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One of the most popular methods to prepare tryptic peptides for bottom-up proteomic analysis is in-gel digestion. To date, there have been few studies comparing various digestion protocols. In this study we compare the efficiency of several popular in-gel digestion protocols along with new pieces of technology that may improve digestion efficiency, using a human epidermoid carcinoma cell lysate protein standard. The efficiency of each protocol will be based on the number of proteins identified, their respective sequence coverage and relative quantitation using spectral counting. The importance of this study lies in its comparison of pre-existing in-gel digestion methods and newly developed technologies. These new technologies introduce the potential for a more cost effective digestion, higher protein yield and an overall reduction in time. The following four protocols will be compared: Shevchenko's overnight protocol (Methods in Molecular Biology 1999;122:383-397), in-gel digestion in a barometric pressure cycler (Pressure Biosciences, Boston, MA), and in-gel digestion in a scientific microwave (CEM, Mathews, NC). In addition several variables will be tested for increased digestion efficiency and keratin contamination including the elimination of vacuum centrifugation and the use of modified and non-modified trypsin. Statistical analysis will be performed on replicate samples to determine if there are any significant differences between protocols.