Lipid Isobaric Mass Tagging for Enhanced Relative Quantification of Unsaturated sn-Positional Isomers

Changes in the levels of lipid sn-positional isomers are associated with perturbation of the physiological environment within the biological system. Consequently, knowing the concentrations of these lipids holds significant importance for unraveling their involvement in disease diagnosis and pathological mechanisms. However, existing methods for lipid quantification often fall short in accuracy due to the structural diversity and isomeric forms of lipids. To address this challenge, we have developed an aziridine-based isobaric tag labeling strategy that allows (i) differentiation and (ii) enhanced relative quantification of lipid sn-positional isomers from distinct samples in a single run. The methodology enabled by aziridination, isobaric tag labeling, and lithiation has been applied to various phospholipids, enabling the determination of the sn-positions of fatty acyl chains and enhanced relative quantification. The analysis of Escherichia coli lipid extracts demonstrated the enhanced determination of the concentration ratios of lipid isomers by measuring the intensity ratios of mass reporters released from sn-positional diagnostic ions. Moreover, we applied the method to the analysis of human colon cancer plasma. Intriguingly, 17 PC lipid sn-positional isomers were identified and quantified simultaneously, and among them, 7 showed significant abundance changes in the colon cancer plasma, which can be used as potential plasma markers for diagnosis of human colon cancer.

Sodium ion adduction was also studied after isobaric tag labeling.In this experiment, diagnostic ions for C=C bond locations and sn-positions were detected during MS 3 (Figure S2).However, the MS 4 spectrum did not reveal any mass reporter ions when fragmenting the diagnostic ions for sn-positions.Conversely, lithium ion-adducted lipids allowed for the observation of reporter ions in MS 4 , attributed to the elevated intensities of lithiated diagnostic ions for sn-positions in MS 3 .Equal concentrations of lithium salt and sodium salt were employed.If sodium is to be used in the method, a higher concentration of sodium salt may be required.

S3. Reaction optimization of aziridination of PC 16:0/20:4 using different concentrations of pyridine, HOSA, and catalyst, and applying different reaction times
100 µM lipid PC 16:0/20:4 was used for reaction optimization.The conversion distributions of the mono-aziridination product (mono-azi), di-aziridination product (di-azi), triaziridination product (tri-azi), and tetra-aziridination product (tetra-azi) were calculated by taking the intensity of the specific product intensity over the sum intensity of all lipid aziridines.The results indicate that the di-azi product was dominant and achieved the highest conversion under the reaction condition of 5 equivalent (eq) of pyridine, 1.5 eq of HOSA, 20% eq catalyst, and 24 hours of reaction time (Figure S3a-d).
In polyunsaturated lipids, such as PC 16:0/20:4, aziridination (azi) occurred at all C=C bonds, resulting in the formation of mono-azi and di-azi products.Subsequent MS 3 analysis of the mono-azi lipid revealed diagnostic ions indicating C=C bond positions at ∆5, ∆8, ∆11, and ∆14 (Figure S3e).The intensities of these diagnostic ions suggested varying reaction efficiencies at different C=C locations.For instance, the C=C bond at ∆5, situated closest to the carbonyl group, exhibited the lowest abundance of diagnostic ion peaks, possibly due to reduced reaction efficiency at this position caused by steric hindrance from the head group.Importantly, the variability in reaction efficiencies at different C=C bond positions does not impact relative quantification, as our method assesses abundance changes of the same lipid across different samples with identical reaction efficiencies.In many reactions, there are notable variations in the efficiency of cis and trans C=C bonds.However, we specifically chose N-H aziridination to label lipid C=C bonds, as this method exhibits minimal disparity in reaction efficiencies, as demonstrated in the referenced paper (40.Angew.Chem. Int. Ed. 2017, 56, 9886-9890).For instance, in Compound 7 with a trans C=C bond, the aziridination yield is 80%, while in Compound 28 with a cis C=C bond, the yield is 77%.These comparable yields underscore the uniform reaction efficiency of both cis and trans compounds, alleviating concerns in our analysis using the chosen tagging method.

S-5
N-H aziridination of lipid C=C bonds has been successfully demonstrated across various lipid classes, including triglycerides, fatty acids, fatty acid esters, phospholipids, and cholesterol ester lipids as presented in this study and in our previous work (Angew.Chem.Int. Ed. 2022, 61, e202207098).This collective evidence underscores the versatility of aziridination as a derivatization method for unsaturated lipids.
Differences in aziridination efficiencies were observed across various lipid classes.For instance, the aziridination yields for PE 32:1 and PG 32:1 were 77% and 94%, respectively, calculated as [Intensity of lipid aziridine]/([Intensity of lipid aziridine] + [Intensity of unreacted lipid]).It is important to emphasize that while there is variability in reaction efficiencies among different lipid classes, this does not affect relative quantification.Our method evaluates changes in abundance for the same lipid across different samples with consistent reaction efficiencies.

Figure S4 .
Figure S4.Full mass spectrum of iTRAQ-labeling reaction mixture of PC 18:1/16:0 under the optimized condition.The product peak was labeled in red.

Figure S7 .Figure S10 .
Figure S7.(a) MS 4 spectra of lithiated isobaric tagged (a) PG 18:1/16:0 and (b) PE 18:1/16:0 after mixing the two samples.The mass reporter ions at m/z 114 and 117 were observed and the mass reporter ion intensity ratio (I114/I117) indicated the concentration ratio of the two lipids at 1:2.Mass reporter ions are labeled in red.