Fig. 5.
Variances in detection sensitivity arise from structural differences and regional total ion intensities. A, B: Structural differences influence detection sensitivity. A: Absolute quantities of NS- and AS-SM4s sulfatides determined in the different renal locations by MALDI on-target and UPLC-ESI-MS2 with the help of an internal NS-sulfatide standard. AS-SM4s values obtained with MALDI on-target are significantly higher than those determined with the UPLC-ESI-MS2 method. B: Relative ratio of AS- over NS-sulfatides as determined for all three methods. Intensities obtained for AS-sulfatides in each region were divided by those obtained for NS-sulfatides within the papillae. Then all AS/NS ratios were normalized to those obtained by UPLC-ESI-MS2 for each region, respectively. Note, both MALDI methods result in AS/NS levels that are twice as high as the ESI method. C, D: Regional differences in sensitivity. Values obtained for AS-sulfatides or for PI(38:4) in papillae (P) and medulla (M) were normalized to corresponding values obtained in the cortical (C) region, which contained the lowest total ion concentrations due to low sulfatide levels. The graphs reflect the relative regional intensities of AS-sulfatides (C) and PI, PI(38:4) (D), as obtained by the different MS techniques. With MALDI IMS, significantly lower values for PI were obtained in the sulfatide rich papillae and medulla as well as significant lower values for AS-sulfatides in the medulla as compared to UPLC-ESI-MS2. Note, this effect reversed when PI was analyzed in sulfatide-deprived kidney, having only residual AS-sulfatides in the medulla. (MALDI IMS Cstf/f Pax8Cre, n = 3; MALDI IMS, n = 3; UPLC-ESI MS2, n = 3; MALDI on-target, n = 3; *P < 0.05; **P < 0.01; ***P < 0.001).