AfCS Research Reports

Stimulation of the AfCS WEHI-231 B-cell receptor with 0.13 mM anti-IgM ligand resulted in robust changes in glycerophospholipid concentrations. Lipid arrays were constructed from 10 sets of samples. Each data set contained four exact replicates of paired samples that included a control (basal) and matched ligand-stimulated sample at each of five time points: 1.5, 3, 6, 15, and 240 minutes. Thus, each array was constructed from 400 samples. The lipid species were identified using both the positive (array 1, supplemental material) and negative (array 2, supplemental material) ESI modes. Excerpts from the positive (A) and negative (B) mode arrays are shown in Fig. 5.

Fig. 5. Excerpts from positive (A) and negative (B) mode lipid arrays. The first column contains lipid species identified by ESI MS/MS. The second column indicates the mass-to-charge ratio (m/z) of the observed compounds. The remaining five columns are for the five time points (1.5, 3, 6, 15, and 240 minutes). The values in these columns represent the significance score, which is the sum of that cell for the 10 individual experiments, with positive numbers representing increasing signal and negative values indicating a decreasing signal. Therefore, an array cell containing the number -8 is interpreted to mean the indicated species was observed to decrease in 8 of the 10 trials and remain stable in the other 2, or that the species decreased in 9 of the experiments and increased in 1 at that time point. These scores are color coded by signal frequency with deep blue or red, indicating an absolute score of 6 or more from a possible 10 (shown highly significant by computer simulation). Lighter shades of red and blue indicate significant stimulations (-5 or 5). Green colored cells, representing a -4 to 4 significance score, indicate statistical stability between basal and stimulated conditions.

In array one, only a few changes in concentration were observed during the 1.5- and 3-minute time points. However, highly significant decreases were observed for many phosphatidylcholine and/or phosphatidylethanolamine species at the 6- and 15-minute time points, with corresponding increases in several lyso-PC compounds. By the fourth hour, the cells had mostly returned to their prestimulated states. A list of lipids having significant or highly significant changes is summarized in Table 3.

The temporal trend in array two was shifted towards the later time points. Little movement was observed during the 1.5-, 3-, or 6-minute experiments. But, clusters of phosphatidylinositols and phosphatidylserines were observed to decrease highly significantly at the 15-minute time point. Corresponding increases in lyso-PI, lyso-PS, and glycerophosphatidic acids were also recorded. A summary of observed changes for the entire array is shown in Table 4.