During cardiac ischemia, human serum albumin's tertiary structure is altered by a yet unknown mechanism within minutes of arterial occlusion. Herein it is shown that ischemic conditions induce changes to albumin's post-translational protein profile that can be quantitatively assayed using mass spectrometry. Profiling albumin post-translational modifications in a model cohort is suggestive of the diagnostic power of an albumin biomarker panel as an ischemic indicator. Specifically, ischemic conditions lead to altered expression of three first domain PTMs on residues Cys34, Ser58, and Cys91 respectively.
The focus of this research has been the use of targeted mass spectrometry approaches to examine the tertiary protein features of albumin in both qualitative and a quantitative contexts. A number of albumin amino acid residues had previously been identified as sites of albumin post-translational modification. However, studies to characterize and profile the human serum albumin protein had previously been limited both in depth and in scope. To determine if preceding efforts had sufficiently and fully characterized the protein, we multiplexed proteomic technologies during characterization efforts. In so doing, we established a profile of endogenous albumin PTMs from which to determine both basal and disease-altered stoichiometry.
To correlate change in albumin's modification expression to specific clinical states, we converted profiling efforts into an assay format using selected reaction monitoring mass spectrometry. In this, we have avoided more canonical techniques or protocols during protein characterization in favor of instrumentation and methods that provide increased speed, throughput, and/or reproducibility during analysis. To this end, we have replaced manual sample preparation steps with an automated system directly interfaced to the mass spectrometer. Integrating a comprehensive stand-alone proteomic workstation into the quantitative mass spectrometric platform expands the overall utility of mass spectrometry with regards to clinical diagnostics. The MS/MS capabilities of the mass spectrometer allow for specific and sensitive measurements in complex biological fluids (like clinical specimens—blood, CSF, urine, etc.). The robotics capabilities of the sample preparation workstation allow for high-throughput, highly reproducible work flows. Automating sample preparation builds quality assessment and measured best practices into mass spectrometric assay design. The end result is a robust, high-throughput pipeline, rigorous in regards to sensitivity of detection, accuracy of measurement, and reproducibility of process.
|Advisor:||Cotter, Robert J.|
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 74/01(E), Dissertation Abstracts International|
|Keywords:||Cardiac ischemia, Posttranslational modifications, Proteomics, Serum albumin|
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