Scientific Publications by FDA Staff

Anal Biochem 2011 Feb 15;409(2):202-12

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis.

Getie-Kebtie M, Lazarev A, Eichelberger M, Alterman M

Here we present a MALDI-TOF/TOF based label-free relative protein quantification strategy that involves SDS/PAGE separation of proteins followed by in-gel trypsin digestion. Main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high pressure-assisted in-gel trypsin digestion method that is based on Pressure Cycling Technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as, number of peaks detected, number of peptides identified, and sequence coverage, and, the digestion time was reduced to 45 min. The gel/mass spectrometry-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the LC/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10% and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins co-migrating during electrophoretic separation.