Figure 6. LC-MS analysis of the CMAH reaction mixture.

Equal numbers of platelets from saline-infused WT mice (A) and 5-HT-infused WT mice (B) were prepared for CMAH enzymatic assay. Platelet lysates were resuspended in enzyme assay buffer containing TX-100 as described in the Methods. The platelet lysate in enzyme reaction buffer was mixed with 22.5 μM substrate (CMP-Neu5Ac) and incubated at 37°C for 45 min²⁸. The reaction mixture was analyzed with LC-MS for the level of Neu5Gc formation, and the neuraminic acid monosaccharides, Neu5Ac and Neu5Gc, were resolved as described in the Methods. Platelets from mice infused with SAL showed a higher peak corresponding to Neu5Ac (blue rectangle), whereas platelets from mice infused with 5-HT predominantly showed Neu5Gc (red rectangle). (C) The LC-MS analysis of blood fractions for CMAH. Equal amounts of protein lysate (60 μg) from the RBC + BC component, or from platelets (as the source of enzyme) were incubated with CMP-Neu5Ac (substrate) and the reaction continued 45 min before analysis by LC-MS to determine conversion of Neu5Ac to Neu5Gc⁴⁴. The highest relative ratio of Neu5Gc to Neu5Ac was formed in the reaction mixture from platelets exposed to elevated levels of plasma 5-HT. (D) Time and substrate concentration-dependence of CMAH. Neu5Gc formed in the CMAH assay described under “Methods” was monitored by LC-MS as a function of incubation time using 60 μg of platelet protein per assay. CMP-Neu5Ac was used as a substrate at a concentration of 11.25 μM and the reaction followed to ensure an enzymatic rather than chemical profile.