Affinity Mass Spectrometry
Our ability to identify specific biopolymers and evaluate their structures defines our current potential to understand biological function. We have developed new mass spectrometric methods with analyte capture and desorp-tion strategies designed to facilitate the detection and structural analysis of specific biological marker proteins, even when present in trace quantities. We refer to this method as affinity mass spectrometry (AMS). Affinity mass spectrometry is defined as the design and /or use of mass spectrometric probe element surfaces that have been transformed from inert sample presenting platforms into active affinity capture and release devices.
Mass spectrometric probes were prepared with surfaces enhanced for affinity capture (SEAC) of lactoferrin. The goal of this investigation was to design and produce new classes of active probe elements for laser desorption time-of-flight mass spectrometry such that the disposable probe elements have surface characteristics that are optimized for DNA- or immunoglobulin-based lactoferrin capture and subsequent desorption/ionization for detection/characterization by time-of-flight mass spectrometry. With this new technology, we achieve the fast and sensitive detection and characterization (e.g., structural intactness and metal ion binding status) of lactof errin in various unf ractionated biological samples. We also evaluated SEAC probe elements to capture or dock predetermined numbers of lactoferrin molecules in predefined areas of the probe element surface to automate quantitative assessments. Enzymatic and chemical modifications were performed on the tethered lactoferrin left on the probe surface after the initial mass spectrometric analysis. We further improved lactoferrin detection sensitivity by thousands of folds by amplifying the bound lactoferrin signal with coupled enzyme reaction. AMS probes with SEAC can also be designed for the detection of cell surface lactoferrin receptors or markers of cellular responses to lactoferrin with equal sensitivity.
KeywordsDiethyl Pyrocarbonate Probe Element Human Lactoferrin Bovine Lactoferrin Capture Device
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