Affinity MALDI-MS of Thrombin

Our first demonstration of aptamer-modified surfaces for affinity MALDI-MS used the thrombin-TBA system described for affinity CE studies in the preceding section (Dick and McGown, 2004). In the MALDI-MS experiments, 3 fiL of sample was applied to the aptamer-coated and scrambled oligonucleotide-coated MALDI probe surfaces and incubated for 30 minutes. The surfaces were then rinsed with buffer, followed by application of the MALDI matrix and MALDI-TOF-MS analysis. 

Experiments were also conducted using pooled human plasma. The predominant, stable form of the protein in plasma is prothrombin, a zymogen that is cleaved to yield the activated thrombin upon appropriate signaling (Blomback and Hanson, 1979). The presence of any detectable thrombin in its active (i.e., cleaved) form is probably due to stabilizers in the pooled plasma preparation. The plasma concentration of prothrombin is about 90mg/L (Blomback and Hanson, 1979), which corresponds to about 2.5 p.M, or 7.5 pmol on the aptamer spot. 

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