U.S. Department of Energy

Pacific Northwest National Laboratory

Separating Lipid Isomers with LC-IMS-MS Measurements to Understand Their Role in Biochemical Processes


Lipids play a vital role in many biochemical processes in biological and environmental systems. While there is great interest in understanding lipidomic processes, distinguishing lipids can be difficult. Unlike the 20 amino acids in peptides, lipids are composed of very similar components such as double bonds and fatty acids which are just arranged at different positions resulting in many lipid isomers with vast functional differences. Since mass spectrometry (MS) is unable to distinguish identical masses, coupling MS with separation techniques such as liquid chromatography and ion mobility spectrometry is promising for distinguishing the isomers. In this study, we utilized LC-IMS-MS to analyze lipid isomers in complex extracts and gain knowledge about the specific roles that lipids have in biochemical processes.    



Lipid standards and complex extracts were analyzed with a Waters HPLC and an Agilent 6560 IMS-QTOF MS platform with both positive and negative polarities to understand whether lipid classes and isomers are separable. Once injected into the nanoESI source, ions were passed through the inlet capillary, focused by a high pressure electrodynamic ion funnel, accumulated in an ion funnel trap before being injected into the IMS drift cell and refocused by a second ion funnel at the drift cell exit prior to QTOF MS detection. Initially, isomeric standards were evaluated manually and then added to LIQUID, the in-house lipid software, to increase its information content. LIQUID was then used for analysis of the complex lipid extracts. 


Preliminary Data 

Over 500 different lipids representing phospholipid, saccharolipid, glycerolipid, sphingolipid and fatty acyl classes were analyzed with the LC-IMS-MS platform. When the LC and IMS dimension were studied independently, it was observed that each lipid class had a distinct slope in the IMS dimension and even subclasses such as phosphatidylcholine and phosphatidylethanolamine could be distinguished. However, if only the LC separation was used, many of the classes eluted together even when long 100-min gradients were employed and the subclasses were not separated under any gradient conditions. The information obtained from the IMS separations illustrated that it is a complementary technique to LC-MS and utilizing all three dimensions (mass, IMS drift time and LC elution time) could be very important in characterizing and identifying unknown lipids.


Lipid isomers consisting of enantiomers, cis/trans double bond orientations and positions, and sn-1/sn-2 chain differences were also analyzed with LC-IMS-MS since these isomers are not distinguished in current LC-MS analyses. In the LC-IMS-MS studies, lipids with cis double bonds were more compact than those with trans orientations, since the cis double bond alignment positions the carbon chains in closer proximity. The location of the double bond also affected the lipid size with centrally located cis double bonds inducing smaller lipid sizes than those with end positions. However, backbone locations for trans orientations could not be quantified since the trans alignment didn’t induce a big change to the lipid backbone. To illustrate this trend, four different bond locations and orientations were studied for the 18:1 fatty acid resulting in cis 11<cis 9< trans 11=trans 9. Enantiomers for ceramides and sn-1 and sn-2 chains in phospholipids were also investigated and found to be separable with IMS. These isomeric trends were then applied to complex lipid extracts and the novel discoveries will be illustrated in the presentation.   


Novel Aspect 

Using IMS-MS to separate isomeric lipidomic classes, enantiomers, cis/trans double bond orientations and positions, and sn-1/sn-2 chain differences

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