Simple LC-MS Method for Differentiation of Isobaric Phosphatidylserines and Phosphatidylcholines with Deuterated Mobile Phase Additives

Abstract

Lipids from different classes sometimes can exhibit the same exact mass upon electrospray ionization; this presents an analytical challenge in lipidomics. In the negative ionization mode, for example, this can occur with phosphatidylcholines (PCs) and phosphatidylserines (PSs), making them indistinguishable in the absence of fragmentation data. PSs are found at low concentrations in biological samples, making MS/MS spectra difficult to obtain. Moreover, while PCs and PSs are distinguishable in the positive mode, PSs do not ionize as well as PCs, and their ionization is suppressed by the PCs. Here, we show that, in the negative ionization mode, substituting protiated LC-MS additives with their deuterated forms provides a way to distinguish PCs and PSs without chemical derivatization. The method described leverages the differential ionization mechanism of PCs and PSs. PCs are ionized via adduction with salts, whereas PSs ionize via hydrogen abstraction. Substituting the salts used for LC-MS with their deuterated form shifts the mass of PCs by the number of deuterium atoms in the salt, while the mass of PSs remains the same. This comparative shift enables their direct differentiation. We demonstrate that the use of deuterated formate shifts the mass of PCs and provides a direct method to distinguish PCs and PSs, even at biologically relevant low concentrations. The utility of the method was established and validated in the simultaneous analysis of PCs and PSs in lipid extracts from isolated liver mitochondria in two different rat strains. Thirteen low concentration PSs were identified that would otherwise not have been distinguishable from low concentration PCs.

Figure 1

LC-MS chromatograms of an equimolar concentrations (32 μM) of PS (17:0/17:0) and PC (17:0/17:0). A: Positive mode analysis of PS (17:0/17:0) and PC (17:0/17:0) B: Negative ion mode analysis of PS(17:0/17:0) and PC (17:0/17:0).

Figure 2

PC (36:1) and PS (39:0) exhibit the same exact mass upon negative mode ionization in the presence of formate. A: The proposed transformation of PC 36:1 after analysis with mobile phases that contain deuterated formate (FA_D). B: The m/z of PS (39:0) is expected to remain the same after analysis with mobile phases that use either deuterated or protiated formate.

Figure 3

Direct infusion negative mode analysis of a mixture of PC (17:0/17:0) and PS (17:0/17:0). A: Analysis of the PC and PS mixture with solvent comprised of protiated ammonium formate (FA_H). Both the M−H ion of the PS and the M+FA-H ion of the PC are observed. B: Analysis of the PC and the PS mixture with a solvent comprised of deuterated formate (FA_D). The m/z of PS (17:0/17:0) remains the same ([M−H]-), but the m/z of PC(17:0/17:0) is shifted by 1 Da (the mass difference between a hydrogen and a deuterium atom) from 806.5945 to 807.6018. The instrument mass error is 5ppm.

Figure 4

The residual protiated peak of a PC [PC(34:1)] is observed at 0.5% of the deuterated peak when LC-MS analysis is performed with >95% deuterated formate and deuterated formic acid.

Figure 5

Comparison of retention times of lipid standards analyzed using either protiated or deuterated formate. A: The separation of a mixture of phospholipids with protiated additives (formate/formic acid). B: The separation of phospholipids with deuterated additives (formate/formic acid). The slight shift observed in retention time can be attributed to injection-to-injection variation.

Figure 6. LC-MS analysis of a liver mitochondrial pool with protiated and deuterated formate and formic acid

A: The phospholipid (PL) and sphingolipid (SL) region of a mitochondrial pool analyzed with protiated additives (formate/formic acid). The retention time and m/z of some peaks are shown. B: The PL and SL region of the same mitochondrial pool analyzed with deuterated additives (formate/formic acid). A mass shift of 1 Da is observed for all LC peaks that contain a choline moiety. Note that the data shown are raw chromatograms and no retention time alignment has been performed. The axes are presented shifted by ~ 30 seconds, and major peaks of interest are linked between panels A and B by arrows to highlight the conserved order of elution. The ion count of the most abundant phospholipid (PC (38:4) at RT 13.99 in deuterated formate was 1.2E7. The ion count of the most abundant PS (PS(38:4) at RT 12.77 was 8.4E4 and of the ion count of the least abundant PS, PS (38:7) at RT 11.29 was 4.6E2 (both in deuterated formate/formic acid).

Figure 7

Analysis of a liver mitochondrial extract with protiated additives (formate/formic acid) or deuterated additives (formate/formic acid) to distinguish isobaric PCs and PSs with the same exact mass. A: The extracted ion chromatogram (XIC) of m/z 782.4978 (+/− 5 ppm) in protiated formate/formic acid shows two peaks with that mass. B: The XIC of m/z 782.4978 (+/− 5ppm) shows a single peak which represents a PS. The mass of the second peak (XIC 783.5040 +/− 5 ppm) is shifted by 1 Da to 783.5026 -- an indication that it is a PC.

Figure 8

Comparison of the four most abundant PSs in rat liver mitochondria of obesity prone (OP) and obesity resistant (OR) rats. The raw data is normalized to the sum of the top hundred most intense ion to emphasize shifts of the relative abundance of these lipids.