Annotation of fragment ions from stable isotope-labeled lipids.

<p>A) Positive FTMS² spectrum of protonated PC 34:2(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H13). The fragment ions identify the molecular lipid species as PC(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H13) 16:1–18:1. B) Positive FTMS² spectrum of protonated PC 32:0(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H6). The fragment ions identify the molecular lipid species as PC 16:0(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H3)-16:0(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H3). C) Negative FTMS² spectrum of deprotonated PI 34:1(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H6). The fragment ions identify the molecular lipid species PI(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref002" target="_blank">2</a>]H6) 16:0–18:1. D) Negative FTMS² spectrum of the formate adduct of Cer 44:0;4(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref013" target="_blank">13</a>]C2[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref015" target="_blank">15</a>]N). The annotated fragment ions identify the molecular species as Cer 18:0;3(+[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref013" target="_blank">13</a>]C2[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.ref015" target="_blank">15</a>]N)/26:0;1. Note that non-annotated fragment ions derive from co-isolated lipids. Fragmentation diagrams for the lipid molecules and indicated fragment ion <i>m/z</i> values are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.s008" target="_blank">S6 Fig</a></b>.</p

CID of lipid molecules produces several types of fragments that can be used for annotating intact lipid molecules at three different levels.

<p>The shorthand notation of the fragment ions is described in the sections: Results and discussion, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.s001" target="_blank">S1 Text</a>. LCFs, lipid class-selective fragments; MLFs, molecular lipid species-specific fragments; DBFs, double bond location-specific fragments.</p

Annotated fragment ion spectra of representative lipid molecules from five different lipid categories.

<p>Fragment ion <i>m/z</i> values are denoted according to the three-step procedure outlined in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.g002" target="_blank">Fig 2</a></b>. The shorthand notation includes nomenclature based on both charged and neutral fragments (separated by “|”) (step 2). Annotation shown in boldface is prioritized based on the guidelines outlined in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.g002" target="_blank">Fig 2</a></b> (step 3). Non-prioritized shorthand notation is occasionally omitted to avoid overly congested mass spectra. The representation of fragment ion <i>m/z</i> values by mass-balanced chemical reactions and fragment structures are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.s005" target="_blank">S3 Fig</a></b> (step 1). A) Negative FTMS² spectrum of deprotonated ACoA 19:0. B) Positive FTMS² spectrum of ammoniated TAG 18:0–18:1–18:2. C) Positive FTMS² spectrum of protonated PE O-18:1p/20:4. D) Negative FTMS² spectrum of deprotonated and doubly charged CL 14:1–14:1–14:-15:1. E) Negative FTMS³ spectrum of FA 18:1 carboxylate anion <i>m/z</i> 281.3 derived from PC 16:0–18:1(9). F) Positive FTMS² spectrum of protonated SM 18:1;2/17:0. G) Negative FTMS² spectrum of deprotonated Cer 18:1;2/17:0;1. H) Positive FTMS² spectrum of ammoniated SE 27:1/19:0 (cholesteryl ester 19:0).</p

Identification of PS 20:4–22:6 in mouse hippocampus.

<p>A) Negative FTMS spectrum of mouse hippocampus. The precursor ion matching deprotonated PS 42:10 is highlighted in boldface. B) Negative FTMS² spectrum of <i>m/z</i> 854.6 with detection of MLFs and LCFs matching PS 20:4–22:6, annotated in boldface.</p

Screenshot of the ALEX¹²³ lipid calculator showing MS² information for the molecular lipid species PS 20:4–22:6 (MS² spectrum is shown in Fig 6).

<p>The application is freely available at <a href="http://www.alex123.info/ALEX123/MS.php" target="_blank">www.alex123.info/ALEX123/MS.php</a>.</p

Outline of three-step procedure for implementing shorthand notation of lipid fragment <i>m/z</i> values.

<p>Step 1: Detected fragment ion <i>m/z</i> values are first recapitulated using mass-balanced chemical reactions showing putative structures of both charged and neutral fragments. Step 2: These fragments are then annotated using fragment type-specific annotation rules (described in detail in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.s001" target="_blank">S1 Text</a></b>). Step 3: Prioritizing the nomenclature to use for shorthand notation of detected fragment ion <i>m/z</i> values is based on fragment type, charge and mass difference between charged fragments and composites of neutral fragments (also described in detail in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.s001" target="_blank">S1 Text</a></b>). Note that the shorthand notation of fragment ion <i>m/z</i> values can be based on combinations of fragment types (i.e. DBFs, MLFs, LCFs and iMLFs).</p

LDA software supports automated annotation of lipid fragment ions.

<p>A) Negative ion mode extracted ion chromatogram of <i>m/z</i> 718.5379±0.013, corresponding to deprotonated PE 34:0 (i.e., synthetic standard PE 17:0–17:0). B) Negative ion mode FTMS² spectrum of <i>m/z</i> 718.5. Fragment ions are automatically annotated by LDA and collectively used to identify the molecular lipid species PE 17:0–17:0. C) Positive ion mode extracted ion chromatogram of <i>m/z</i> 675.5897±0.013, corresponding to sodiated DAG 38:0 (i.e., synthetic standard DAG 18:0–20:0). D) Positive ion mode FTMS² spectrum of <i>m/z</i> 675.6. Fragment ions are automatically annotated by LDA and collectively used to identify the molecular lipid species DAG 18:0–20:0.</p

Use mass-balanced chemical reactions to recapitulate lipid fragmentation.

<p>A) Negative FTMS² spectrum of <i>m/z</i> 836.5, corresponding to the acetate adduct of PC 18:3–18:3. The precursor ion is annotated at the lipid species level (i.e. PC 36:6) since that the composition of FA moieties cannot be inferred from the <i>m/z</i> value. Prioritized shorthand notation of fragment <i>m/z</i> values are in boldface and implemented according to the three-step procedure shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.g002" target="_blank">Fig 2</a></b>. Non-prioritized (redundant) shorthand notation is shown non-boldface and separated from the prioritized shorthand notation by “|”. B) Overview of fragmentation pathways for [PC 18:3–18:3+CH3COO]^- with putative structures of neutral and charged fragments. Note that each chemical reaction is mass-balanced (i.e. the total mass of all fragments equal the mass of the intact precursor molecule). Each structure is represented with charge, monoisotopic mass, shorthand notation and fragment type. Note that neutral (shown on the right) and charged (shown on the left) fragments are prefixed with and without a minus sign “-“, respectively. The annotations shown in boldface (prioritized) are based on annotation rules outlined in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188394#pone.0188394.g002" target="_blank">Fig 2</a></b> (step 3). LCF, lipid class-selective fragment; MLF, molecular lipid species-specific fragment, iMLF, intermediate MLF.</p