Yamane Mototeru
Department of Biochemistry, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, 160, Tokyo, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jan 5;783(1):181-90. doi: 10.1016/s1570-0232(02)00637-2.
I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC-thermospray mass spectrometry. The HPLC-thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa(+) and MH(+)-H(2)O ions. As the other minor ions, MK(+), MH(+) and m/z 282 ions were detected. Although the preponderance of MNa(+) ions compared with the MH(+)-H(2)O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH(+)-H(2)O ions based on the elimination of the hydroxyl group introduced at the alpha-position of acyl-portion compared with the MNa(+) ions was detected in alpha-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-D-sphingosine (f=0.45), N-palmitoyl-D-sphingosine (f=0.40), N-stearoyl-D-sphingosine (f=0.39), N-nervonoyl-D-sphingosine (f=0.39) and N-lignoceroyl-D-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa(+)/MH(+)-H(2)O ions corresponding to several omega-hydroxyacyl-ceramides were detected.
我开发了一种简单的方法，可通过高效液相色谱-热喷雾质谱法同时分析培养细胞亚细胞组分中的神经酰胺。神经酰胺标准品的高效液相色谱-热喷雾质谱图的特征在于MNa(+)和MH(+)-H₂O离子的高强度。作为其他次要离子，检测到了MK(+)、MH(+)和m/z 282离子。尽管在非羟基脂肪酸神经酰胺中检测到MNa(+)离子相对于MH(+)-H₂O离子占优势，但在α-羟基脂肪酸神经酰胺中，检测到基于酰基部分α位引入的羟基消除后的MH(+)-H₂O离子相对于MNa(+)离子占优势。在使用N-辛酰鞘氨醇作为内标对真实神经酰胺进行校准时，每种神经酰胺与内标的峰面积比与每种神经酰胺的已知量之间存在近似线性关系。每种神经酰胺的因子(f)计算如下：N-油酰-D-鞘氨醇(f = 0.45)、N-棕榈酰-D-鞘氨醇(f = 0.40)、N-硬脂酰-D-鞘氨醇(f = 0.39)、N-神经酰-D-鞘氨醇(f = 0.39)和N-木蜡酰-D-鞘氨醇(f = 0.35)。在A549和HepG2细胞的亚细胞组分中，尽管每毫克蛋白质中的神经酰胺种类含量在核膜组分、7000g沉淀组分和100000g沉淀组分中较高，但大部分神经酰胺种类集中在核膜组分中。此外，该方法应用于猪角质层总神经酰胺的温和碱性水解产物，检测到了对应于几种ω-羟基酰基神经酰胺的MNa(+)/MH(+)-H₂O离子。
