MICROWAVE-ASSISTED PROTEOMICS Jennie R. Lill,* Elizabeth S. Ingle, Peter S. Liu, Victoria Pham, and Wendy N. Sandoval Protein Chemistry Department, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 Received 13 November 2006; received (revised) 29 January 2007; accepted 5 February 2007 Published online 1 May 2007 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20140 State-of-the-art proteomic analysis has recently undergone a rapid evolution; with more high-throughput analytical instru- mentation and informatic tools available, sample preparation is becoming one of the rate-limiting steps in protein characteriza- tion workflows. Recently several protocols have appeared in the literature that employ microwave irradiation as a tool for the preparation of biological samples for subsequent mass spectro- metric characterization. Techniques for microwave-assisted bio- catalyzed reactions (including sample reduction and alkylation, enzymatic and chemical digestion, removal and analysis of post- translational modifications and characterization of enzymes and protein-interaction sites) are described. This review summarizes the various approaches undertaken, instrumentation employed, and reduction in overall experimental time observed when microwave assistance is applied. # 2007 Wiley Periodicals, Inc., Mass Spec Rev 26:657–671, 2007 Keywords: microwave; proteomics; mass spectrometry I. INTRODUCTION Proteomic analysis by mass spectrometry has undergone a revolution during the past decade. New technologies have emerged that allow higher-throughput, increased sensitivity, and improved characterization of bio-molecules. This rapid evolution has been due to technology development, including introduction of higher-flow rate and more sensitive chromato- graphy systems (Plumb et al., 2004), faster scanning and more sensitive mass spectrometric instrumentation (Schwartz, Senko, & Syka, 2002), increased mass accuracy for more confident characterization validation and quantitation (Hu et al., 2005) and increased bioinformatic power for database searching, charac- terization of post-translational modifications (PTMs), and protein annotation. Consequently, sample preparation may now be the limiting factor in terms of throughput and recovery for many protein characterization protocols. To address the issue of time-consuming sample preparation, several methodologies have been described whereby lengthy enzymatic incubation protocols were drastically reduced. These protocols included the use of immobilized enzymes (Massolini & Calleri, 2005; Duan et al., 2006), acid-labile surfactants (Umar et al., 2005), and other protocols designed to speed up the proteolytic or chemical cleavage of proteins or removal of PTMs (Papac et al., 1998). However, significant efforts are still underway to find even faster and more widely applicable mass spectrometric compatible techniques. More than two decades ago, the world of synthetic chemistry embraced microwave technology to increase reaction yields and decrease incubation times in their synthesis protocols (Kappe, 2004). Just a few years later, microwave technology began to emerge in protocols of a more bio-analytical nature. Such preliminary techniques included the exposure of antigens for immunohistochemical staining in embedded tissue samples (Patterson & Bulard, 1980; Hafajee & Leong, 2004; Emersen et al., 2006; Temel et al., 2006), increased acid hydrolysis of proteins for amino acid analysis (Gilman & Woodward, 1990; Chiou & Wang, 1990; Chen, Chiou, & Wang, 1991; Davidson, 1996), increased catalysis of enzymatic (Pramanik et al., 2002) and chemical cleavages for peptide mapping (Zhong, Marcus, & Li, 2005; Hua, Low, & Sze, 2005), and increased removal of PTMs for improved protein characterization (Lee et al., 2005a,b; Sandoval et al., 2007). This review summarizes the evolution of microwave irradiation as a proteomic tool for higher throughput sample preparation. Current literature is explored, a comparison is made to conventional analyses, and advantages and potential future of microwave-assisted biological sample preparation for mass spectrometric analysis are discussed. II. MICROWAVE TECHNOLOGY Microwaves are an electromagnetic radiation with wavelengths between 0.01 and 1 m and occupy the electromagnetic spectrum between infrared and radio waves. They occupy the frequency range between 0.3 and 30 GHz; however for commercial microwaves, a narrower range around 2.5 GHz is typically employed (Pozar, 1997). The theory of microwave irradiation was predicted in 1864 (Maxwell, 1888) and first physically demonstrated to exist in 1888 (Buchwald, 1994). The magnetron, a high-voltage system used to generate microwave energy, was invented during the Second World War as part of a radar detection system. Microwave irradiation as a heating method was discovered in 1946, and the first commercial domestic micro- waves were introduced in the 1950s. In 1978, the first commercial microwave for laboratory use was introduced by CEM. These laboratory-targeted microwaves have evolved over the past 20 Mass Spectrometry Reviews, 2007, 26, 657– 671 # 2007 by Wiley Periodicals, Inc. ———— *Correspondence to: Jennie R. Lill, Protein Chemistry Department, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: Jlill@gene.com