PROTEOLYTIC 18 O-LABELING STRATEGIES FOR QUANTITATIVE PROTEOMICS Masaru Miyagi* and K.C. Sekhar Rao Case Center for Proteomics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 Received 10 May 2006; received (revised) 28 August 2006; accepted 12 September 2006 Published online 3 November 2006 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20116 A number of proteomic techniques have been developed to quantify proteins in biological systems. This review focuses on the quantitative proteomic technique known as ‘‘proteolytic 18 O-labeling.’’ This technique utilizes a protease and H 2 18 O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein’s expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein–protein interactions. The present review discusses various aspects of the 18 O-labeling technique, including: its history, the advantages and disadvantages of the proteolytic 18 O-labeling technique compared to other techniques, enzymatic considerations, the problem of variable incorporation of 18 O atoms into peptides with a discussion on recent advancements of the technique to overcome it, computational tools to interpret the data, and a review of the biological applications. # 2006 Wiley Periodicals, Inc., Mass Spec Rev 26:121–136, 2007 Keywords: 18 O-labeling; quantitative proteomics; compara- tive proteomics; mass spectrometry; oxygen-18; carboxyl oxygen exchange reaction I. INTRODUCTION The completion of the genome sequencing of a multitude of organisms, the emergence of new technologies in mass spectrometry, and the development of computational tools that link mass spectrometry data with protein sequence databases for the identification and covalent structural analysis of proteins have together fostered unprecedented opportunities to study proteins on a large scale. Proteins are identified by using these tools by analyzing peptides that are generated from the proteins by enzymatic hydrolysis or chemical fragmentation. However, the mere identification of a protein expressed in a biological system is not sufficient to answer most biological questions. More and more, increasingly, quantitative answers are required (e.g., changes in a protein’s expression levels). Therefore, develop- ment of quantitative proteomic methods is of critical importance for the advancement of proteomic research. Although there are a number of quantitative proteomic methods that have been developed and have been applied to biological samples, further development of these techniques are required to address existing limitations. No technique currently satisfies all the demands required for accurate proteomic determinations. The value of the information obtained in proteomic studies continues to drive the use of these techniques even with their shortcomings. Even though proteolytic 18 O-labeling is not yet a common technique compared to other quantitative proteomic techniques, it has the potential to be useful and to become a widely used technique. There are two major drawbacks to the technique: (1) variable incorporation of 18 O atoms into peptides when trypsin was used as a catalyst (Julka & Regnier, 2004); and (2) lack of broad-based computational tools. However, recent advance- ments in the 18 O-labeling techniques and development of computational tools show considerable promise to overcome these problems. This review will focus on the recent advances of the proteolytic 18 O-labeling technique. II. CURRENT QUANTITATIVE PROTEOMIC METHODS—BRIEF OVERVIEW All current proteomic methods that quantify unknown proteins are relative methods: meaning protein amounts determined in a sample are measured relative to the amounts of the same proteins in other samples. Absolute quantification of proteins can be done by using isotopically labeled synthetic peptides and mass spectrometry (Gerber et al., 2003); however, this goal requires foreknowledge of the target proteins and preparation of isotopically-labeled synthetic peptides for each of the target proteins. This need precludes the use of this technique in global quantitative proteomic analysis that seek to identify and quantify unknown proteins in a sample. A number of quantitative proteomic methods have been developed as shown in Figure 1. There are two primary strategies used in current quantitative proteomics: two-dimensional gel electrophoresis (2D-PAGE)-based methods and mass spectro- metric methods based on isotope labeling strategies. A brief overview of different quantitative proteomic methods that discusses their strengths and limitations is given below. More Mass Spectrometry Reviews, 2007, 26, 121– 136 # 2006 by Wiley Periodicals, Inc. ———— Contract grant sponsor: National Institutes of Health; Contract grant numbers: EY014020, RR016741, RR017699. *Correspondence to: Masaru Miyagi, Case Center for Proteomics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, BRB 928, Cleveland, OH 44106. E-mail: masaru.miyagi@case.edu