Date of Graduation
Eberly College of Arts and Sciences
Aaron T. Timperman.
The performance of saw-tooth gradient for comprehensive proteome analysis is evaluated using model proteins and a low molecular weight human serum by reverse-phase high performance liquid chromatography. The peak splitting and number of peptides detected is used to characterize the effective peak capacity of the saw-tooth gradient as a function of solvent strength change (Delta&phis;). Peak splitting demonstrated in experimental result as one single peptide or protein detected in more than one saw-tooth step. No peak splitting is observed directly from chromatograms for two model proteins by decreasing the solvent strength change (Delta&phis;) from 5.0%, 1.0%, to 0.1%. The numerical comparisons of the peak area, peak width and peak asymmetry from saw-tooth gradients with the same parameter from the linear gradient show no peak splitting for both model proteins. Anthracene does not have peak splitting, but a significant reduction in peak area is observed. It is indicated that the S values proportional with the molecular weight significantly affect the observed effective peak capacities. Peak capacity of the saw-tooth gradient is also explored by a LMW (≤30kDa) human serum on an online capillary-HPLC-ESI-FTICR MS by decreasing the Delta&phis; from 1.0%, 0.5%, 0.2%, to 0.1% in the 26.0%-27.0% solvent B range. When the Delta&phis; decreases from 0.5% to 0.1%, the number of protein splitting increases from 5 to 28 while a higher increase in the number of total peptide signals detected is observed from 21 to 67 when Delta&phis; decreases from 1.0% to 0.1%. In fact, more peptide signals are observed with the saw-tooth gradient than the long linear gradient. Moreover, no new proteins are detected in the isocratic holding segment. Overall, the saw-tooth gradient provides a promising first dimension separation technique for a two-dimension separation with an effective peak capacity of several hundred at 0.1% Delta&phis;.
Cai, Guimei, "Evaluating the effective peak capacity of a saw-tooth gradient for reverse-phase high performance liquid chromatography separation of proteins and peptides" (2007). Graduate Theses, Dissertations, and Problem Reports. 1819.