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CN-121994943-A - Structure annotation method for analyzing lipid sn-and C=C positions based on liquid chromatography-organic ionic electron collision excitation mass spectrum

CN121994943ACN 121994943 ACN121994943 ACN 121994943ACN-121994943-A

Abstract

The invention discloses a structure annotation method for analyzing lipid sn-and C=C positions based on liquid chromatography-organic ion electron collision excitation mass spectrometry. The method realizes simultaneous analysis of polar lipids and neutral lipids by one sample injection by adding a proper amount of 0.1mM sodium acetate during lipid re-dissolution. Meanwhile, a method for automatically annotating complex organic ion electron collision excitation mass spectrograms is developed, and comprises the steps of (1) extracting mass spectrum information in original data, (2) identifying lipid subclasses, (3) matching lipid total compositions, (4) pairing sn-positions, (5) locating C=C positions, and (6) determining annotation levels. The annotation accuracy of the present annotation method at the sn-and c=c isomer levels was 100% and 82.3%, respectively, for 34 lipid standards in serum. 1312 sn-isomers and 1033 c=c isomers were co-injected from mixed plasma samples of healthy people and patients with mild cognitive impairment of alzheimer's disease. In a word, the lipid fine structure analysis method developed by the invention has the characteristics of simple pretreatment, high coverage and high efficiency in annotating the lipid fine structure.

Inventors

  • XU GUOWANG
  • CHEN YAO
  • HU CHUNXIU
  • YANG JUN
  • WANG XINXIN
  • ZHANG YUQING

Assignees

  • 中国科学院大连化学物理研究所

Dates

Publication Date
20260508
Application Date
20241108

Claims (7)

  1. 1. The method for analyzing the structural annotation of the sn-and C=C positions of the lipid based on liquid chromatography-organic ion electron collision excitation mass spectrum is characterized by comprising the following steps: (1) Pretreating an analysis sample to obtain a lipid extract in the sample; (2) Re-suspending the lipid extract with a solution containing 0.1mM sodium acetate to obtain a solution of lipid extract to be sampled; (3) Acquiring the original data of the lipid extract in the step (2) by adopting a liquid chromatography-organic ion electron collision excitation mass spectrometry combined method; (4) The structural positions of lipids sn-and c=c were annotated according to the raw mass spectral data of the obtained sample lipid extract.
  2. 2. The structure annotation method as claimed in claim 1, wherein: the sample in step (1) includes, but is not limited to, plasma, serum, tissue, cells, yeast, microalgae, and the like.
  3. 3. The structure annotation method as claimed in claim 1, wherein: when the samples in the step (1) are blood plasma, serum, tissues and cells, the pretreatment process comprises the steps of adding 10-100 mu L of the samples, 100-1000 mu L of methanol and 0.5-5mL of methyl tertiary butyl ether into a centrifuge tube, shaking and uniformly mixing for 5-15min, then adding 100-1000 mu L of water, swirling for 1-3min to form a two-phase system, centrifuging for 10-30min at 8000-15000g, and taking supernatant to freeze-dry to obtain lipid extracts in the samples; When the samples in the step (1) are yeast and microalgae, the pretreatment process comprises the steps of adding 10-100 mu L of the samples and 100-1000 mu L of methanol into a centrifuge tube, grinding for 2min at 25HZ frequency by a tissue grinder, adding 0.5-5mL of methyl tertiary butyl ether, shaking and mixing for 5-15min, adding 100-1000 mu L of water, swirling for 1-3min to form a two-phase system, centrifuging for 10-30min at 8000-15000g, taking supernatant and freeze-drying to obtain the lipid extract in the samples.
  4. 4. The structure annotation method as claimed in claim 1, wherein: The 0.1mM sodium acetate solution in step (2) is 30% -95% (v/v) aqueous organic solvent, wherein the organic solvent includes, but is not limited to, one or two of acetonitrile, isopropanol, methanol, etc.
  5. 5. The structure annotation method as claimed in claim 1, wherein: The liquid chromatography condition in the step (3) is that the mobile phase A is 30% -90% (v/v) acetonitrile water solution containing ammonium acetate, and the mobile phase B is 30% -90% (v/v) isopropanol acetonitrile solution containing ammonium acetate; The organic ion electron impact excitation mass spectrum in the step (3) is an EAD cleavage mode under the electron beam pressure of 10eV, and data acquisition is carried out under a positive ion mode.
  6. 6. The method of claim 1, wherein the step (4) annotates the structural positions of lipids sn-and c=c based on the obtained sample lipid mass spectrum information, comprising the steps of: 1) Extracting lipid spectrogram information according to the original data of the lipid extract, wherein the lipid spectrogram information comprises the mass-to-charge ratio of parent ions in the primary lipid spectrogram information and the mass-to-charge ratio and relative intensity of neutron ions in the secondary lipid spectrogram information; 2) Identifying lipid subclasses, namely marking the classes of phosphatidylcholine, sphingomyelin and phosphatidylethanolamine lipids as known classes, and marking the classes of ceramide, diglyceride and triglyceride lipids as unknown classes; 3) Matching the lipid total composition, namely constructing an index library comprising known categories and unknown categories to match the total composition, matching the lipid precursor ion charge ratios of the known categories and the unknown categories according to the theoretical molecular mass in the index library, annotating the lipid total composition, annotating the lipid with the precursor ion charge ratio by finding the closest theoretical molecular mass in the same subclass for the lipid marked with the subclass, and annotating the lipid with the unknown subclass by finding the minimum difference between the actual parent ion mass charge ratio and the theoretical parent ion mass charge ratio in the index library of the unknown categories; 4) Determining sn-position, namely performing characteristic fragment ion matching according to the established sn-position index library, annotating lipid sn-position, wherein for phosphatidylcholine, phosphatidylethanolamine and diglyceride lipid, the relative intensity of fragment ions generated by fragmentation between C1-C2 of the glycerogelatin skeleton is lower than that of fragment ions generated by fragmentation between C1-O and C2-O, so that the characteristic fragment ions between C1-C2 can be used for determining sn-position; For sphingomyelin and ceramide lipids, odd-numbered and even-numbered acyl chain fragments in the organic ion electron impact excitation spectrum can be used for solving the arrangement problem of two chains on a framework and sn-2; For triglyceride lipids, the co-loss of sn-1 or sn-3 and sn-2 fatty acid chains produces a pair of double peaks differing in mass by 2.01Da, the chain length and double bond number of sn-1 or sn-3 are calculated through the double peaks, meanwhile, the double loss of sn-1 and sn-3 fatty acyl groups causes the sn-2 fragment ions to be in a unimodal state, thereby determining the position of sn-2; 5) The c=c position is located by observing a mass difference of 26.02Da in the presence of a double bond on the fatty acyl chain instead of 28.02Da from a carbon-carbon single bond, thus locating the lipid double bond position; 6) The annotation Level was determined by classifying the annotation results, classifying the lipids annotated at both the c=c and sn-positions as Level 1, assigning only the lipid annotated at the sn-position or c=c position as Level 2, defining the lipid not annotated at the sn-position and c=c position but inferred from the total composition as Level 3.
  7. 7. The method of claim 1, wherein, The lipid is lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, diglyceride and triglyceride.

Description

Structure annotation method for analyzing lipid sn-and C=C positions based on liquid chromatography-organic ionic electron collision excitation mass spectrum Technical Field The present invention relates to the fields of analytical chemistry, software development and clinical medicine. In particular, the invention relates to an analytical method for establishing a high-coverage liquid chromatography-organic ion electron impact excitation tandem mass spectrum and a method for developing an organic ion electron impact excitation spectrum for automatically annotating lipid isomers, and a group of different lipid isomers can be found by using the method to distinguish a healthy group from an early-stage Alzheimer disease group. Background Lipids play a vital role in a variety of cellular functions, such as constituting cell membranes, transmitting biological nerve signals, and providing energy for cellular activities. Lipid database LIPIDMAPS has recorded 40000 lipid molecules, which are divided into eight major classes and numerous subclasses. Taking Phosphatidylcholine (PC) lipid molecules as an example, complete identification requires elucidation of six structural planes, 1) lipid subclasses, 2) acyl chain composition, 3) sn-position of acyl or ether chains on glycerol backbone, 4) position of carbon-carbon double bond (c=c), 5) substituted functional group class and position (e.g. methylation and hydroxylation), 6) stereoisomers (cis/trans isomerism) of carbon-carbon double bond. The difference in molecular structure of the lipid isoforms determines the difference in their properties, resulting in differences in the biological functions they perform. For example, increasing the omega-3/omega-6 ratio has been shown to have beneficial effects on a variety of diseases, including cardiovascular disease, cancer, inflammation, and autoimmune disease. In view of the importance of lipid structure, the acquisition of detailed structural information across lipid classes is critical to advance lipidomics. Liquid chromatography-mass spectrometry (LC-MS) has become a powerful tool for lipidomic analysis, enabling accurate characterization and quantification of lipids in complex biological samples. However, conventional dissociation techniques, such as Collision Induced Dissociation (CID) or high energy collision dissociation (HCD), do not distinguish between sn-isomerism and c=c isomerism in lipids. In recent years, many mass spectrometry-based analytical techniques have greatly improved our ability to distinguish lipid isomers. These reactions include Patern co-Buchi (PB) and epoxidation reactions, which accurately determine the position of C=C by integrating the C=C derivatization with CID or HCD. In addition, novel ion activation/dissociation techniques are also of implication in mapping the sn-and c=c positions of lipids, including ozone induced dissociation (OzID), ultraviolet photodissociation (UVPD), and electron impact excitation of organic ions (EIEIO). Advances in these methods not only broaden the scope of lipidomic analysis, but also reveal the link between lipid isomer composition and disease pathology. However, to date, only a few lipid structure characterization strategies have been able to analyze neutral and polar lipids simultaneously in one experiment, impeding the progress of larger-scale lipidomic studies. It is well known that accurate structural characterization of complex lipids requires complex separation techniques and automated data analysis tools. To date, only a limited number of tools have been developed to analyze lipid complex structural data. For example LipidOA has proven to be an effective tool for locating the position of the glycerophospholipid double bond by PB-MS/MS data. In addition, the MS-DIAL 5.0 software provides a powerful function that can process MS data in CID and EIEIO modes. In addition, research has also been conducted to develop an undisclosed self-programming offline analysis software for decoding lipid information collected from EIEIO using differential ion mobility mass spectrometry. However, existing lipid analysis tools are often limited to single lipid class or identification of c=c positions, which results in poor utilization of lipid structure information. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a structure annotation method for analyzing the sn-and C=C positions of lipid based on liquid chromatography-organic ion electron impact excitation mass spectrometry, and the analysis parameters of the liquid chromatography-organic ion electron impact excitation tandem mass spectrometry (LC-EIEIO-MS/MS) method are optimized, so that the simultaneous analysis of polar and neutral lipids in one sample injection is realized. The resulting integrated lipid mass spectrometry data was then annotated using an efficient annotation method provided by the present invention to locate the sn-position and c=c position of the complex li