Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry

Abstract

A lipidomics strategy, combining high resolution reversed-phase liquid chromatography (RPLC) with high resolution quadrupole time-of-flight mass spectrometry (QqTOF), is described. The method has carefully been assessed in both a qualitative and a quantitative fashion utilizing human blood plasma. The inherent low technical variability associated with the lipidomics method allows to measure 65% of the features with an intensity RSD value below 10%. Blood plasma lipid spike-in experiments demonstrate that relative concentration differences smaller than 25% can readily be revealed by means of a t-test. Utilizing an advanced identification strategy, it is shown that the detected features mainly originate from (lyso-)phospholipids, sphingolipids, mono-, di- and triacylglycerols and cholesterol esters. The high resolution offered by the up-front RPLC step further allows to discriminate various isomeric species associated with the different lipid classes. The added value of utilizing a Jetstream electrospray ionization (ESI) source over a regular ESI source in lipidomics is for the first time demonstrated. In addition, the application of ultra high performance LC (UHPLC) up to 1200 bar to extend the peak capacity or increase productivity is discussed.

Introduction

A novel trend in life sciences lies in the global non-targeted analysis of biomolecules which has led to the disciplines of genomics, transcriptomics, proteomics and metabolomics. Genomics has initiated this omics cascade and the concept of unbiased analysis subsequently attracted a vast amount of researchers to the omics field. The potentials of these relatively new disciplines are indeed enormous as they might impact on biomarker discovery, drug discovery/development and system knowledge [1], [2], [3], [4], amongst others.

The realization that lipids not only serve as building materials of membranes and energy providers but are also involved in biological processes such as signaling, cell–cell interactions, etc. and, moreover, are linked to diseases such as diabetes, obesity, atherosclerosis, Alzheimer, etc., has led to the emergence of the discipline of lipidomics [4], [5], [6], [7], [8], [9]. Lipidomics, often regarded as a subset of metabolomics, aims at the comprehensive measurement of the lipids present in a cell, tissue, biological fluid, etc. and the concomitant detection of the lipid responses to various stimuli, e.g. disease and pharmaceutical treatment. This holistic approach, simultaneously measuring hundreds of species, is revolutionary since it allows to reveal differences between conditions without a priori knowledge.

As part of our lipidomics platform, we recently described the analysis of the fatty acids in a single drop of human blood using capillary gas chromatography–mass spectrometry (GC–MS). A retention time locked database was constructed with more than 100 fatty acids and related substances [10]. It is, however, of equal importance to study the intact lipids. The researcher is confronted with a substantial complexity originating from an enormous structural diversity and dynamic range. Mass spectrometry, particularly using electrospray ionization (ESI), is the principal enabling technology to tackle the lipidome [9], [11], [12], [13], [14]. The complexity of the sample under investigation evidently benefits from the use of high resolution, accurate mass and tandem mass spectrometric equipment. Therefore, FT-ICR, orbitrap and (Q)-TOF MS systems are dominating the lipidomics literature [15], [16], [17], [18], [19], [20]. Triple quadrupole instruments [21], [22], [23] have as well proven their value for the class-specific detection through precursor ion and neutral loss scanning. A number of researchers solely rely on mass spectrometry to measure the lipidome and in the so-called shotgun lipidomics approach intra-source separation is exploited to widen the lipidome coverage [19], [20], [22], [24]. Impressive results have been reported but one is inevitably confronted with the fact that the mass spectrometer can only tolerate a certain complexity, has a limited in-spectrum dynamic range and is sensitive towards ion suppression. This, combined with the knowledge that lipid structures often come in a variety of isomers, which are difficult, if not impossible, to distinguish solely relying on mass spectrometry, justifies the combined use of chromatography and mass spectrometry. Various reports describe the combination of either reversed-phase or normal-phase HPLC with mass spectrometry and the combination of both in multidimensional set-ups has been reported as well [15], [16], [17], [18], [25], [26], [27], [28]. It is obvious that, in recent years, the field of lipid analysis has substantially been reshaped and revitalized largely driven by advances in mass spectrometry and chromatography [29]. Nevertheless, the field is in a continuous search/quest to further mine the lipidome, in a comparative/quantitative fashion, with the ultimate goal to widen our biological knowledge. Further contributing to this search, the present paper reports on a lipidomics method, applied on human blood plasma, utilizing high resolution reversed-phase liquid chromatography hyphenated to Jetstream ESI-QqTOF mass spectrometry. To demonstrate the power, both the qualitative and quantitative aspect of the method are carefully assessed.