach on mature 3T3-L1 adipocytes, we were able to discriminate between rapidly and slowly metabolised proteins. In our isolate, proteins of the cytoskeleton appeared to be slowly metabolised, whereas components of the extracellular matrix, in particular collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) showed rapid accumulation of newly synthesized proteins. Both proteins appeared to be metabolised in the same ratio as they are present in collagen fibers, i.e. 2:1 (COL1A1: COL1A2). In addition, functionally related proteins were also readily labeled. Taken together, we have shown that a combination of stable isotope labeling and protein profiling by gel electrophoresis and MALDI-TOF analysis can simultaneously provide information on the identity and relative metabolic rate of proteins in eukaryotic cells in a simple, nonhazardous and rapid-throughput way. Keywords: Adipocytes / Matrix-assisted laser desorption/ionization-time of flight mass spectrometry / Protein turnover / Stable isotopes / Two-dimensional gel electrophoresis Received: February 2, 2004; revised: April 16, 2004; accepted: May 1, 2004 Proteomics 2004, 4, 3855–3863 3855 1 Introduction Differential gel electrophoresis is commonly accepted as a powerful technique to study changes in gene expres- sion at the protein level. Traditionally, the composition of various protein isolates is compared after separation on 1-DE or 2-DE gels as band- or spot-patterns, respec- tively. Quantitative differences observed in this way are the reflection of differences in the expression of individual genes, which can be identified using mass spectrometric analysis of the respective proteins. Differences in protein concentration as seen on gels can in principle be ascrib- ed to alternative mechanisms of regulation including changes in protein turnover, PTM and transition to or from an organelle or a cellular substructure which is not repre- sented in the protein isolate [1, 2]. To be able to dis- criminate between these alternatives, protein profiling needs to be followed by other analytical assays [3]. Protein turnover is a result of protein synthesis and breakdown. In general, labeling experiments are per- formed to quantify these processes [4]. The rate of incor- Correspondence: Professor Dr. Edwin Mariman, Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, PO Box 616, NL- 6200 MD, Maastricht, The Netherlands E-mail: e.mariman@hb.unimaas.nl Fax: 131-43-3670976 Abbreviations: COL1A1, collagen type I alpha 1; COL1A2 col- lagen type I alpha 2  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200400861