Profiling of Low-Abundance Branched-Chain Fatty Acids via Radical Directed Dissociation Tandem Mass Spectrometry
More
Advanced Search
  • Free to Read
  • Editors Choice
Article

Profiling of Low-Abundance Branched-Chain Fatty Acids via Radical Directed Dissociation Tandem Mass Spectrometry
Click to copy article linkArticle link copied!

  • Ruijun Jian
    Ruijun Jian
    MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China
    More by Ruijun Jian
    Orcidhttps://orcid.org/0009-0003-7744-6860
  • Shengzhuo Wang
    Shengzhuo Wang
    MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China
    More by Shengzhuo Wang
  • Lipeng Qiao
    Lipeng Qiao
    MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China
    More by Lipeng Qiao
  • Xue Zhao*
    Xue Zhao
    College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot, 010070 Inner Mongolia China
    *Email: [email protected]
    More by Xue Zhao
    Orcidhttps://orcid.org/0009-0001-8686-2773
  • Yu Xia*
    Yu Xia
    MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China
    *Email: [email protected]
    More by Yu Xia
    Orcidhttps://orcid.org/0000-0001-8694-9900
Open PDFSupporting Information (1)

Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2026, 37, 3, 782–789
Click to copy citationCitation copied!
https://doi.org/10.1021/jasms.6c00001
Published February 18, 2026

Copyright © 2026 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved. This publication is licensed under these Terms of Use.

Request reuse permissions

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Branched-chain fatty acids (BCFAs) are key components of the bacterial lipidome, playing a role in regulating membrane fluidity and permeability. In mammals, BCFAs occur at much lower concentrations, and their functions remain largely unexplored. Conventional lipid analysis methods, employing collision-induced dissociation (CID)-tandem mass spectrometry (MS/MS), often fail to locate methyl branching, as fragmentation rarely occurs around the branching site. Here, we introduce a bifunctional derivatization reagent, 1-(8-methoxy-5-quinolinyl) methanamine (MeO-QN), for pinpointing methyl branching in BCFAs with high sensitivity. MeO-QN enhances ionization efficiency of derivatized BCFAs in positive ion mode due to its quinoline moiety and serves as a precursor for radical-directed dissociation (RDD). Upon CID, the quinoline-O radical (QN-O•) is generated, which subsequently induces RDD along the fatty acyl chain and forms a characteristic 28 Da spacing indicative of the branching point. By integrating this MS/MS method with reversed-phase liquid chromatography, we have developed a sensitive analytical workflow, detecting BCFAs at sub-nM levels in mammalian samples. We detected the rarely reported n-5 methyl branched fatty acid (FA 16:0;12Me) in pooled human plasma. We also observed significantly reduced even-chain isobranched fatty acids in breast cancer cells (MDA-MB-468) versus normal breast cells (MCF-10A), suggesting its potential in cancer biomarker discovery.

This publication is licensed for personal use by The American Chemical Society.

Copyright © 2026 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved.

Subjects

what are subjects

Keywords

what are keywords

Introduction

Click to copy section linkSection link copied!
Fatty acids (FAs) are the building blocks of a variety of complex lipid molecules, (1) and they play key roles in cell energy metabolism as well as signal transduction. (2) Branched-chain fatty acids (BCFAs) are a subclass of FAs, featuring fatty acyl chains attached with one or more methyl branches. Originating predominantly from gut bacteria, milk, and dairy products, BCFAs have attracted growing research interest due to their potential health-promoting properties. (3) Nevertheless, BCFAs are found in relatively low concentrations, constituting less than 1% of the total fatty acid content in mammalian tissues (4,5) with the exception of the skin and vernix caseosa. There is a strong need for the development of sensitive analytical methods for their identification and quantitation.
Gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) is the most commonly used method for the analysis of FAs once they are derivatized into fatty acid methyl esters (FAMEs). However, it struggles to distinguish isomeric BCFAs due to the absence of characteristic ions indicative of methyl branches and the frequent coelution of these isomers in GC. (6) To address this limitation, Ran-Ressler et al. coupled tandem mass spectrometry (MS/MS) with EI-MS, (7) allowing for confident identification of methyl esters of saturated BCFAs. The combination of EI-MS/MS and covalent adduct chemical ionization mass spectrometry (CACI-MS) further enabled complete structural elucidation of monounsaturated BCFAs. (8) More recently, Wang et al. developed a chemical ionization (CI)-based methodology termed “three ion monitoring”, which showed improved sensitivity for BCFAs as compared to previously described methods. (9)
With the development of electrospray ionization (ESI), fatty acids are increasingly analyzed via ESI-MS/MS, employing collision-induced dissociation (CID). However, decarboxylation and dehydration are the dominant fragmentation pathways when analyzed in negative ion mode, thereby providing limited insights into chain modifications. (10) Alternatively, higher-energy CID (>50 eV) initiates charge-remote fragmentation (CRF) in the presence of a fixed positive charge, which can be incorporated to FAs via derivatization, (11) e.g., N-(4-aminomethylphenyl)pyridinium (AMPP) (12) and gas-phase ion/ion charge inversion. (13) CRF induces fragmentations along the fatty acyl chain, which can be used to locate the anteisoconfiguration of methyl branching. However, it fails to distinguish between iso- and straight-chain isomers. On the other hand, radical-directed dissociation (RDD) generates rich intrachain cleavages on fatty acyls, (14) thereby facilitating more effective identification of chain modifications. RDD can be initiated from ultraviolet photodissociation (UVPD) of lipids ions consisting of labile chromatophores, such as carbon–iodine (C–I) bond. (15) Blanksby group developed a fixed-charge photolabile reagent, I-AMPP+, and successfully coupled this derivatization approach with 266 nm UVPD into a liquid chromatography (LC)-MS workflow. (16) McLuckey group employed gas-phase conversion of even-electron deprotonated FAs to radical cations by using [Mg(Terpyridine-TEMPO)2]2+ and [Mg(tributyl-terpyridine)2]2+ as reagents ions. They demonstrated the capability to localize the site(s) of methyl branching via CID of the thus-formed radical cations. (14,17) Our group previously developed a CID-triggered RDD approach for lipid structural analysis. FAs were derivatized by O-benzylhydroxylamine (O-BHA); subsequent CID of the lithium ion adduct of the O-BHA derivatized FAs formed lipid nitroxide radical, leading to fragments indicative of the methyl branching. (18) However, the requirement for the addition of lithium ions to enhance RDD poses a limitation when coupled with reversed-phase liquid chromatography (RPLC).
Herein, we have developed a new RDD reagent, 1-(8-methoxy-5-quinolinyl) methanamine (MeO-QN), which enables both charge switching and CID-triggered RDD for the localization of the methyl branching of BCFAs. When coupled with RPLC-MS, our method achieved subnanometer sensitivity for BCFAs, outperforming conventional CID and existing RDD approaches in both detection limits and structural specificity. This method was further applied to profile free and total FAs at the methyl branching level in human plasma and breast cancer cells.

Experimental Section

Click to copy section linkSection link copied!

Lipid Nomenclature

The structural annotation of FAs follows the shorthand notation proposed by LIPIDMAPS. (19) BCFAs are annotated according to the n-nomenclature, which starts from the methyl end and indicates the total number of carbon atoms in the backbone. A fatty acid with a methyl branch on the penultimate carbon (n-2) is designated as an iso-BCFA, abbreviated as “i-”, whereas one with a methyl branch on the antepenultimate carbon (n-3) is classified as an anteiso-BCFA, abbreviated as “a-”. For instance, 15-methylhexadecanoic acid is denoted as FA 16:0;15Me and is also referred to as “i-17:0”. The prefix “n-” denotes a straight chain or normal chain FAs.

Materials

All fatty acid standards were acquired from Cayman Chemical (Ann Arbor, MI, USA). The organic solvents and water used were of HPLC-MS grade and were sourced from Fisher Scientific (Ottawa, ON, Canada). MeO-QN was obtained from Fluorochem Ltd. (Derbyshire, UK). The reagents 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU) and 1-Hydroxybenzotriazole (HOBt) were purchased from Bidepharm (Shanghai, China). Pooled human plasma added with anticoagulant lithium heparin was obtained from Innovative Research, Inc. (Novi, MI, USA). MCF-10A and MDA-MB-468 cell lines were acquired from the American Type Culture Collection (ATCC) in Manassas, VA, USA. Further details regarding cell culture and pretreatment can be found in the Supporting Information.

Free Fatty Acid (FFA) Extraction and Total Fatty Acid (TFA) Extraction

FFAs in pooled human plasma were extracted using the acidic H2O/MeOH/isooctane procedure. (20) The plasma sample (100 μL with 2.5 nmol FA 18:0-D4 added) was extracted by a solution containing 200 μL of water, 500 μL of methanol, 25 μL of HCl and 1.5 mL of isooctane. The sample was vortexed for 10 min and centrifuged at 8000 rpm for 5 min to collect the upper layer. The extraction process was repeated once, and organic layers were combined for drying under nitrogen flow.
TFA in cell lines were obtained by total lipid extraction via methyl tert-butyl ether (MTBE) protocol followed by saponification. (21) Cells (5 × 105) were extracted by a solution consisting of 500 μL of water, 600 μL of methanol, and 2 mL of MTBE with 5 nmol of FA 18:0-D4 added. After 10 min vortex, the mixture was centrifuged at 10,000 rpm for 10 min and the upper layer was collected. The same amount of MTBE was added to the remaining mixture, and the extraction process was repeated once. The upper layers were combined and dried under nitrogen flow. The extracted lipids were saponified in 500 μL MeOH/15% KOH (50/50, v/v) at 37 ◦C for 30 min. The solution was acidified to pH < 5 by adding 1 N HCl dropwise and subsequently extracted by 1.5 mL isooctane for two times. The combined extracts of FAs were dried under nitrogen flow.
Fatty acids are ubiquitously present in laboratoryware, solvents, and extraction backgrounds. Blank samples spiked with internal standards (IS), were prepared. Background interference was calculated based on the ratio of extracted ion peak area of a given FA to that of IS, denoted as (AFA/AIS)blank, where the subscript “blank” indicates the blank solutions used for background correction. For the relative quantitation of FAs in biological samples, all reported FA/AIS values were corrected by a subtraction of (AFA/AIS)blank.

Derivatization by MeO-QN

FA standards or dried lipid extracts were dissolved in 60 μL of ACN. HATU (22) (20 μL, 20 mM in ACN) and HOBt (20 μL, 20 mM in ACN) were added to the FA solution and the mixture was vortexed for 5 min. Then MeO-QN (20 μL, 30 mM ACN) was added to the solution. The above solution was incubated at 60 °C for 1 h and then cooled to room temperature before being dried under nitrogen flow. The derivatized FAs were redissolved in 1 mL of ACN before RPLC-MS analysis.

RPLC-MS analysis

Most data were obtained by a triple quadrupole/linear ion trap hybrid mass spectrometer QTRAP 4500 (Sciex, Toronto, Canada) coupled with an Exion LC AC system. The CORTECS C18 column was used (90Å, 1.6 μm, 2.1 mm × 150 mm, Waters, Milford, MA, USA) for separation. Mobile phase A was 10 mM HCO2NH4 in ACN/H2O (60/40, v/v) and mobile phase B was 10 mM HCO2NH4 in ACN/IPA/H2O (50/45/5, v/v/v). A flow rate of 0.25 mL/min was used. The LC gradient was set as follows: 10% B at 0–6 min, 10%–95% B at 6–36.5 min, 95%B at 36.5–42 min, and 10% B at 42.5–45 min. The injection volume was 2 μL per run. Source parameters were set as follows: ESI voltage (+4500 V/-4500 V), curtain gas (30 psi), interface heater temperature (450 °C), and nebulizing gas (GS1, 50 psi; GS2, 50 psi). MS2 CID of fatty acid derivatives were acquired on a QTRAP 4500 mass spectrometer using both beam-type CID and ion-trap CID. Ion-trap CID was employed for the identification of complex samples. For beam-type CID, the applied acquisition mode was Enhanced Product Ion (EPI), whereas that for ion-trap CID was MS/MS/MS (MS (3)), in which the second precursor was set identical to the first precursor. Detailed experimental parameters are provided in Tables S1 and S2. For quantification, we employed precursor ion scan for m/z 158, with the m/z range set at 300–600, a scan rate of 200 Da/s, and the collision energy (CE) adjusted to 55 eV. The high-resolution CID spectra of MeO-QN were acquired on a quadrupole time-of flight (Q-TOF) mass spectrometer (X500R, Sciex, Toronto, Canada) with CE set as 25 eV and MS resolving power around 30,000.

Quantum Chemical Computation

Density functional theory (DFT) computations were performed using the Gaussian 16 suite of programs for geometry optimizations and vibrational frequencies of MeO-QN. The M062X functional, which yields precise results for small molecules, was adopted with the def2tzvp basis sets. The geometries were optimized to a minimum at 0 K. The bond dissociation energy was calculated by the difference between the enthalpy (vibrational frequency corrected at 298 K) of the radical product and that of the fracture reactant.

Results and Discussion

Click to copy section linkSection link copied!

CID Triggered Quinoline-O· RDD

Although the bond dissociation energy (BDE) of the C–O bond in a neutral MeO-QN molecule is as high as 63.7 kcal/mol (Figure 1a), MS2 CID of [MeO-QN+H]+ (Figure 1b) produces QN-O• radical ions (m/z 174.080) at relatively high abundance through loss of a methyl radical (•CH3). DFT calculation reveals that upon protonation an intramolecular hydrogen bond of 2.19 Å is formed between the O atom of MeO-QN and the proton on the quinoline nitrogen, creating a stable five-membered ring (Figure S1a). Consequently, the BDE of the O–CH3 decreased to 48.5 kcal/mol after protonation. In the MS2 CID spectrum of [MeO-QN+H]+, we also observed fragment peaks at m/z 173.073 from CH4 loss and m/z 158.061, denoted as “Q” in Figure 1c, corresponding to sequential losses of •CH3 and •NH2. Additionally, the fragment at m/z 146.061 results from the sequential losses of •CH3 and CO, leading to ring closure.

Figure 1

Figure 1. (a) BDE of the C–O bond in MeO-QN before and after protonation. (b) MS2 CID spectrum of [MeO-QN+H]+. (c) Derivatization of FA by MeO-QN and proposed fragmentation pathways of derivatized FA ([FA# + H]+) under CID.

High Resolution Image
Download MS PowerPoint Slide
We then investigated whether QN-O• could induce carbon–carbon bond cleavages within the fatty acyl chain, referred to later as the intrachain cleavages. CRF can produce intrachain fragmentation patterns (23) similar to those caused by RDD. (18) To distinguish between the fragments resulting from these two types of fragmentation, we employed fully deuterated FA 17:0-D33 as a model compound. The derivatization efficiency of the fatty acid was found to be quantitative, reaching 100% (Figure S2). We then compared the MS/MS behavior of MeO-QN derivatized FA ([FA 17:0-D33# + H]+) under beam-type and ion-trap CID conditions. Although the FA QN-O• radical cation (m/z 459) was not present, we observed a series of fragments with 16 Da spacing (CD2), starting from the methyl end (m/z 441, 425, ..., and 233) under both CID conditions (Figure S3a-c, series I). These fragments should arise from RDD processes in which QN-O• abstracts a D atom along the acyl chain, leading to radical propagation and subsequent intrachain cleavage (Scheme S1). Additionally, we observed another fragment ion series with 16 Da spacing of (CD2), e.g., m/z 390, 374, ..., and 246, but at much lower abundance. These ions should result from CRF (Figure S3a, series II, Scheme S1), which occur 5–6 carbons away from the methyl terminus and increase in abundance as it approaches the carbonyl end. Fragments generated at the same site via CRF are 14 Da larger than those from RDD of FA 17:0-D33 because RDD requires an additional loss of the nondeuterated methyl group. We found that CRF was more abundant in beam-type CID while it was almost absent in ion-trap CID. Therefore, ion-trap CID was chosen in later studies for the analysis of BCFAs.

Locating Methyl Branching via QN-O• RDD

FA n-17:0 and its two branched-chain isomers, FA i-17:0 and FA a-17:0, were employed to explore whether QN-O• induced RDD can be used to distinguish BCFAs. The ion-trap CID spectra of these MeO-QN derivatized isomers are compared in Figure 2a-c. For FA n-17:0, a series of fragments spaced by 14 Da, ranging from m/z 411 to m/z 215, is formed from sequential losses of 15 Da (-•CH3) and •CnH2n+1 (Figure 2a). The continuous ion series, however, is interrupted by a distinct spacing of 28 Da (C2H4), due to cleavage of C–C bonds adjacent to the branching site, i.e., m/z 383, 411 pair for i-17:0 (Figure 2b) and m/z 369, 397 for a-17:0 (Figure 2c). This 28 Da spacing is a signature to identify and locate methyl branching. Interestingly, the lower m/z fragment in the ion pair exhibits higher ion abundance than the larger m/z one. This might result from the preferred loss of a more stable secondary carbon-centered radical (i.e., an isopropyl radical) than a primary carbon-centered radical.

Figure 2

Figure 2. Ion-trap CID spectra of MeO-QN derivatized FAs and observed fragmentation sites of protonated FA-QN-O•. (a) [n-17:0#+H]+, (b) [i-17:0#+H]+, (c) [a-17:0#+H]+, and (d) [FA 16:0;3Me,7Me,11Me,15Me#+H]+.

High Resolution Image
Download MS PowerPoint Slide
Besides the distinct 28 Da spacing pattern for the two BCFAs, the three isomers shared other common fragments ranging from m/z 150 to m/z 230. The fragment ion at m/z 229 is likely generated from C3–C4 cleavage, forming a conjugated enone ion. The fragment at m/z 216 is a radical cation, resulting from C2–C3 cleavage, while [MeO-QN+H]+ (m/z 189) is formed from amide bond cleavage. The fragment at m/z 158, denoted as the “Q” fragment, is the base fragment peak in all three isomers. This fragment was later used for quantitation by MS/MS. We also performed beam-type CID for the derivatives of the three fatty acids (Figure S4a-c); however, the RDD fragments were formed at much lower abundance (Figure S 4d). In addition to monomethyl-BCFAs, we utilized an isoprenoid fatty acid, phytanic acid (FA 16:0;3Me,7Me,11Me,15Me), as a model of polymethyl-BCFAs. CID induced RDD provides signature 28 Da peak spacings for n-2, n-6, n-10, and n-14 methyl branches, respectively (Figure 2d). These findings demonstrate that QN-O• RDD is suitable for locating methyl branches in monomethyl and polymethyl BCFAs.

Analysis of Other Types of Chain Modifications

We also tested whether QN-O• RDD could locate other chain modifications such as C═C, cyclopropane, and hydroxyl modification. However, we found that it was difficult to distinguish between C═C and cyclopropyl alcohol (Figure S6a-c). The energies required for proton migration from quinoline to the C═C and the cyclopropane group are approximately 35 and 47 kcal/mol, respectively (Table S1 (24)). They are less than the BDE of the C–O bond in protonated MeO-QN, therefore suppressing the RDD fragmentation around these two types of chain modifications. For hydroxy fatty acids, loss of H2O dominates with minimum RDD detected in positive ion mode (Figure S6d).

An RPLC-MS/MS Workflow for Profiling of BCFAs from Biological Samples

The QN-O• RDD MS/MS method was further integrated into an RPLC-MS/MS workflow. Briefly, the FA isomers were separated by RPLC; during subsequent MS/MS experiments, the RDD fragments were used for their identification, while PIS of the Q fragment (m/z 158) was used for the relative quantification of FAs. It should be noted that the gas-phase basicity of the quinoline is 220.2 kcal/mol, (24) even higher than that of pyridine (214.6 kcal/mol). Consequently, the ion signals of MeO-QN derivatized FAs in positive ion mode ESI are boosted, significantly enhancing the detection sensitivity (Figure S7a). Figure 3a shows the extracted ion chromatogram (XIC, PIS of m/z 158) of the linear and branched-isomer groups of FA 15:0 and 17:0. The resolution between the iso- and anteiso-forms exceeds half-peak separation (R= 1.2). We used FA d4–18:0 (5 μM) as an internal standard to create calibration curves for five fatty acids prepared in standard solutions with different branching locations, lengths, and degrees of saturation. The linear dynamic range is about 4 orders of magnitude, ranging from 5 nM to 25 μM. The limit of identification (LOI), established by setting RDD fragments with a signal-to-noise ratio (S/N) exceeding 3, is 0.2 nM for linear and single-methyl branched fatty acids and 1 nM for phytanic acid (Figure 3b, Figure S8), significantly better than the previously reported nitroxide radical RDD approach. (18)

Figure 3

Figure 3. (a) XIC from PIS of m/z 158 for equimolar mixtures of FA 15:0 and FA 17:0 isomers. (b) Calibration curves and limit of identification (LOI) for various fatty acids employing PIS of m/z 158 (IS: FA d4–18:0).

High Resolution Image
Download MS PowerPoint Slide
The workflow for profiling BCFAs from complex biological samples is summarized in Scheme 1. First, free fatty acids (FFA) or total fatty acids (TFA) obtained through saponification are derivatized using MeO-QN. Two separate injections were performed to enable quantitation and identification of the FAs. After separation by RPLC, we performed PIS of m/z 158 in positive ion mode to obtain a profiling of the fatty acid composition. Subsequently, we utilized QN-O• RDD to determine the branched-chain location for each chromatographic peak. For chromatographic peaks that were not fully separated, we employed PeakFit version 4.12 software for deconvolution. The deconvolution process utilized the residual method with the peak type set to Gaussian.

Scheme 1

Scheme 1. Workflow for the Analysis of Fatty Acids with Precision at the Branched-Chain Level Using QN-O• RDD
High Resolution Image
Download MS PowerPoint Slide

Profiling of FFAs from Human Plasma

Circulating FFAs provide fuel for physiologic activities and participate in important signaling events. (25) There is growing evidence that BCFAs are associated with inflammation (26) and they play important roles in regulating energy metabolism. (27) The QN-O• RDD workflow was applied to analyze FFAs in pooled human plasma. We found that contamination of fatty acids from solvent was unavoidable even though LC-MS quality-level solvent was used (SI, Figure S9). Due to the inevitability and instability of fatty acid contamination, (28) we set up blank extractions for background correction for analysis between each sample. Furthermore, for the identification of fatty acids in biological samples, confirmation of their presence was only made when AFA/AIS exceeded three times the background level (AFA/AIS) Blank.
We identified a total of 52 FFAs down to the branching location level, including 17 saturated straight-chain FAs, 13 saturated BCFAs, and 22 unsaturated FAs (Figure 4a). These subgroups of FAs exhibit a concentration span of 4 orders of magnitude, with all saturated BCFAs at sub-μM concentrations. Compared with FFAs recently reported by Menzel et al., (29) we revealed the existence of very long-chain FAs (VLCFAs), such as n-25:0, n-26:0 and FA i-24:0. Furthermore, we identified FA 16:0;12Me, the n-5 methyl-branched isomer among three other major isomers of FA 17:0. The XIC of [FA 17:0#+H]+ (m/z 441) from human plasma is shown in Figure 4b. High quality MS2 CID data were obtained for the four most abundant elution peaks. The RDD data of the peak eluted at 21.0 min are shown in Figure 4c as an example. The characteristic 28 Da interval between ion pairs m/z 341 and m/z 369 suggests the presence of the n-5 methyl branching. The n-5 isomer accounts for 2% of total FA 17:0 and elutes earliest than the iso- (21.5 min), anteiso- (21.2 min), and straight chain (22.2 min) isomers on RPLC (Figure 4b). Our data also reveal that odd-chain FAs exist as straight chain, iso-, and anteiso-isomers, whereas even-chain FAs occur exclusively in the straight chain and iso-form. It is hypothesized that BCFAs in humans are derived from dietary sources, predominantly dairy products, ruminant milk, and other foods of ruminant origin.

Figure 4

Figure 4. (a) Profiling of FFAs in human plasma (IS: FA 18:0-D4, 50 μ M). (b) XIC of [FA 17:0#+H]+ (m/z 441) in human plasma. (c) Ion trap CID spectrum of [FA 17:0#+H]+ eluted at 21.0 min in panel b.

High Resolution Image
Download MS PowerPoint Slide

Profiling of TFAs in Human Breast Cancerous and Normal Cells

Recently, BCFAs, such as i-15:0, have been found to exhibit anticancer properties. (30,31) We employed the human breast cancer cell line MDA-MB-468 and the normal breast epithelial cell line MCF-10A as models to investigate the differences in the total fatty acid composition at the branched chain level between cancerous and normal cells. When normalized to the total fatty acid content, unsaturated fatty acids exhibit significant changes (Figure 5a, Figure S11). In cancer cells, the levels of most monounsaturated (FA 18:1) and poly unsaturated fatty acids with a degree of saturation higher than 3 (FA 20:4, FA 22:5, FA 22:6) were significantly increased, likely due to enhanced desaturase enzyme activity in lipid de novo synthesis. (32) In contrast, the levels of FA 18:2 and FA 20:2 were significantly reduced, with FA 14:2 and FA 26:2 undetectable in cancer cells. Among 5 groups of saturated BCFAs (Figure 5b), the relative compositions of FA i-16:0 and FA i-18:0 were 0.7% and 0.3% in the cancer cells, exhibiting significant decreases relative to 1.8% and 0.9% found in normal cells. Toda et al. reported that FA i-16:0 showed inhibitory effects on fatty acid synthesis, thus leading to the death of breast cancer cells. (33) The mechanistic explanation for the reduced levels of even-chain BCFAs in breast cancer cells remains unclear; however, they may offer novel insights into human health assessment and disease-related analysis.

Figure 5

Figure 5. (a) Volcano plot of TFA at the sum composition level in breast cancer cells and (b) comparisons of BCFA isomer compositions in breast cancer cells (MDA-MB-468) to normal breast epithelial cells (MCF-10A).

High Resolution Image
Download MS PowerPoint Slide

Conclusions

Click to copy section linkSection link copied!
In summary, we have developed a novel approach for analysis of BCFAs by utilizing MeO-QN as a charge-switching reagent and radical precursor for CID-triggered RDD MS/MS. This method significantly improves the sensitivity and confidence for the BCFA analysis from complex lipidomes, even at sub-nM concentrations. We confirmed the presence of the uncommon n-5 methyl branched fatty acid (FA 16:0;12Me) in pooled human plasma. Furthermore, the observed decrease of FA i-16:0 and FA i-18:0 in breast cancer cells compared to normal breast cells might provide new insight into the investigation of metabolic remodeling in breast cancer.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.6c00001.

  • Experimental details; figures of optimized MeO-QN structure through DFT calculations, mass spectra of MeO-QN derivatized FAs, and background contaminations, tables of QTRAP 4500 parameters and gas basicity of functional groups (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Authors
  • Authors
    • Ruijun Jian - MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China;  Orcidhttps://orcid.org/0009-0003-7744-6860
    • Shengzhuo Wang - MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China
    • Lipeng Qiao - MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biological, Department of Chemistry, Tsinghua University, Beijing 100084, China
  • Author Contributions

    Conceptualization, R.J., Y.X.; investigation, R.J., Y.X.; theoretical calculation, S.W.; data processing, R.J., L.Q..; writing─original draft preparation, R.J., Y.X.; writing─review and editing, R.J., X.Z., and Y.X.; supervision, Y.X. All authors have read and agreed to the published version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

Financial support from the National Natural Science Foundation of China (no. 22225404, 22074075) and the National Key R&D Program of China (2023YFA0913902) is greatly appreciated.

References

Click to copy section linkSection link copied!

This article references 33 other publications.

  1. 1
    Rustan, A. C.; Drevon, C. A., Fatty Acids: Structures and Properties. In Encycl. Life Sci. , 2005.  DOI: 10.1038/npg.els.0003894 .
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Currie, E.; Schulze, A.; Zechner, R.; Walther, T. C.; Farese, R. V. Cellular Fatty Acid Metabolism and Cancer. Cell Metab 2013, 18, 153– 161,  DOI: 10.1016/j.cmet.2013.05.017
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Taormina, V. M.; Unger, A. L.; Schiksnis, M. R.; Torres-Gonzalez, M.; Kraft, J. Branched-Chain Fatty Acids─An Underexplored Class of Dairy-Derived Fatty Acids. Nutrients 2020, 12, 2875– 2890,  DOI: 10.3390/nu12092875
    Google Scholar
    There is no corresponding record for this reference.
  4. 4
    Martínez-Montoro, J. I.; Núñez-Sánchez, M. Á.; Martinez-Sanchez, M. A.; Balaguer-Román, A.; Fernández-Ruiz, V. E.; Ferrer-Gómez, M.; Sledzinski, T.; Frutos, M. D.; Fernández-García, J. C.; Mika, A.; Ramos-Molina, B. Hepatic and serum branched-chain fatty acid profile in patients with nonalcoholic fatty liver disease: A case–control study. Obesity 2023, 31, 1064– 1074,  DOI: 10.1002/oby.23711
    Google Scholar
    There is no corresponding record for this reference.
  5. 5
    Mika, A.; Stepnowski, P.; Kaska, L.; Proczko, M.; Wisniewski, P.; Sledzinski, M.; Sledzinski, T. A comprehensive study of serum odd- and branched-chain fatty acids in patients with excess weight. Obesity 2016, 24, 1669– 1676,  DOI: 10.1002/oby.21560
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Wang, Z.; Yang, T.; Brenna, J. T.; Wang, D. H. Fatty acid isomerism: analysis and selected biological functions. Food Funct 2024, 15, 1071– 1088,  DOI: 10.1039/D3FO03716A
    Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Ran-Ressler, R. R.; Lawrence, P.; Brenna, J. T. Structural characterization of saturated branched chain fatty acid methyl esters by collisional dissociation of molecular ions generated by electron ionization. J. Lipid Res. 2012, 53, 195– 203,  DOI: 10.1194/jlr.D020651
    Google Scholar
    There is no corresponding record for this reference.
  8. 8
    Wang, D. H.; Wang, Z.; Brenna, J. T. Gas Chromatography Chemical Ionization Mass Spectrometry and Tandem Mass Spectrometry for Identification and Straightforward Quantification of Branched Chain Fatty Acids in Foods. J. Agric. Food. Chem. 2020, 68, 4973– 4980,  DOI: 10.1021/acs.jafc.0c01075
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Wang, D. H.; Wang, Z.; Chen, R.; Kothapalli, K. S. D.; Brenna, J. T. Very Long-Chain Branched-Chain Fatty Acids in Chia Seeds: Implications for Human Use. J. Agric. Food. Chem. 2020, 68, 13871– 13878,  DOI: 10.1021/acs.jafc.0c05612
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Kerwin, J. L.; Wiens, A. M.; Ericsson, L. H. Identification of fatty acids by electrospray mass spectrometry and tandem mass spectrometry. J. Mass Spectrom. 1996, 31, 184– 192,  DOI: 10.1002/(SICI)1096-9888(199602)31:2<184::AID-JMS283>3.0.CO;2-2
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Randolph, C. E.; Beveridge, C. H.; Iyer, S.; Blanksby, S. J.; McLuckey, S. A.; Chopra, G. Identification of Monomethyl Branched-Chain Lipids by a Combination of Liquid Chromatography Tandem Mass Spectrometry and Charge-Switching Chemistries. J. Am. Soc. Mass Spectrom. 2022, 33, 2156– 2164,  DOI: 10.1021/jasms.2c00225
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Wang, M.; Han, R. H.; Han, X. Fatty Acidomics: Global Analysis of Lipid Species Containing a Carboxyl Group with a Charge-Remote Fragmentation-Assisted Approach. Anal. Chem. 2013, 85, 9312– 9320,  DOI: 10.1021/ac402078p
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Randolph, C. E.; Foreman, D. J.; Betancourt, S. K.; Blanksby, S. J.; McLuckey, S. A. Gas-Phase Ion/Ion Reactions Involving Tris-Phenanthroline Alkaline Earth Metal Complexes as Charge Inversion Reagents for the Identification of Fatty Acids. Anal. Chem. 2018, 90, 12861– 12869,  DOI: 10.1021/acs.analchem.8b03441
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Shenault, D. S. M.; Fabijanczuk, K. C.; Murtada, R.; Finn, S.; Gonzalez, L. E.; Gao, J.; McLuckey, S. A. Gas-Phase Ion/Ion Reactions to Enable Radical-Directed Dissociation of Fatty Acid Ions: Application to Localization of Methyl Branching. Anal. Chem. 2024, 96, 3389– 3401,  DOI: 10.1021/acs.analchem.3c04510
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Pham, H. T.; Ly, T.; Trevitt, A. J.; Mitchell, T. W.; Blanksby, S. J. Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry. Anal. Chem. 2012, 84, 7525– 7532,  DOI: 10.1021/ac301652a
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Narreddula, V. R.; Boase, N. R.; Ailuri, R.; Marshall, D. L.; Poad, B. L. J.; Kelso, M. J.; Trevitt, A. J.; Mitchell, T. W.; Blanksby, S. J. Introduction of a Fixed-Charge, Photolabile Derivative for Enhanced Structural Elucidation of Fatty Acids. Anal. Chem. 2019, 91, 9901– 9909,  DOI: 10.1021/acs.analchem.9b01566
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Nsiah, S. T.; Fabijanczuk, K. C.; McLuckey, S. A. Structural characterization of fatty acid anions via gas-phase charge inversion using Mg(tri-butyl-terpyridine)22+ reagent ions. Rapid Commun. Mass Spectrom. 2024, 38, e9741  DOI: 10.1002/rcm.9741
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Jian, R.; Zhao, X.; Lin, Q.; Xia, Y. Profiling of branched-chain fatty acids via nitroxide radical-directed dissociation integrated on an LC-MS/MS workflow. Analyst 2022, 147, 2115– 2123,  DOI: 10.1039/D2AN00266C
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Liebisch, G.; Fahy, E.; Aoki, J.; Dennis, E. A.; Durand, T.; Ejsing, C. S.; Fedorova, M.; Feussner, I.; Griffiths, W. J.; Kofeler, H.; Merrill, A. H., Jr.; Murphy, R. C.; O’Donnell, V. B.; Oskolkova, O.; Subramaniam, S.; Wakelam, M. J. O.; Spener, F. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures. J. Lipid Res. 2020, 61, 1539– 1555,  DOI: 10.1194/jlr.S120001025
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Quehenberger, O.; Armando, A. M.; Dennis, E. A. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography–mass spectrometry. BBA-Mol. Cell Biol. L. 2011, 1811, 648– 656,  DOI: 10.1016/j.bbalip.2011.07.006
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Matyash, V.; Liebisch, G.; Kurzchalia, T. V.; Shevchenko, A.; Schwudke, D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid Res. 2008, 49, 1137– 1146,  DOI: 10.1194/jlr.D700041-JLR200
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. Advantageous Applications of Azabenzotriazole (Triazo1opyridine)-based Coupling Reagents to Solid-phase Peptide Synthesis. J. Chem. Soc., Chem. Commun. 1994, 2, 201– 203,  DOI: 10.1039/c39940000201
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Gross, M. L. Charge-remote fragmentations: method, mechanism and applications. Int. J. Mass Spectrom. Ion Processes 1992, 118–119, 137– 165,  DOI: 10.1016/0168-1176(92)85060-D
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Hunter, E. P. L.; Lias, S. G. Evaluated Gas Phase Basicities and Proton Affinities of Molecules: An Update. J. Phys. Chem. Ref. Data 1998, 27, 413– 656,  DOI: 10.1063/1.556018
    Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Yu, F.; Zong, B.; Ji, L.; Sun, P.; Jia, D.; Wang, R. Free Fatty Acids and Free Fatty Acid Receptors: Role in Regulating Arterial Function. Int. J. Mol. Sci. 2024, 25, 7853,  DOI: 10.3390/ijms25147853
    Google Scholar
    There is no corresponding record for this reference.
  26. 26
    Yan, Y.; Wang, Z.; Greenwald, J.; Kothapalli, K. S. D.; Park, H. G.; Liu, R.; Mendralla, E.; Lawrence, P.; Wang, X.; Brenna, J. T. BCFA suppresses LPS induced IL-8 mRNA expression in human intestinal epithelial cells. Prostag. Leukotr. Ess. 2017, 116, 27– 31,  DOI: 10.1016/j.plefa.2016.12.001
    Google Scholar
    There is no corresponding record for this reference.
  27. 27
    Katare, P. B.; Tingstad, R. H.; Beajani, S. T.; Indseth, J. P.; Telle-Hansen, V. H.; Myhrstad, M. C. W.; Rustan, A. C.; Eide, L.; Witczak, O.; Aas, V. Divergent effects of monomethyl branched-chain fatty acids on energy metabolism and insulin signaling in human myotubes. J. Lipid Res. 2025, 66, 100764– 100775,  DOI: 10.1016/j.jlr.2025.100764
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Chen, D.; Zhao, S.; Ma, G.; Li, L.; Li, L. An Analytical Strategy for Reliable Metabolome Analysis of Clinical Leftover Sera Using Timed Aliquoting. Anal. Chem. 2025, 97, 25655– 25663,  DOI: 10.1021/acs.analchem.5c04801
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    Menzel, J. P.; Young, R. S. E.; Benfield, A. H.; Scott, J. S.; Wongsomboon, P.; Cudlman, L.; Cvačka, J.; Butler, L. M.; Henriques, S. T.; Poad, B. L. J.; Blanksby, S. J. Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome. Nat. Commun. 2023, 14, 3940,  DOI: 10.1038/s41467-023-39617-9
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Roy, R.; Roseblade, A.; Rawling, T. Expansion of the structure-activity relationship of branched chain fatty acids: Effect of unsaturation and branching group size on anticancer activity. Chem. Phys. Lipids 2020, 232, 104952– 104959,  DOI: 10.1016/j.chemphyslip.2020.104952
    Google Scholar
    There is no corresponding record for this reference.
  31. 31
    Gozdzik, P.; Magkos, F.; Sledzinski, T.; Mika, A. Monomethyl branched-chain fatty acids: Health effects and biological mechanisms. Prog. Lipid Res. 2023, 90, 101226– 101236,  DOI: 10.1016/j.plipres.2023.101226
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Nagarajan, S. R.; Butler, L. M.; Hoy, A. J. The diversity and breadth of cancer cell fatty acid metabolism. Cancer Metab. 2021, 9, 2– 29,  DOI: 10.1186/s40170-020-00237-2
    Google Scholar
    There is no corresponding record for this reference.
  33. 33
    Wongtangtintharn, S.; Oku, H.; Iwasaki, H.; Toda, T. Effect of Branched-Chain Fatty Acids on Fatty Acid Biosynthesis of Human Breast Cancer Cells. J. Nutr. Sci. Vitaminol. 2004, 50, 137– 143,  DOI: 10.3177/jnsv.50.137
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article has not yet been cited by other publications.

Go to
Get e-Alerts
Get e-Alerts

Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2026, 37, 3, 782–789
Click to copy citationCitation copied!
https://doi.org/10.1021/jasms.6c00001
Published February 18, 2026

Copyright © 2026 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved. This publication is licensed under these Terms of Use.

Request reuse permissions

Article Views

759

Altmetric

-

Citations

-
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. (a) BDE of the C–O bond in MeO-QN before and after protonation. (b) MS2 CID spectrum of [MeO-QN+H]+. (c) Derivatization of FA by MeO-QN and proposed fragmentation pathways of derivatized FA ([FA# + H]+) under CID.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Ion-trap CID spectra of MeO-QN derivatized FAs and observed fragmentation sites of protonated FA-QN-O•. (a) [n-17:0#+H]+, (b) [i-17:0#+H]+, (c) [a-17:0#+H]+, and (d) [FA 16:0;3Me,7Me,11Me,15Me#+H]+.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. (a) XIC from PIS of m/z 158 for equimolar mixtures of FA 15:0 and FA 17:0 isomers. (b) Calibration curves and limit of identification (LOI) for various fatty acids employing PIS of m/z 158 (IS: FA d4–18:0).

    High Resolution Image
    Download MS PowerPoint Slide

    Scheme 1

    Scheme 1. Workflow for the Analysis of Fatty Acids with Precision at the Branched-Chain Level Using QN-O• RDD
    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. (a) Profiling of FFAs in human plasma (IS: FA 18:0-D4, 50 μ M). (b) XIC of [FA 17:0#+H]+ (m/z 441) in human plasma. (c) Ion trap CID spectrum of [FA 17:0#+H]+ eluted at 21.0 min in panel b.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. (a) Volcano plot of TFA at the sum composition level in breast cancer cells and (b) comparisons of BCFA isomer compositions in breast cancer cells (MDA-MB-468) to normal breast epithelial cells (MCF-10A).

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 33 other publications.

    1. 1
      Rustan, A. C.; Drevon, C. A., Fatty Acids: Structures and Properties. In Encycl. Life Sci. , 2005.  DOI: 10.1038/npg.els.0003894 .
      There is no corresponding record for this reference.
    2. 2
      Currie, E.; Schulze, A.; Zechner, R.; Walther, T. C.; Farese, R. V. Cellular Fatty Acid Metabolism and Cancer. Cell Metab 2013, 18, 153– 161,  DOI: 10.1016/j.cmet.2013.05.017
      There is no corresponding record for this reference.
    3. 3
      Taormina, V. M.; Unger, A. L.; Schiksnis, M. R.; Torres-Gonzalez, M.; Kraft, J. Branched-Chain Fatty Acids─An Underexplored Class of Dairy-Derived Fatty Acids. Nutrients 2020, 12, 2875– 2890,  DOI: 10.3390/nu12092875
      There is no corresponding record for this reference.
    4. 4
      Martínez-Montoro, J. I.; Núñez-Sánchez, M. Á.; Martinez-Sanchez, M. A.; Balaguer-Román, A.; Fernández-Ruiz, V. E.; Ferrer-Gómez, M.; Sledzinski, T.; Frutos, M. D.; Fernández-García, J. C.; Mika, A.; Ramos-Molina, B. Hepatic and serum branched-chain fatty acid profile in patients with nonalcoholic fatty liver disease: A case–control study. Obesity 2023, 31, 1064– 1074,  DOI: 10.1002/oby.23711
      There is no corresponding record for this reference.
    5. 5
      Mika, A.; Stepnowski, P.; Kaska, L.; Proczko, M.; Wisniewski, P.; Sledzinski, M.; Sledzinski, T. A comprehensive study of serum odd- and branched-chain fatty acids in patients with excess weight. Obesity 2016, 24, 1669– 1676,  DOI: 10.1002/oby.21560
      There is no corresponding record for this reference.
    6. 6
      Wang, Z.; Yang, T.; Brenna, J. T.; Wang, D. H. Fatty acid isomerism: analysis and selected biological functions. Food Funct 2024, 15, 1071– 1088,  DOI: 10.1039/D3FO03716A
      There is no corresponding record for this reference.
    7. 7
      Ran-Ressler, R. R.; Lawrence, P.; Brenna, J. T. Structural characterization of saturated branched chain fatty acid methyl esters by collisional dissociation of molecular ions generated by electron ionization. J. Lipid Res. 2012, 53, 195– 203,  DOI: 10.1194/jlr.D020651
      There is no corresponding record for this reference.
    8. 8
      Wang, D. H.; Wang, Z.; Brenna, J. T. Gas Chromatography Chemical Ionization Mass Spectrometry and Tandem Mass Spectrometry for Identification and Straightforward Quantification of Branched Chain Fatty Acids in Foods. J. Agric. Food. Chem. 2020, 68, 4973– 4980,  DOI: 10.1021/acs.jafc.0c01075
      There is no corresponding record for this reference.
    9. 9
      Wang, D. H.; Wang, Z.; Chen, R.; Kothapalli, K. S. D.; Brenna, J. T. Very Long-Chain Branched-Chain Fatty Acids in Chia Seeds: Implications for Human Use. J. Agric. Food. Chem. 2020, 68, 13871– 13878,  DOI: 10.1021/acs.jafc.0c05612
      There is no corresponding record for this reference.
    10. 10
      Kerwin, J. L.; Wiens, A. M.; Ericsson, L. H. Identification of fatty acids by electrospray mass spectrometry and tandem mass spectrometry. J. Mass Spectrom. 1996, 31, 184– 192,  DOI: 10.1002/(SICI)1096-9888(199602)31:2<184::AID-JMS283>3.0.CO;2-2
      There is no corresponding record for this reference.
    11. 11
      Randolph, C. E.; Beveridge, C. H.; Iyer, S.; Blanksby, S. J.; McLuckey, S. A.; Chopra, G. Identification of Monomethyl Branched-Chain Lipids by a Combination of Liquid Chromatography Tandem Mass Spectrometry and Charge-Switching Chemistries. J. Am. Soc. Mass Spectrom. 2022, 33, 2156– 2164,  DOI: 10.1021/jasms.2c00225
      There is no corresponding record for this reference.
    12. 12
      Wang, M.; Han, R. H.; Han, X. Fatty Acidomics: Global Analysis of Lipid Species Containing a Carboxyl Group with a Charge-Remote Fragmentation-Assisted Approach. Anal. Chem. 2013, 85, 9312– 9320,  DOI: 10.1021/ac402078p
      There is no corresponding record for this reference.
    13. 13
      Randolph, C. E.; Foreman, D. J.; Betancourt, S. K.; Blanksby, S. J.; McLuckey, S. A. Gas-Phase Ion/Ion Reactions Involving Tris-Phenanthroline Alkaline Earth Metal Complexes as Charge Inversion Reagents for the Identification of Fatty Acids. Anal. Chem. 2018, 90, 12861– 12869,  DOI: 10.1021/acs.analchem.8b03441
      There is no corresponding record for this reference.
    14. 14
      Shenault, D. S. M.; Fabijanczuk, K. C.; Murtada, R.; Finn, S.; Gonzalez, L. E.; Gao, J.; McLuckey, S. A. Gas-Phase Ion/Ion Reactions to Enable Radical-Directed Dissociation of Fatty Acid Ions: Application to Localization of Methyl Branching. Anal. Chem. 2024, 96, 3389– 3401,  DOI: 10.1021/acs.analchem.3c04510
      There is no corresponding record for this reference.
    15. 15
      Pham, H. T.; Ly, T.; Trevitt, A. J.; Mitchell, T. W.; Blanksby, S. J. Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry. Anal. Chem. 2012, 84, 7525– 7532,  DOI: 10.1021/ac301652a
      There is no corresponding record for this reference.
    16. 16
      Narreddula, V. R.; Boase, N. R.; Ailuri, R.; Marshall, D. L.; Poad, B. L. J.; Kelso, M. J.; Trevitt, A. J.; Mitchell, T. W.; Blanksby, S. J. Introduction of a Fixed-Charge, Photolabile Derivative for Enhanced Structural Elucidation of Fatty Acids. Anal. Chem. 2019, 91, 9901– 9909,  DOI: 10.1021/acs.analchem.9b01566
      There is no corresponding record for this reference.
    17. 17
      Nsiah, S. T.; Fabijanczuk, K. C.; McLuckey, S. A. Structural characterization of fatty acid anions via gas-phase charge inversion using Mg(tri-butyl-terpyridine)22+ reagent ions. Rapid Commun. Mass Spectrom. 2024, 38, e9741  DOI: 10.1002/rcm.9741
      There is no corresponding record for this reference.
    18. 18
      Jian, R.; Zhao, X.; Lin, Q.; Xia, Y. Profiling of branched-chain fatty acids via nitroxide radical-directed dissociation integrated on an LC-MS/MS workflow. Analyst 2022, 147, 2115– 2123,  DOI: 10.1039/D2AN00266C
      There is no corresponding record for this reference.
    19. 19
      Liebisch, G.; Fahy, E.; Aoki, J.; Dennis, E. A.; Durand, T.; Ejsing, C. S.; Fedorova, M.; Feussner, I.; Griffiths, W. J.; Kofeler, H.; Merrill, A. H., Jr.; Murphy, R. C.; O’Donnell, V. B.; Oskolkova, O.; Subramaniam, S.; Wakelam, M. J. O.; Spener, F. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures. J. Lipid Res. 2020, 61, 1539– 1555,  DOI: 10.1194/jlr.S120001025
      There is no corresponding record for this reference.
    20. 20
      Quehenberger, O.; Armando, A. M.; Dennis, E. A. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography–mass spectrometry. BBA-Mol. Cell Biol. L. 2011, 1811, 648– 656,  DOI: 10.1016/j.bbalip.2011.07.006
      There is no corresponding record for this reference.
    21. 21
      Matyash, V.; Liebisch, G.; Kurzchalia, T. V.; Shevchenko, A.; Schwudke, D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid Res. 2008, 49, 1137– 1146,  DOI: 10.1194/jlr.D700041-JLR200
      There is no corresponding record for this reference.
    22. 22
      Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. Advantageous Applications of Azabenzotriazole (Triazo1opyridine)-based Coupling Reagents to Solid-phase Peptide Synthesis. J. Chem. Soc., Chem. Commun. 1994, 2, 201– 203,  DOI: 10.1039/c39940000201
      There is no corresponding record for this reference.
    23. 23
      Gross, M. L. Charge-remote fragmentations: method, mechanism and applications. Int. J. Mass Spectrom. Ion Processes 1992, 118–119, 137– 165,  DOI: 10.1016/0168-1176(92)85060-D
      There is no corresponding record for this reference.
    24. 24
      Hunter, E. P. L.; Lias, S. G. Evaluated Gas Phase Basicities and Proton Affinities of Molecules: An Update. J. Phys. Chem. Ref. Data 1998, 27, 413– 656,  DOI: 10.1063/1.556018
      There is no corresponding record for this reference.
    25. 25
      Yu, F.; Zong, B.; Ji, L.; Sun, P.; Jia, D.; Wang, R. Free Fatty Acids and Free Fatty Acid Receptors: Role in Regulating Arterial Function. Int. J. Mol. Sci. 2024, 25, 7853,  DOI: 10.3390/ijms25147853
      There is no corresponding record for this reference.
    26. 26
      Yan, Y.; Wang, Z.; Greenwald, J.; Kothapalli, K. S. D.; Park, H. G.; Liu, R.; Mendralla, E.; Lawrence, P.; Wang, X.; Brenna, J. T. BCFA suppresses LPS induced IL-8 mRNA expression in human intestinal epithelial cells. Prostag. Leukotr. Ess. 2017, 116, 27– 31,  DOI: 10.1016/j.plefa.2016.12.001
      There is no corresponding record for this reference.
    27. 27
      Katare, P. B.; Tingstad, R. H.; Beajani, S. T.; Indseth, J. P.; Telle-Hansen, V. H.; Myhrstad, M. C. W.; Rustan, A. C.; Eide, L.; Witczak, O.; Aas, V. Divergent effects of monomethyl branched-chain fatty acids on energy metabolism and insulin signaling in human myotubes. J. Lipid Res. 2025, 66, 100764– 100775,  DOI: 10.1016/j.jlr.2025.100764
      There is no corresponding record for this reference.
    28. 28
      Chen, D.; Zhao, S.; Ma, G.; Li, L.; Li, L. An Analytical Strategy for Reliable Metabolome Analysis of Clinical Leftover Sera Using Timed Aliquoting. Anal. Chem. 2025, 97, 25655– 25663,  DOI: 10.1021/acs.analchem.5c04801
      There is no corresponding record for this reference.
    29. 29
      Menzel, J. P.; Young, R. S. E.; Benfield, A. H.; Scott, J. S.; Wongsomboon, P.; Cudlman, L.; Cvačka, J.; Butler, L. M.; Henriques, S. T.; Poad, B. L. J.; Blanksby, S. J. Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome. Nat. Commun. 2023, 14, 3940,  DOI: 10.1038/s41467-023-39617-9
      There is no corresponding record for this reference.
    30. 30
      Roy, R.; Roseblade, A.; Rawling, T. Expansion of the structure-activity relationship of branched chain fatty acids: Effect of unsaturation and branching group size on anticancer activity. Chem. Phys. Lipids 2020, 232, 104952– 104959,  DOI: 10.1016/j.chemphyslip.2020.104952
      There is no corresponding record for this reference.
    31. 31
      Gozdzik, P.; Magkos, F.; Sledzinski, T.; Mika, A. Monomethyl branched-chain fatty acids: Health effects and biological mechanisms. Prog. Lipid Res. 2023, 90, 101226– 101236,  DOI: 10.1016/j.plipres.2023.101226
      There is no corresponding record for this reference.
    32. 32
      Nagarajan, S. R.; Butler, L. M.; Hoy, A. J. The diversity and breadth of cancer cell fatty acid metabolism. Cancer Metab. 2021, 9, 2– 29,  DOI: 10.1186/s40170-020-00237-2
      There is no corresponding record for this reference.
    33. 33
      Wongtangtintharn, S.; Oku, H.; Iwasaki, H.; Toda, T. Effect of Branched-Chain Fatty Acids on Fatty Acid Biosynthesis of Human Breast Cancer Cells. J. Nutr. Sci. Vitaminol. 2004, 50, 137– 143,  DOI: 10.3177/jnsv.50.137
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.6c00001.

    • Experimental details; figures of optimized MeO-QN structure through DFT calculations, mass spectra of MeO-QN derivatized FAs, and background contaminations, tables of QTRAP 4500 parameters and gas basicity of functional groups (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.