In-Silico-Generated Library for Sensitive Detection of 2-Dimethylaminoethylamine Derivatized FAHFA Lipids Using High-Resolution Tandem Mass Spectrometry

Abstract

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a family of recently discovered lipids with important physiological functions in mammals and plants. However, low detection sensitivity in negative ionization mode mass spectrometry makes low-abundance FAHFA challenging to analyze. A 2-dimethylaminoethylamine (DMED) based chemical derivatization strategy was recently reported to improve the MS sensitivity of FAHFAs by labeling FAHFAs with a positively ionizable tertiary amine group. To facilitate reliable, high-throughput, and automatic annotation of these compounds, a DMED-FAHFA in silico library containing 4290 high-resolution tandem mass spectra covering 264 different FAHFA classes was developed. The construction of the library was based on the heuristic information from MS/MS fragmentation patterns of DMED-FAHFA authentic standards, and then, the patterns were applied to computer-generated DMED-FAHFAs. The developed DMED-FAHFA in silico library was demonstrated to be compatible with library search software NIST MS Search and the LC–MS/MS data processing tool MS-DIAL to guarantee high-throughput and automatic annotations. Applying the in silico library in Arabidopsis thaliana samples for profiling FAHFAs by high-resolution LC–MS/MS enabled the annotation of 19 DMED-FAHFAs from 16 families, including 3 novel compounds. Using the in silico library largely decreased the false-positive annotation rate in comparison to low-resolution LC–MS/MS. The developed library, MS/MS spectra, and development templates are freely available for commercial and noncommercial use at .

Analytical Methods for the Determination of Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) in Biological Samples, Plants and Foods

“…Two years later, the same group described a faster protocol for the endogenous FAHFA measurements [17], employing positive pressure (nitrogen) to push solvents though a Strata SI-1 silica SPE cartridge (500 mg silica, 3 mL, Phenomenex, Torrance, CA, USA). By this modification, they shortened the SPE step time from 4 h to 1 h. Other methods described the use of a Hysphere C8 catridge [18], a HyperSep silica SPE column (500 mg, 6 mL, Thermo Scientific, Waltham, MA, USA) [15,[19][20][21], or a strong anion exchange (SAX) SPE-cartridge (1 mL, 50 mg, Weltech, Wuhan, China) [22][23][24].…”

“…Upon DMED labeling, the detection sensitivities of FAHFAs increased and the limits of detections (LODs) of labeled FAHFAs ranged from 0.01 to 0.14 pg. This derivatization approach has been used by the same group in their more recent studies [23,24].…”

Chemical Labeling Assisted Detection and Identification of Short Chain Fatty Acid Esters of Hydroxy Fatty Acid in Rat Colon and Cecum Contents

“…The fragment ion peak at m / z 448 was generated from the neutral loss of the dimethylamino group or –NH(CH 3 ) 2 ( m / z 45 Da) of the molecular ion. The loss of dimethylamino moiety is commonly observed for the DMED-labeled FAHFAs [ 16 ], and it was observed to be the most predominant ion for all the identified SFAHFAs. The product ion at m / z 406 was annotated as the loss of a neutral ketene or CH 2 CO ( m / z 42 Da) from the fragment ion m / z 448 [M+H–NH(CH 3 ) 2 ] + , which is normally observed for acetic acid derivatives [ 18 ].…”

“…However, FAHFAs are low abundant and these techniques are limited by the low detection sensitivity and poor isomer separation. An effort to overcome these problems by chemical labeling experiments using n-[4-(aminomethyl) phenylpyridinium] (AMPP) [ 15 ], 2-dimethylaminoethylamine (DMED) [ 10 , 16 ] were established. In particular, DMED labeling of carboxyl moiety of FAHFAs was found to be advantageous because of enhanced detection sensitivities from 7–72 folds [ 10 ].…”

“…In particular, DMED labeling of carboxyl moiety of FAHFAs was found to be advantageous because of enhanced detection sensitivities from 7–72 folds [ 10 ]. Moreover, the DMED labeling technique improved the identification of many isomeric species, and their in-silico generated libraries were established [ 16 ].…”