Rapid quantitative analysis of human serum sphingomyelin species using MALDI-TOF mass spectrometry with lipid hydrolase treatment

• We developed a simple and quantitative method for analysis of human serum sphingomyelin species.
• Sphingomyelin species were measured by lipid hydrolase treatment and MALDI-TOF mass spectrometry.
• MALDI-TOF mass spectrometry revealed 15 SM species containing fatty acids ranging from C15 to C24.
• The SM species composition of 10 healthy young subjects showed a similar profile.
• This measurement procedure will be applicable in clinical laboratory studies.

BackgroundSphingomyelin (SM) is a key component of extracellular membranes and lipoproteins, and plays roles in cell signaling and as a component of lipoproteins. SM species differ in terms of fatty acid (FA) composition. However, no simple, rapid, quantitative assay for identifying different SM species has yet been reported. In this study, lipid hydrolase treatment and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify serum SM species.MethodsSera were collected from healthy young individuals. To identify SM species, sera were treated with phospholipase A2 and lipoprotein lipase, and lipids were extracted using the standard chloroform/methanol (2/1 v/v) method.ResultsWe detected 15 peaks from serum using MALDI-TOF MS, which were assigned to SM species bound with FA components ranging from C15:0 to C24:2. The most prominent serum SM species was SM [C16:0], which accounted for approximately 26% of serum SM. Some SM species contained an odd-carbon FA (C15, C21, and C23), and these accounted for approximately 4% of serum SM. The reproducibility of major SM species within and between application positions on MS-sample plate was CV = 3.0%–7.9% and CV = 3.1%–6.8%, respectively. The concentration and dilution ratio were linearly related. The SM species composition of 10 healthy young subjects showed a similar profile.ConclusionsWe developed a rapid, and quantitative method for identifying serum SM species using lipid hydrolase treatment and MALDI-TOF MS. This method will be suitable for clinical laboratory studies to examine the associations between SM species and disease states.