Strategies to Improve/Eliminate the Limitations in Shotgun Lipidomics

Abstract

Direct infusion-based shotgun lipidomics is one of the most powerful and useful tools in comprehensive analysis of lipid species from lipid extracts of various biological samples with high accuracy/precision. However, despite many advantages, the classical shotgun lipidomics suffers some general dogmas of limitations, such as ion suppression, ambiguous identification of isobaric/isomeric lipid species, and ion source-generated artifacts, restraining the applications in analysis of low-abundance lipid species, particularly those less ionizable or isomers that yield almost identical fragmentation patterns. This article reviews the strategies (such as modifier addition, prefractionation, chemical derivatization, charge feature utilization) that have been employed to improve/eliminate these limitations in modern shotgun lipidomics approaches (e.g., high mass resolution mass spectrometry-based and multidimensional mass spectrometry-based shotgun lipidomics). Therefore, with the enhancement of these strategies for shotgun lipidomics, comprehensive analysis of lipid species including isomeric/isobaric species is achieved in a more accurate and effective manner, greatly substantiating the aberrant lipid metabolism, signaling trafficking, and homeostasis under pathological conditions.

Keywords: in-source fragmentation; ion suppression; isobaric/isomeric species; multidimensional mass spectrometry; shotgun lipidomics.

Fig. 1.

A summary of the strategies for reduction of ion suppression in MDMS-SL. Many strategies have been exploited to reduce ion suppression through enhancement of the signals of molecular ions of selected lipid class(es) in MDMS-SL analysis. These include, but are not limited to, (1) adding a suitable modifier to enhance ionization responses of non-polar lipids; (2) exploiting more sensitive fragments to detect lipid species of a class or a subclass; (3) developing different derivatization methods for different moieties present in different lipid classes to selectively enhance ionization efficiency and/or to generate informative, sensitive, and specific fragment ions; and (4) using unique charge properties or prefractionation to analyze a particular lipid class(es) (e.g., cardiolipin as doubly-charged ions or lysolipids with prefractionation).

Fig. 2.

Representative product-ion ESI-MS spectra of lithiated PC, lysoPC and SM species after CID. Product-ion ESI-MS mass spectra of lithiated PC 16:0/18:1 (a), lithiated 1-palmitoyl-sn-3-phosphocholine (b), and lithiated SM d18:1/18:0 (c) were performed on a QqQ mass spectrometer. Collision activation was conducted with collision energy of 32, 22, and 32 eV, respectively, and gas pressure at 1.0 mTorr.

Fig. 3.

Representative tandem mass spectrum of Fmoc-derivatized phoshoethanolamine-containing lipids in the lipid extract of mouse retinas by NLS of the Fmoc moiety. Tandem mass spectra of Fmoc-PE (inset A) and Fmoc-lysoPE (inset B) were acquired by NLS of 222.2 Da (i.e., Fmoc moiety) on a QqQ mass spectrometer with collision energy of 30 eV and collision gas pressure of 1.0 mTorr. Inset C shows the presence of many very low-abundance PE species in the region. IS denotes internal standard ^[28].

Fig. 4.

Negative ion ESI-MS analysis of a lipid extract of mouse myocardium with a Thermo Scientific TSQ ESI mass spectrometer. The expanded mass spectrum clearly shows the presence of many doubly-charged ions corresponding to cardiolipin molecular species. IS, internal standard.

Fig. 5.

Schematic illustration of the methylation reaction with TMS-diazomethane for analysis of isomeric PG and BMP species. Representative structures of PG and BMP with and without methylation (Panel A). Full-scan mass analysis (Panel B) and neutral loss scan of 203 Da after methylation (Panel C) ^[41].

Fig. 6.

Representative scheme of the methylation reaction of PIP2 with TMS-diazomethane and their resultant ions in MS and tandem MS analyses.