Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies

Abstract

Phospholipids serve as central structural components in cellular membranes and as potent mediators in numerous signaling pathways. There are six main classes of naturally occurring phospholipids distinguished by their distinct polar head groups that contain many unique molecular species with distinct fatty acid composition. Phospholipid molecular species are often expressed as isobaric species that are denoted by the phospholipid class and the total number of carbon atoms and double bonds contained in the esterified fatty acyl groups (e.g., phosphatidylcholine 34:2). Techniques to separate these molecules exist, and each has positive and negative attributes. Hydrophilic interaction liquid chromatography uses polar bonded silica to separate lipids by polar head group but not by specific molecular species. Reversed phase (RP) chromatography can separate by fatty acyl chain composition but not by polar head group. Herein we describe a new strategy called differential ion mobility spectrometry (DMS), which separates phospholipid classes by their polar head group. Combining DMS with current LC methods enhances phospholipid separation by increasing resolution, specificity, and signal-to-noise ratio. Additional application of specialized information-dependent acquisition methodologies along with RP chromatography allows full isobaric resolution, identification, and compositional characterization of specific phospholipids at the molecular level.

Keywords: glycerophospholipids; lipid metabolism; lipid profiling.

Fig. 1.

Separation of phospholipid classes by HILIC-UPLC. Multiplex analysis of purified phospholipids (A) and human serum (B) using PIS and NLS in positive and negative ion mode. Top panel shows HILIC chromatographic separation and mass spectrometric detection of phospholipid/sphingolipid classes by either precursor m/z 184, 241, 264 or neutral loss m/z 141, 172, 185, 98. Middle panel shows NLS m/z 141 in positive ion mode specific for PE and LPE. Bottom panel shows summed mass spectral (MS) data from NLS m/z 141 in positive ion mode shown indicated in gray above. Inset: magnification from m/z 762 to m/z 774.

Fig. 2.

Separation of phospholipid and sphingolipid classes by DMS. DMS of purified phospholipids/sphingolipids in positive mode and negative mode. Top panels (A, B) show total ion current of DMS separation and MS detection using EMS and the trap function. COV voltage was ramped from −25 to 10. Bottom panels show summed mass spectral data from COV 1.7 to 2.3 specific for SM (C) or COV 2.4 to 3.8 specific for PI (D) indicated in gray above. DMS of human serum in positive mode or negative mode. Top panels (E, F) show total ion current of DMS separation and MS detection using the trap function. COV was ramped from −25 to 10. Bottom panels show summed mass spectral data from COV 1.7 to 2.3 specific for SM (G) or COV 2.4 to 3.8 specific for PI (H) indicated in gray above.

Fig. 3.

Application of DMS combined with HILIC-UPLC to enhance sensitivity. HILIC-based chromatographic separation of lipids extracted from human serum in positive ion mode (left panels) or negative ion mode (right panels) without DMS (A) or with DMS (B). Selected COV values were specific for each phospholipid class.

Fig. 4.

Application of DMS combined with RP-UPLC to enhance selectivity. A: RP-UPLC separation of lipids extracted from human serum in positive ion mode (left panels) or negative ion mode (right panels) without DMS (A) or with DMS (B). Top panel of A shows total ion current of RP chromatogram by EMS scan. Bottom panel shows mass spectral data of EMS scan. Top panel of B shows total ion current of RP chromatograms by EMS scan with application of COV values specific for each phospholipid class (sum of 14 scans). Bottom panel shows three selected EMS scans with COV voltages of 0.3, 2.5, and −5.0 specific for PC, SM, and Cer in positive ion mode or −3.0, −2.9, and 3.0 for PC, PA, and PI in negative ion mode. Also shown are mass spectral data.

Fig. 5.

Combination of RP-UPLC and DMS using IDA reveals molecular species. Top panels show RP-UPLC separation of PE using NLS m/z 141 without DMS (A) or with DMS (B). COV of −4.2 specific for PE was selected. Middle panels show extracted m/z of 744.5 from NLS m/z 141. Bottom panels show enhanced product ion (EPI) scans of the areas indicated in gray above. Inset: magnification from m/z 250 to m/z 325. Of note, the NLSs were performed in the positive ion mode (+); the IDA was performed in the negative ion mode (−).