Method for specific separation and enrichment of acid sphingolipid and glycosphingolipid in human serum
A technology for enriching human and glycosphingolipids, applied in material separation, analytical materials, measurement devices, etc., can solve problems such as low ionization efficiency, in-source fragmentation, low abundance, and difficult detection
Active Publication Date: 2019-07-30
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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However: Due to the low ionization efficiency and in-source fragmentation of the phosphorylated sphingolipid components S1P and C1P in mass spectrometry, as well as the structural diversity and low abundance of glycosphingolipids in body fluids, phosphorylation, sulfation Analysis of glycosphingolipids and glycosphingolipids is a great challenge in sphingolipidomics research
To solve this technical challenge of the difficulty in the detection of these specially modified sphingolipids, we developed a method to comprehensively improve the detection sensitivity of these sphingolipids, that is, to use titanium dioxide (TiO 2 ) technology to specifically enrich acidic sphingolipids and glycosphingolipids
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Embodiment 1
[0090] The method for preparing a sphingolipid sample in serum by using titanium dioxide comprises the following steps:
[0091] (1) Take 50 μL of serum from each patient and transfer it to a glass tube. After adding 1.0 mL of methanol and 0.5 mL of chloroform, 10 μL of C12-sulfatide ester solution (2.5 μM) was added, and ultrasonically oscillated at room temperature for 30 seconds. The mixture was incubated overnight at 48°C (first extraction).
[0092] (2) After cooling to room temperature, 75 μL potassium hydroxide methanol solution (1M) was added and shaken in a water bath at 37° C. for 2 hours. Let cool to room temperature and add acetic acid to neutralize. Centrifuge at 3000 rpm for 10 minutes at high speed, and take the supernatant.
[0093] (3) Add 0.5 mL of methanol, 1.0 mL of chloroform and 0.5 mL of isopropanol to the precipitate, oscillate for 30 seconds, and centrifuge at 3000 rpm for 10 minutes at high speed, and take the supernatant.
[0094] (4) Add 1 mL of...
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The invention discloses a method for specific separation and enrichment of acid sphingolipid and glycosphingolipid in human serum. The method realizes a sample preparation step of complex sphingolipidomics in serum through a titanium dioxide column separation technology, and comprises separation of neutral sphingolipid and the acid sphingolipid, and separation of the acid sphingolipid and the glycosphingolipid. The method provided by the invention can separate the neutral sphingolipid, the glycosphingolipid and the acid sphingolipid (such as phosphorylated sphingolipid and sulfatide), and consequently, realizes removal of nature determination and quantification to other minute amount even trace amount of sphingolipid (the glycosphingolipid and the phosphorylated sphingolipid) by high abundance neutral sphingolipid in mass spectrum. According to a titanium dioxide enrichment method in the method provided by the invention, eight biomarkers are detected from a clinical specimen, whereinthe biomarkers comprise ceramide in the neutral sphingolipid, S1P and C1p in the acid sphingolipid and the glycosphingolipid. And the eight biomarkers can distinguish Early and middle and advanced stage patients well.
Description
technical field [0001] The invention relates to the field of separation of biological samples, in particular to a method for specifically separating and enriching acidic sphingolipids and glycosphingolipids in human serum. Background technique [0002] Lung cancer is the main cause of cancer death in the world, its incidence rate is increasing year by year, and its case fatality rate ranks first among all kinds of tumors. However, due to the lack of obvious and specific clinical manifestations of its early lesions, it is difficult to find, and most patients are already in the middle and late stages when they see a doctor, which delays the best time for treatment. Because it is difficult to obtain pathological tissue, diagnosis and treatment are often delayed, which puts forward higher requirements for the methods and means of clinical diagnosis of lung cancer. There are two main types of lung cancer: small cell lung cancer and non-small cell lung cancer (NSCLC) (1). NSCLC ...
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IPC IPC(8): G01N30/06G01N30/08G01N30/14G01N30/02G01N30/72
CPCG01N30/02G01N30/06G01N30/08G01N30/14G01N30/72G01N2030/027G01N2030/062G01N2030/065
Inventor 孟琼黄炳培赵新保孟亚明孔祥展陈逸钿
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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