Intact protein separations by using slip flow with nano-liquid chromatography-mass spectrometry
Abstract
Proteins represent the biggest challenge in analytical separations mainly because they have slow diffusion that hinders mass transfer as well as multiple isoforms due to post-translational modifications. In this work, 470 nm silica particles were used to make a significant improvement in protein separations, in order to speed disease detection and drug development. Slip flow has become a topic of interest in reversed-phase liquid chromatography because it gives a flow enhancement that facilitates the usage of submicrometer particles, providing a large improvement in separation efficiency. Moreover, slip flow provides an additional improvement in efficiency by reducing the velocity distribution in the mobile phase. A capillary densely packed with 470 nm silica particles with a laser pulled tip is shown to provide higher peak capacity and sensitivity in the separation of intact proteins by reversed-phase liquid chromatography-mass spectrometry (LC-MS). LCMS provides the capability of on-line identification, which is desirable for biomarker discovery and protein drug characterization. Model proteins were studied: ribonuclease A, trypsin inhibitor, and carbonic anhydrase, where the latter had impurities of superoxide dismutase and ubiquitin. The proteins were well separated at room temperature with negligible peak tailing. Separation of a cell lysate with a 60 min gradient showed extremely high peak capacities of 750 and above for a peptide and relatively homogeneous proteins. The chromatograms exhibited excellent reproducibility in retention time. Several applications of slip flow nano-LC-MS were presented in this thesis: I) ubiquitylation study; II) sialylated Lewis X bearing protein analysis for cancer biomarker discovery; III) RRM2 characterization; IV) peptide separations. These applications demonstrated that slip flow nano-LC-MS platform has practical value in real sample analysis. The ultra-efficiency provides faster analysis and better resolution, which could help resolve challenging analytical problems.
Degree
Ph.D.
Advisors
Wirth, Purdue University.
Subject Area
Analytical chemistry|Biochemistry|Physical chemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.