Fast quantitative 2D NMR for metabolomics and lipidomics: A tutorial

Nuclear magnetic resonance (NMR) is a well-known analytical technique for the analysis of complex mixtures. Its quantitative capability makes it ideally suited to metabolomics or lipidomics studies involving large sample collections of complex biological samples. To overcome the ubiquitous limitation of spectral overcrowding when recording 1D NMR spectra on such samples, the acquisition of 2D NMR spectra allows a better separation between overlapped resonances while yielding accurate quantitative data when appropriate analytical protocols are implemented. Moreover, the experiment duration can be considerably reduced by applying fast acquisition methods. Here, we describe the general workflow to acquire fast quantitative 2D NMR spectra in the “omics” context. It is illustrated on three representative and complementary experiments: UF COSY, ZF-TOCSY with nonuniform sampling, and HSQC with nonuniform sampling. After giving some details and recommendations on how to apply this protocol, its implementation in the case of targeted and untargeted metabolomics/lipidomics studies is described.     

Feature selection for OPLS discriminant analysis of cancer tissue lipidomics data

The mass spectrometry‐based molecular profiling can be used for better differentiation between normal and cancer tissues and for the detection of neoplastic transformation, which is of great importance for diagnostics of a pathology, prognosis of its evolution trend, and development of a treatment strategy. The aim of the present study is the evaluation of tissue classification approaches based on various data sets derived from the molecular profile of the organic solvent extracts of a tissue. A set of possibilities are considered for the orthogonal projections to latent structures discriminant analysis: all mass spectrometric peaks over 300 counts threshold, subset of peaks selected by ranking with support vector machine algorithm, peaks selected by random forest algorithm, peaks with the statistically significant difference of the intensity determined by the Mann‐Whitney U test, peaks identified as lipids, and both identified and significantly different peaks. The best predictive potential is obtained for OPLS‐DA model built on nonpolar glycerolipids (Q² = 0.64, area under curve [AUC] = 0.95); the second one is OPLS‐DA model with lipid peaks selected by random forest algorithm (Q² = 0.58, AUC = 0.87). Moreover, models based on particular molecular classes are more preferable from biological point of view, resulting in new explanatory mechanisms of pathophysiology and providing a pathway analysis. Another promising features for OPLS‐DA modeling are phosphatidylethanolamines (Q² = 0.48, AUC = 0.86).     

脂质组学分析中样品前处理技术的研究进展

脂质不仅是细胞膜的主要组成部分,还参与一些生命活动如能量存储、信号传导等,在生命体中发挥着重要作用。近年来,越来越多的研究表明脂质的变化与一些重大疾病的发生发展密切相关,脂质组学研究对理解疾病的发生机制及过程具有重要意义。在脂质分析过程中,由于样品基质的干扰或被分析物浓度的限制,通常需要对样品进行前处理,以得到最佳的分析性能。该文综述了脂质组学分析中的样品前处理技术,包括脂质的提取方法（如液液萃取、固相萃取等）和针对不同类脂质的化学衍生化技术在各领域,尤其是生命分析和代谢组学中的应用,并对脂质组学分析中的样品前处理技术的发展进行了展望。     

Recent progresses of derivatization approaches in the targeted lipidomics analysis by mass spectrometry

Lipidomics plays an essential role in the development of an improved understanding of lipids metabolism and the identification of new biomarkers or therapeutic targets of related diseases. The strong analytical power of mass spectrometry and its rapid developments in the respect of instruments and techniques have significantly accelerated the emerging lipidomics and related application fields in biology, medicine, and pharmacy. The strategy of chemical derivatization can remarkably improve the shortcomings of mass spectrometric analytical technologies of shotgun lipidomics and liquid chromatography mass spectrometry, and in the past decade many related studies have been reported for fatty acids, glycerophospholipids, sphingomyelins, monoglycerides, diacylglycerols, long‐chain bases, steroids, and so on. Therefore, this review will focus on new chemical derivatization approaches about the research progresses of shotgun‐based and liquid chromatography mass spectrometry–based targeted lipidomics (from 2005 to July 2019, most of reports emerged in the past 5 years), and put forward the problems and prospects in this field. It is expected to be helpful for the design and synthesis of new derivatization reagents, especially the outstanding stable isotope labeling derivatization reagents, and the development and application of new chemical derivatization strategies and matched mass spectrometric analysis methods.     

功能脂质组质谱分析

近年来,鉴于脂质在疾病发生发展过程中的重要作用,功能脂质组研究受到广泛关注.质谱技术是功能脂质组分析的主要手段,本文将介绍用于功能脂质组分析的质谱技术,概述国内功能脂质组质谱分析的研究进展,并提出问题和展望,以期可以在现有方法上进行改进提高,用以发现更多的功能脂质,进而用于发展疾病生物标志物及治疗靶标以及研究疾病机理等,并期待推进功能脂质组学研究的发展.     

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon–carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP − H]⁻, generates FA anions, [FA − H]⁻. The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)₃]²⁺, to form charge inverted complex cations of the form [FA − H + MgPhen₂]⁺. CID of the complex generates product ion spectral patterns that allow for the identification of carbon–carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.     

Ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry-based lipidomics reveals key lipid molecules as potential therapeutic targets of Polygonum cuspidatum against hyperlipidemia in a hamster model

Polygonum cuspidatum is a homology of traditional medicine and functional food widely distributed around the world. Our previous study on the hyperlipidemic animal model demonstrated that Polygonum cuspidatum was effective in ameliorating hyperlipidemia, which is characterized by lipid disorders. Herein, the regulatory effect of Polygonum cuspidatum on lipid metabolism needs to be known if its hypolipidemic mechanism is desired to clarify. In this study, an ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry-based lipidomic strategy was first applied to investigate the lipidomic patterns of high-fat diet-induced hyperlipidemic hamsters when treated with Polygonum cuspidatum. The results showed that Polygonum cuspidatum improved the lipidomic profile of hyperlipidemia. A total of 65 differential lipids related to the hypolipidemic effect of Polygonum cuspidatum were screened out and identified, and these differential lipids covered various categories, such as phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, sphingomyelins and so on. Orally administrated Polygonum cuspidatum restored these differential lipids back to normal or nearly normal levels. This study adopted lipidomics to reveal the key lipid molecules as potential therapeutic targets of Polygonum cuspidatum against hyperlipidemia, which would provide a scientific basis for its clinical application.     

High‐resolution multiple reaction monitoring method for quantification of steroidal hormones in plasma

Multiple reaction monitoring (MRM) is one of the most powerful modes of analysis in liquid chromatographic tandem mass spectrometry for quantification of low‐concentration metabolites in biological samples. The advances in mass spectrometry enabled the development of high‐resolution multiple reaction monitoring (MRM^HR) and became suitable for the more specific analysis of target analytes. This is important for lipidomic studies and contributes in the medical and pharmaceutical fields, primarily in investigating alterations in cells or fluids relevant to various diseases. Therefore, this work proposes the development of the MRM^HR method for quantification of circulating steroids. We focused on the determination of corticosterone, 11‐dehydrocorticosterone (11‐DHC), cortisol, cortisone, aldosterone, and progesterone concentration in serum, by using 129sv male mice exposed to chronic unpredictable stress to validate the quantification. The method was conducted according to the ANVISA normative, adopting a coefficient of variation, as well as relative standard deviation and relative error lower than 15% in linearity, intraday and interday precision, and accuracy. For cortisol, corticosterone, and their inert metabolites (cortisone and 11‐DHC), the lower limit of quantification was 3.9 ng· mL⁻¹, while that for progesterone and aldosterone was 7.8 and 15.6 ng· mL⁻¹, respectively. MRM^HR analysis showed that animals submitted to stressors have 4.5 times more corticosterone in their serum than nonstressed mice. However, 11‐DHC concentration does not vary significantly in response to stress for these animals. The results indicate that the method can be applied for quantification of steroids in several biological samples, such as human plasma.     

Supercritical fluid chromatography coupled to mass spectrometry for lipidomics

Supercritical fluid chromatography (SFC) has experienced a particular revival in recent years thanks to the development of robust and efficient commercial systems. Because of its physico‐chemical properties, supercritical carbon dioxide (CO₂) mixed with cosolvents and additives is particularly suitable for SFC to allow the elution of compounds of different polarities and more particularly complex lipids. Hyphenation with mass spectrometry (MS) is increasingly described in the literature but still requires many further developments in order to be as user‐friendly as coupling with liquid chromatography. The basic concepts of SFC and MS hyphenation will be first considered. Then a representative example of method development in lipidomics will be introduced. In conclusion, the challenges and future needs in this field of research will be discussed.     

Seasonal variations in the profile of main phospholipids in Mytilus galloprovincialis mussels: A study by hydrophilic interaction liquid chromatography–electrospray ionization Fourier transform mass spectrometry

A systematic characterization of phosphatidylcholines and phosphatidylethanolamines in mussels of sp Mytilus galloprovincialis was performed by high‐efficiency hydrophilic interaction liquid chromatography combined with electrospray ionization and Fourier transform mass spectrometry (FTMS), based on a quadrupole‐Orbitrap hybrid spectrometer. The FTMS/MS experiments under high collisional energy dissociation conditions, complemented by low‐energy collisionally induced dissociation MSⁿ (n  = 2,3) experiments, performed in a linear ion trap mass spectrometer, were exploited for structural elucidation purposes. The described approach led to an unprecedented characterization of the mussel phospholipidome, with 185 phosphatidylcholines and 131 phosphatidylethanolamines species recognized, distributed among diacylic, plasmanylic, and plasmenylic forms. This was the starting point for the evaluation of the effects of season (in particular, of sea temperature) on the profile of those phospholipids. To this aim, a set of mussel samples retrieved from commercial sources in different periods of the year was considered. Principal component analysis revealed a clear separation between samples collected in periods characterized by cold, intermediate, or warm sea temperatures, respectively. In particular, an enrichment in phospholipids containing unsaturated side chains was observed in mussels collected from cold seawaters (winter‐early spring), thus confirming the general model previously elaborated to explain the adaptation of marine invertebrates, including some bivalve molluscs, to low temperatures. On the other hand, relevant levels of plasma(e)nylic and acylic phospholipids bearing either saturated or non‐methylene‐interrupted side chains were found in mussels collected in warm seawaters (typical of summer and early autumn, at Italian latitudes). This finding opened interesting perspectives towards the development of strategies able to prevent global warming–related mussel losses in aquacultural plants.