Abstract
One of the most popular methods to prepare tryptic peptides for bottom-up proteomic analysis is in-gel digestion. To date, therehave 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. Theefficiency of each protocol will be based on the average 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), a modification of the Modified Shevchenko’s overnight protocol in which we removed the vacuum centrifugation steps, 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 andthe use of modified and unmodified trypsin. Statistical analysis will be performed on replicate samples to determine if there are any significant differences between protocols.