Honggang Nie

Lipid profiling of rat peritoneal surface layers by online normal- and reversed-phase 2D LC QToF-MS

An online, two-dimensional (2D) liquid chromatography (LC) quadrupole time-of-flight mass spectrometry (QToF-MS) method was developed for lipid profiling of rat peritoneal surface layers, in which the lipid classes and species could be simultaneously separated in one injection with a significantly increased sensitivity. Different lipid classes were separated on a normal-phase column in the first dimension and lipid molecular species were separated on a reversed-phase column in the second dimension, so that the ion suppression effects were reduced while the detection sensitivity was improved. Identified were 721 endogenous lipid species from 12 lipid classes, in which 415 structures were confirmed using tandem mass spectra, and the other 306 lipid molecular species were identified by accurate masses. The linearity, limit of detection, and repeatability were all satisfactory. The method was applied to the investigation of the lipid changes in rat peritoneal surface layer after peritoneal dialysis, and 32 potential lipid biomarkers were identified, as their concentrations in the dosed group were 2.2–12.5 times of those in the control group. The results revealed that this 2D LC-MS system was a promising tool for lipid profiling of complex biological samples.

Recent advances of chromatography and mass spectrometry in lipidomics

Lipidomics, as a novel branch of metabolomics, which is aimed at comprehensive analysis of lipids and their biological roles with respect to health and diseases, has attracted increased attention from biological and analytical scientists. As a result of the complexity and diversity of lipids, accurate identification and efficient separation are required for lipidomics analysis. Mass spectrometry (MS) and chromatography have been extensively developed in the past few decades and hold a distinguished position in qualification and separation science. They are powerful and indispensable tools for lipidomics. Herein, we present the recent advancement of MS, chromatography, and their hyphenation technologies in lipidomics.

Development of high internal phase emulsion polymeric monoliths for highly efficient enrichment of trace polycyclic aromatic hydrocarbons from large-volume water samples

In this work, polymerized high internal phase emulsion (polyHIPE) monoliths were prepared and applied as monolithic adsorbent materials for proconcentration of trace polycyclic aromatic hydrocarbons (PAHs) from large-volume water samples. The monolithic polyHIPE columns were prepared by in situ polymerization of the continuous phase of a high internal phase emulsion (HIPE) containing styrene (STY), divinylbenzene (DVB) and glycidyl methacrylate (GMA) in pipette tips, and the resulting STY/DVB/GMA polyHIPE monoliths exhibited highly interconnected porosity and large surface areas, making them excellent candidates as adsorbents for enrichment of trace aromatic compounds. The prepared STY/DVB/GMA polyHIPE monoliths were applied to the determination of trace PAHs in environmental water samples by combing with high performance liquid chromatography-fluorescence detection (HPLC-FLD). Under the optimized experimental conditions, the polyHIPE monoliths could effectively enrich trace 13 PAHs from 500mL of water samples, the mean recoveries at four spiked levels were ranged from 80.7% to 115.0% with the relative standard deviations (RSDs) lower than 14%, and the detection limits (LODs) were ranged from 4.0 to 228pg/L. In addition, the prepared polyHIPE monolith was stable enough for more than 200 replicate extraction cycles without measurable loss of performance on the enrichment of PAHs, and good column-to-column repeatability was obtained with RSD less than 13%. The proposed method was applied to simultaneous analysis of 13 PAHs in water samples with satisfactory recoveries.

Analysis of aristolochic acids by CE-MS with carboxymethyl chitosan-coated capillary.

A CE‐MS method for rapid determination of aristolochic acid‐I and aristolochic acid‐II (AA‐II) in traditional Chinese medicines and biological samples was described in the present paper. AA‐I and AA‐II can be baseline separated within 6 min by CE‐MS with carboxymethyl‐chitosan‐coated capillary. CZE conditions including pH, concentration of buffer, applied voltage, and capillary temperature were systematically investigated, and the composition and flow rate of sheath liquid were also optimized for CE‐MS. Furthermore, the CE‐UV method without any additives in BGE solution was established and compared with the CE‐MS method. The results showed that the two methods could achieve satisfactory separation efficiency, repeatability, and linearity, while the LOD was 0.6 μg/mL for CE‐UV and 0.05 μg/mL for CE‐MS. Compared with the CE‐UV method, the sensitivity of CE‐MS was significantly improved, in addition to the structure information provided by MS detection at the same time. As an application example, a spiked sample in human serum was analyzed by the CE‐MS method, indicating that the new CE‐MS method can be applied to analyze AAs in biological samples.

Simultaneous determination of jasmonic acid epimers as phytohormones by chiral liquid chromatography–quadrupole time-of-flight mass spectrometry and their epimerization study

Jasmonic acid (JA) is an essential plant hormone involved in plant development and defense system. There are four stereoisomeric forms of JA and they act quite differently in vivo. In this work, a normal phase liquid chromatography-quadrupole time-of-flight mass spectrometry (NPLC-QTOF-MS) method using cellulose tris (4-methylbenzoate) coated silica gel as the chiral stationary phase was first established for the simultaneous discrimination and direct analysis of all the four JA stereoisomers without need of derivatization. A non-endogenous JA stereoisomer was introduced as the internal standard to ensure the reliability of the developed method. Satisfactory results were obtained in terms of sensitivity (limit of detection, 0.5 ng mL(-1) or 2.4 fmol), linearity (R(2)=0.9996) and repeatability (run-to-run RSD of migration time and peak area, 0.37% and 5.9%, respectively, n=6). Endogenous rise of two natural JA stereoisomers was detected in tobacco leaves and their variations in response to mechanical wounding were monitored. In addition, the configurational stability of JA stereoisomers was investigated using the stereoisomerically pure forms which were not commercially available but easily obtained by our semi-preparative chiral LC method. Experimental evidence indicated that both of the two naturally existing JA stereoisomers were putative signals for wounding response, and the epimerization between them was not a spontaneous process simply promoted by the thermodynamical instability as expected before.

Interface for Online Coupling of Surface Plasmon Resonance to Direct Analysis in Real Time Mass Spectrometry

The online coupling of surface plasmon resonance (SPR) with mass spectrometry (MS) has been highly desired for the complementary information provided by each of the two techniques. In this work, a novel interface for direct and online coupling of SPR to direct analysis in real time (DART) MS was developed. A spray tip connected with the outlet of the SPR flow solution was conducted as the sampling part of the DART-MS, with which the online coupling interface of SPR-MS was realized. Four model samples, acetaminophen, metronidazole, quinine, and hippuric acid, dissolved in three kinds of common buffers were used in the SPR-DART-MS experiments for performance evaluation of the interface and the optimization of DART conditions. The results showed consistent signal changes and high tolerance of nonvolatile salts of this SPR-MS system, demonstrating the feasibility of the interface for online coupling of SPR with MS and the potential application in the characterization of interaction under physiological conditions.

Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency

Serotonin is an important neurotransmitter that regulates a wide range of physiological, neuropsychological, and behavioral processes. Consequently, serotonin deficiency is involved in a wide variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and depression. The pathophysiological mechanisms underlying serotonin deficiency, particularly from a lipidomics perspective, remain poorly understood. This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipidomic profiling of tryptophan hydroxylase 2 knockout (Tph2−/−) mice. Using a high-throughput normal-/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (NP/RP 2D LC–QToF-MS) method, 59 lipid biomarkers encompassing glycerophospholipids (glycerophosphocholines, lysoglycerophosphocholines, glycerophosphoethanolamines, lysoglycerophosphoethanolamines glycerophosphoinositols, and lysoglycerophosphoinositols), sphingolipids (sphingomyelins, ceramides, galactosylceramides, glucosylceramides, and lactosylceramides) and free fatty acids were identified. Systemic oxidative stress in the Tph2−/− mice was significantly elevated, and a corresponding mechanism that relates the lipidomic findings has been proposed. In summary, this work provides preliminary findings that lipid metabolism is implicated in serotonin deficiency.

Study on Variation of Lipids during Different Growth Phases of Living Cyanobacteria Using Easy Ambient Sonic-Spray Ionization Mass Spectrometry

Lipids are important components of cell membranes and thylakoids in cyanobacteria, and they play vital roles in various biological activities. Real-time tracing of the variation of membrane lipids can provide insights of the physiological status of cyanobacterial cells. In this work, easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was utilized to investigate the changes of acidic lipids in unicellular (Synechocystis 6803, Synechococcus 7002) and filamentous (Anabaena 7120) cyanobacteria during different growth phases. A sqdX mutant with a reduced synthesis of sulfoquinovosyl diacylglycerol (SQDG) was constructed to verify the acquired data of EASI-MS. Principal component analysis (PCA) was performed to compare the acquired data, enabling the discrimination of different species of cyanobacteria in day-to-day analysis. The results showed that the three representative cyanobacteria and their growth status can be easily determined on the basis of the lipids components detected by EASI-MS. Very interestingly, significant decreases of the ratios of SQDG/PG and dramatic changes of the unsaturation level of lipids were observed in different culture times in these cyanobacteria, and these two unique characters can be used describe the aging of cyanobacteria.

Lipidomic analysis of plasma in patients with lacunar infarction using normal-phase/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry

AbstractStroke is a major cause of mortality and long-term disability worldwide. The study of biomarkers and pathogenesis is vital for early diagnosis and treatment of stroke. In the present study, a continuous-flow normal-phase/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF/MS) method was employed to measure lipid species in human plasma, including healthy controls and lacunar infarction (LI) patients. As a result, 13 lipid species were demonstrated with significant difference between the two groups, and a “plasma biomarker model” including glucosylceramide (38:2), phosphatidylethanolamine (35:2), free fatty acid (16:1), and triacylglycerol (56:5) was finally established. This model was evaluated as an effective tool in that area under the receiver operating characteristic curve reached 1.000 in the discovery set and 0.947 in the validation set for diagnosing LI patients from healthy controls. Besides, the sensitivity and specificity of disease diagnosis in validation set were 93.3% and 96.6% at the best cutoff value, respectively. This study demonstrates the promising potential of NP/RP 2D LC-QToF/MS-based lipidomics approach in finding bio-markers for disease diagnosis and providing special insights into the metabolism of stroke induced by small vessel disease. Graphical abstractFlow-chart of the plasma biomarker model establishment through biomarker screening and validation

Lipid profiling of cyanobacteria Synechococcus sp. PCC 7002 using two‐dimensional liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two-dimensional (normal- and reversed-phase) liquid chromatography online coupled to quadrupole time-of-flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd-chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.