Identification of major sphingolipid chain lengths involved in chemoresistance, which are regulated by BCR in primary CLL cells. LC-ESI-MS/MS with differentiation between distinct fatty acyl subspecies of sphingolipids after BCR stimulation for 24 hours was applied (n = 8). (A) Ceramide levels were significantly reduced after BCR engagement compared with native controls, especially of the most highly abundant C16:0 and C24:1 (C16:0, P < .0001; C24:1, P = .0010). (B) IgM stimulation caused a significant increase in glucosylceramide (C16:0, P = .0004; C24:1, P = .0012). (C) Sphingomyelin levels showed no differences between native and IgM-stimulated samples. (D) Ceramide/glucosylceramide ratio based on mass spectrometric data: A higher quotient of the ratio reveals an increase of ceramide, whereas a lower ratio indicates higher glucosylceramide levels. BCR cross-linking reverted the ratio toward pro-survival glucosylceramide associated with a lower ratio. Results of LC-ESI-MS/MS are expressed as mean ± SEM of 8 independent experiments. Statistics are calculated by unpaired t test.