Synthesis of 13 C-Labeled Iodoacetanilide and Application to Quantitative Peptide Analysis by Isotope Differential Mass Spectrometry Satomi Niwayama, a, * Sadamu Kurono b and Hiroyuki Matsumoto b, * a Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, USA b Department of Biochemistry and Molecular Biology, NSF EPSCoR Oklahoma Biotechnology Networtk Laser Mass Spectrometry Facility, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA Received 26 February 2003; accepted 28 April 2003 Abstract— 13 C-Labeled and unlabeled iodoacetanilides have been synthesized for covalent modification of the sulfhydryl groups of cysteine residues in proteins or peptides. A combination of these reagents, coupled with mass spectrometry, is a powerful tool for quantitative analysis of peptides and hence proteins. # 2003 Elsevier Ltd. All rights reserved. Quantitative analysis of proteins is an essential part of proteomics, which studies proteomes, a set of proteins expressed under certain physiological conditions. Examples of conventional methods for quantification include densitometry of the gel and counting radio- activities that are incorporated by metabolic labeling. 1 3 Recently, stable isotope labeling followed by mass spectrometry analysis has been emerging as a powerful technology for more accurate quantification of proteins. This technique is based on the assumption that the iso- topically labeled molecule and its parent species behave similarly and their ionization efficiencies are the same. 4 Most common and classical approaches have relied on incorporation of specific isotope atoms such as 15 N, 18 O, or D into growing cells in the isotope-enriched media or into the digested peptides during the proteo- lytic cleavage of proteins. However, such methods require a long processing time and are laborious, and may not be applicable to more complex systems such as whole animals. In contrast, chemical modifications by covalent labeling on specific amino acid residues using isotope-labeled reagents followed by mass spectrometry analysis is versatile and convenient, as it can handle any protein extracts. The pioneering work by Aebersold et al. utilized deuterium-labeled isotope-coded affinity tags (ICATs). 5 Their method applies a biotinylated con- jugate of iodoacetamide, which is known to be a specific sulfhydryl group modifier in cysteine residues in pep- tides, and its deuterated derivative. In this method, the biotinyl moiety is used to affinity-purify the resulting peptides that are carrying the modified cysteine residues, making the analysis of whole proteins feasible. If, how- ever, each protein is separated and displayed on a two- dimensional (2-D) gel electrophoresis, the affinity puri- fication of the cysteine-modified peptides will be unne- cessary, making the entire protocol significantly simpler compared to the ICAT method. We have developed a prototype of such a method using isotope-labeled alkylmaleimides. 6 The combination of isotope-labeled and unlabeled chemical modification of specific amino acid residues followed by 2-D gel electropheresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) makes the whole process handier and more economical compared to the ICAT method. Here, we report synthesis of another sulfhydryl-group- specific modifier, 13 C 6 -labeled and unlabeled iodoaceta- nilides (IAA), and the application of this modifier to quantitative analysis of peptides in the context of our proteomics research. We believe that the success of this work signifies success in quantification of tryptic peptides, hence proteins (Scheme 1). 0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0960-894X(03)00503-1 Bioorganic & Medicinal Chemistry Letters 13 (2003) 2913–2916 *Corresponding author. Tel.: +1-405-744-6594; fax: +1-405-744- 6007; e-mail: niwayama@biochem.okstate.edu (S. Niwayama); tel.: +1-405-271-2227; fax: +1-405-271-3139; e-mail: hiro-matsumoto@ ouhsc.edu (H. Matsumoto).