Semester
Summer
Date of Graduation
2007
Document Type
Thesis
Degree Type
MS
College
Eberly College of Arts and Sciences
Department
Chemistry
Committee Chair
Aaron T. Timperman.
Abstract
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;.
Recommended Citation
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.
https://researchrepository.wvu.edu/etd/1819