Aptamer-Enhanced Laser Desorption/Ionization for Affinity Mass Spectrometry

Abstract

The thrombin-binding DNA aptamer was used for affinity capture of thrombin in MALDI-TOF-MS. The aptamer was covalently attached to the surface of a glass slide that served as the MALDI surface. Results show that thrombin is retained at the aptamer-modified surface while nonspecific proteins, such as albumin, are removed by rinsing with buffer. Upon application of the low-pH MALDI matrix, the G-quartet structure of the aptamer unfolds, releasing the captured thrombin. Following TOF-MS analysis, residual matrix and protein can be washed from the surface, and buffer can be applied to refold the aptamers, allowing the surface to be reused. Selective capture of thrombin from mixtures of thrombin and albumin and of thrombin and prothrombin from human plasma was demonstrated. This simple approach to affinity capture, isolation, and detection holds potential for analysis, sensing, purification, and preconcentration of proteins in biological fluids.

Affinity Capture and Detection of Immunoglobulin E in Human Serum Using an Aptamer-Modified Surface in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

“…DNA-coated spots were covalently attached to fused silica slides as previously described [8,9]. The surface of the glass plate was first cleaned and activated by rinsing with methanol, water, and sodium hydroxide.…”

“…A recent addition to the field is the use of aptamer-modified surfaces for affinity protein capture and detection in Matrix-Assisted Laser Desorption-Ionization Mass Spectroscopy (MALDI-MS) [8]. In previous work, we demonstrated proof-of-principle of aptamer surfaces for affinity MALDI-MS using the model system of thrombin capture by the G-quartet DNA thrombinbinding aptamer [8]. The approach was subsequently applied in a non-aptameric system of insulin capture from nuclear extracts of cell lysates by a genomic DNA sequence that forms a G-quadruplex [9].…”

An aptamer-functionalized chemomechanically modulated biomolecule catch-and-release system

“…26,27 Importantly, this aptamer is known to reversibly denature and lose affinity for thrombin at low pH (Fig.S1). 32 Similarly, PAAc--co--PAAm is a well--studied, biocompatible hydrogel that contracts at pH < pKa=4.25 and swells at pH > pKa (Fig.1b). 17 We anticipated that linking these two pH--responsive components with flexible epoxy microstructures (10μm width, 2μm length, 18μm height, 5μm spacing) in a microfluidic system (see SI Fig.…”

“…To experimentally test the system's ability to selectively capture thrombin from a mixture of proteins, a solution containing a mixture of thrombin and transferrin, an iron--binding plasma glycoprotein, as well as a mixture of thrombin and bovine serum albumin (BSA) was introduced into the microfluidic channel with aptamer--decorated and unfunctionalized microstructures, as well as microstructures functionalized with a scrambled DNA sequence that does not bind thrombin. 32,37 The resulting top and bottom fluids were collected separately and analyzed by PAGE gel. The thrombin aptamer--decorated system selectively captured thrombin from the top fluid and released it in the bottom solution for both mixtures, while both BSA and transferrin were retained in the top layer; additionally, all the thrombin, transferrin and BSA were retained in the top layer in the control systems (Fig.5a, b).…”

Aptamer‐Modified Surfaces for Affinity Capture and Detection of Proteins in Capillary Electrophoresis and Maldi–Mass Spectrometry

“…We recently added aptamers to the protein capture toolbox for affinity MALDI-MS (Dick and McGown, 2004;Cole et al, 2007). In our approach, the aptamers are attached to fused-silica MALDI probe surfaces in spots approximately 1 cm in diameter using the same covalent attachment method that we used to modify the fused-silica capillary surfaces in the affinity CE studies described above.…”