18O Stable Isotope Labeling in MS-based Proteomics

Xiaoying Ye Brian Luke Thorkell Andresson Josip Blonder A variety of stable isotope labeling techniques have been developed and used in mass spectrometry (MS)-based proteomics, primarily for relative quantitation of changes in protein abundances between two compared samples, but also for qualitative characterization of differentially labeled proteomes. Differential 16O/18O coding relies on the 18O exchange that takes place at the C-terminal carboxyl group of proteolytic fragments, where two 16O atoms are typically replaced by two 18O atoms by enzyme-catalyzed oxygen-exchange in the presence of H218O. The resulting mass shift between differentially labeled peptide ions permits identification, characterization and quantitation of proteins from which the peptides are proteolytically generated. This review focuses on the utility of 16O/18O labeling within the context of mass spectrometry-based proteome research. Different strategies employing 16O/18O are examined in the context of global comparative proteome profiling, targeted subcellular proteomics, analysis of post-translational modifications and biomarker discovery. Also discussed are analytical issues related to this technique, including variable 18O exchange along with advantages and disadvantages of 16O/18O labeling in comparison with other isotope-coding techniques. - INTRODUCTION A major goal of proteomics is to develop methods enabling the systematic quantitation of protein abundances within the cell/tissue or the comparative measurement of changes in protein abundances between two different states (e.g. healthy versus disease). Therefore, mass spectrometry (MS)-based approaches that quantify changes in protein abundances play an important role in systems biology, improving our understanding of fundamental biological processes or facilitating the identification of specific protein biomarkers [1]. The absolute quantitation of proteins using isotopically labeled synthetic peptides is typically employed in an experimental setting in which proteins of interest are known and physical changes in their abundances are expected to be regulated by particular stimuli or pathological processes. To identify and quantify unknown proteins presumably implicated in certain physiological or pathological responses, global quantitative profiling techniques that measure changes in protein abundances between two samples are required. Corresponding author. Dr Josip Blonder, Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick Inc., NCI at Frederick, P.O. Box B, Frederick, MD 21702-1201, USA. Tel: 1 301 846 7211; Fax: 1 301 846 6037; E-mail: XiaoyingYe is the Postdoctoral Fellow of the Laboratory of Proteomics and Analytical Technologies. Her current research interests involve the development of isotopic-labeled and label-free methods for quantitative proteomics and the application of these techniques to cancer biomarker research and protein complex identification. Brian Luke is a Senior Scientist at the Advanced Biomedical Computing Center. His research includes developing new algorithms for the analysis of large genomic and proteomic datasets. Thorkell Andresson is the Associate Director of Proteomics, Laboratory of Proteomics and Analytical Technologies. His current research interests involve utilizing mass spectrometry to study proteinprotein interaction and protein complex formation under both normal and pathophysiological conditions, with emphasis on quantitative and stoichiometric assessment of these vital physiological processes. Josip Blonder is the Head of Quantitative Proteomics, Laboratory of Proteomics and Analytical Technologies. His current research interests include the development of mass spectrometry techniques for quantitative analysis of membrane proteins and cancer biomarker discovery using clinically relevant specimens. Differential stable isotope labeling that relies on isotope incorporation at the protein or peptide level is primarily employed in the realm of liquid chromatography-mass spectrometry (LC-MS)-based, shotgun proteomics. Recent developments in stable isotope labeling and LC-MS offer significant advantages over 2D-PAGE-based comparative proteomics, including better coverage/quantitation of membrane proteins, proteins with extreme molecular weight and/or pI. Currently, two distinct techniques are used for the incorporation of stable isotopes into the proteome of interest: (i) in vivo labeling, which is accomplished metabolically by supplying the cell/ organism of interest with nutrients highly enriched in stable isotopes [2], using simultaneous anabolic isotope incorporation into all cellular proteins; (ii) in vitro stable isotope labeling, which relies on chemical [3, 4] or enzymatic incorporation of isotopes into the proteome of interest at the protein and/or peptide level [5] after cell lysis or tissue homogenization. Although the 16O/18O labeling is not the most commonly used isotope-tagging technique, its simplicity and instantaneous applicability to clinically relevant and amount-limited samples make this technique easily applicable for protein biomarker discovery that relies on MS-based profiling of human specimens. These specimens typically include tissues obtained by laser-capture microdissection or biofluids obtained by a variety of biopsy procedures. This review focuses on recent developments in the realm of enzyme-mediated 16O/18O stable isotope labeling and its overall utility in MS-based proteomics. PRINCIPLE AND PRACTICE OF 16O/18O LABELING Enzyme-facilitated 18O labeling is a simple technique for tagging peptides in the presence of H218O. It typically relies on class-2 proteases (e.g. trypsin) to catalyze the exchange of two 16O2 atoms for two 18O2 atoms at the C-terminal carboxyl group of proteolytic peptides, resulting in a mass shift of 4 Da between singly charged, differentially labeled peptide ions observed in MS1 mode (Figure 1). The first study describing an enzyme-catalyzed oxygen exchange in the presence of H218O was reported in 1951 by Sprinson and Rittenberg [6], while MS spectra obtained by Antonov et al. using electronbeam MS explicitly showed a mass shift resulting from enzyme-catalyzed 18O incorporation at the carboxylic group of proteolytic peptides [7]. Desiderio and Kai employed enzyme-catalyzed 18O exchange for the preparation of internal standards for MS-based quantitation of peptides in biological extracts [8]. Mirgorodskaya et al. and Stewart et al. [9, 10] proposed the use of 16O/18O labeling for MS-based quantitation of proteins; the application of this technique as an effective quantitative solutionbased, shotgun proteomic tool was first reported by Yao et al. [5]. Coupling the SDSPAGEbased quantitative approach with post-digestion 18O exchange for differential proteomics of protein complexes was first proposed by Bantscheff et al. [11]. 16O/18O labeling has also been used for nonquantitative proteomic (...truncated)