Quantification of Signaling Lipids by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI MS/MS)

Lipids, such as phosphoinositides (PIPs) and diacylglycerol (DAG), are important signaling intermediates involved in cellular processes such as T cell receptor (TCR)-mediated signal transduction. Here we report identification and quantification of PIP, PIP2 and DAG from crude lipid extracts. Capitalizing on the different extraction properties of PIPs and DAGs allowed us to efficiently recover both lipid classes from one sample. Rapid analysis of endogenous signaling molecules was performed by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS), employing lipid class-specific neutral loss and multiple precursor ion scanning for their identification and quantification. Profiling of DAG, PIP and PIP2 molecular species in primary human T cells before and after TCR stimulation resulted in a two-fold increase in DAG levels with a shift towards 1-stearoyl-2-arachidonoyl-DAG in stimulated cells. PIP2 levels were slightly reduced, while PIP levels remained unchanged.


Introduction
Diacylglycerols (DAGs) and phosphoinositides (PIPs) are low abundant lipid classes in cellular membranes whose abundance is temporally and spatially tightly regulated. For the bio-synthesis of PIPs, the precursor phosphatidylinositol (PI) needs to be transported from the ER to different organelles, where PIPs are metabolically generated and interconverted by the enzymatic action of several kinases and phosphatases [1][2][3]. Dependent on the position and extent of phosphorylation, seven distinct phosphoinositide classes are known: PI(3)P, PI(4)P, PI(5)P, PI(3,4)P 2 , PI(3,5)P 2 , PI(4,5)P 2 and PI(3,4,5)P 3 , with PI(4)P and PI(4,5)P 2 being the most abundant forms. Some phosphoinositide classes can be regarded as organelle markers since they are predominantly synthesized and located at specific membranes, such as PI(3)P at early endosomes, PI(4)P at the Golgi complex and PI(4,5)P 2 and PI (3,4,5)P 3 at the plasma membrane [4]. The phosphorylated headgroup of PIPs binds to effector proteins and thereby triggers signaling and activation processes. Several domains have been described to specifically interact with different types of PIPs. These domains include pleckstrin homology (PH), phagocyte oxidase (PX), epsin N-terminal homology (ENTH), C2 and FYVE zinc finger domains [4,5]. For example, at the trans-Golgi network PI(4)P binds to a variety of proteins, such as epsinR, AP-1, FAPP1, FAPP2, OSPB and to CERT, indicating important functional roles of PIPs in vesicular transport [6]. Plasma membrane-located PI(4,5)P 2 is involved in a plethora of processes, such as endo-and exocytosis, plasma membrane cytoskeleton interactions, and signal transduction [4].
Adaptive immunity requires the activation of T cells in order to accomplish the response towards a specific antigen. Stimulation of T cells is achieved by antigenic ligation of the T cell receptor (TCR) in the presence of costimulation [7,8]. Activated T cells produce e.g. cytokines, which are required for the modulation of other immune cells. The expression of many cytokines is triggered through the activation of transcription factors via intracellular signaling pathways induced by TCR ligation. These intracellular signaling cascades involve, among other factors, the activation of phospholipase C γ1 (PLCγ1), which cleaves PI(4,5)P 2 to generate the second messengers DAG and inositol-1,4, 5-triphosphate (IP 3 ) [9]. DAG activates protein kinase C θ (PKCθ), a critical player of the NF-κB pathway, and contributes to AP-1 activation via Ras/ERK [9]. IP 3 on the other hand triggers Ca 2+ -release from the ER leading to store-operated calcium entry and NFAT activation [10]. Ca 2+ signals further contribute to NF-κB activation. Activation of all three transcription factors (AP-1, NF-κB and NFAT) is needed for the expression of certain cytokines, such as the Th1-type-cytokines interleukin-2 (IL-2) and interferon -γ (IFN-γ) [8].
To study signaling processes in T cells, which are accompanied by subtle changes in DAG and PIP 2 , sensitive methods for the identification and quantification of these lipids are required. Conventional approaches for the analysis of PIPs are based on metabolic labeling with myo-[ 3 H]-inositol followed by TLC-or HPLC-analysis [11][12][13]. DAG is traditionally analyzed by the DAG kinase assay [14] or by GC-MS after chemical derivatization [15,16]. Nevertheless, these methods are time-and sample-consuming and, furthermore, show limitations in the resolution of lipid classes and lipid species.
The combination of ESI [17] and (tandem) mass spectrometry was an important progress in the field of structural and quantitative lipid analysis [18][19][20][21][22][23][24][25]. ESI together with lipid class-specific (multiple)-precursor and neutral loss scanning on tandem mass spectrometers enabled the identification and quantification of lipid classes and lipid species directly from crude lipid mixtures. Consequently, the concept of shotgun-lipidomics arose [26][27][28][29][30]. However, in the past, ESI required relatively high amounts of starting material since lipid extracts were infused at flow rates in the µL/min-range. The replacement of the ESI source by a nano-ESI source was an imperative step forward in terms of sample consumption, thus allowing the sensitive analysis of lipid extracts at flow rates in the nL/min-range [27,29,[31][32][33][34][35][36].
Within the last years a variety of methods have been reported allowing the analysis of PIPs by mass spectrometry [37]. ESI-MS/MS has been applied for the identification [38] and structural elucidation of PIPs [39]. Quantification of PIPs was demonstrated by ESI-MS/MS after LC separation [40][41][42] or by direct infusion of lipid extracts [43]. However, there is currently no method available that facilitates the quantification of PIPs by nano-ESI-MS/MS. Furthermore, ESI-MS/MS has been demonstrated for DAG quantification after chromatographic separation [44] or by direct infusion after derivatization [45]. Quantification of positively charged DAG ammonium adducts by nano-ESI-MS/MS was recently demonstrated by neutral loss scanning [34] and, additionally, multiple precursor ion scanning (MPIS) was reported to be applicable for DAG quantification in positive ion mode [29].
Although a variety of methods for the mass spectrometric analysis of PIPs and DAG is currently available, all approaches consider both lipid classes separately with respect to their analysis. In this work, a method for the simultaneous identification and quantification of the signaling intermediates DAG, PIP and PIP 2 is presented. The approach takes advantage of the different extraction properties of these structurally diverse lipid classes. By performing a two-step extraction, both lipid classes can be isolated from one sample at the same time. Nano-ESI MS/MS in combination with internal standards and lipid class-specific scanning was used for the identification and quantification of endogenous signaling lipids. As a proof of principle the method was applied to the profiling of DAG, PIP and PIP 2 molecular species in primary human T cells before and after TCR stimulation.

Extraction of PIPs
Due to their polar headgroups, PIPs are not sufficiently recovered from biological membranes by conventional extraction procedures such as Folch [46] or Bligh and Dyer [47]. Therefore, modified extraction protocols were introduced decades ago [48] and continuously optimized and applied for the analysis of PIPs [49][50][51][52]. A variety of these methods employ acidification of extraction solvents to protonate phosphate groups and thereby facilitate the disruption of ionic interactions with proteins. Furthermore, protonation supports partitioning into the organic phase, which increases the recovery of low abundant PIPs. In contrast to PIPs, DAGs, as neutral lipids, are efficiently extracted by conventional methods.
To make the chemically diverse lipids DAGs and PIPs accessible for simultaneous mass spectrometric analysis, a previously described two-step extraction procedure for the selective enrichment of PIPs was performed [52]. With the first extraction apolar lipids such as DAGs are extracted with a mixture of chloroform/methanol (neutral extraction). Subsequently, negatively charged PIPs, which to a substantial part reside in the membrane, are recovered by an acidic extraction using a mixture of chloroform/methanol/HCl. In order to determine the extraction behavior of endogenous PIPs, HeLa cells were labeled with myo-[ 3 H]-inositol and subjected to a two-step lipid extraction. 3 H-inositol-labeled lipids in neutral and acidic extracts were separated by TLC and visualized by autoradiography (Figure 1a). While most of 3 H-PI was recovered with the neutral extraction, the phosphoinositides 3 H-PIP and 3 H-PIP 2 were predominantly present in the acidic extract. Quantification of the spot intensities revealed that PI was recovered to ~84% in the neutral extract, and to ~16% in the acidic extract ( Figure 1b). In contrast, PIP and PIP 2 were enriched with ~58% and ~79% in the acidic extract. It is of note that we observed a different extraction behavior of endogenous PIPs compared to exogenously added PIP and PIP 2 standards. Whereas endogenous PIP and PIP 2 were protected to more than 50% from the neutral extraction, exogenously added PIP and PIP 2 standards were recovered already to ~50% in the neutral extraction (data not shown). The different extraction behavior of endogenous and exogenous PIPs is an important issue when considering at what step during the extraction process PIP and PIP 2 standards should be spiked to the sample to allow for correct mass spectrometric quantification. Taken together, the labeling experiments demonstrated that the two-step extraction provides an easy and fast approach to enrich PIPs from cellular membranes. Importantly, the initial neutral extraction recovers DAG and depletes the membrane pellet from abundant lipids such as phosphatidylcholine (PC), phosphatidylethanolamine and PI, which otherwise could cause ion suppression and thus reduced ionization efficiency of PIPs during mass spectrometric analysis.

Mass Spectrometric Characterization of DAGs and PIPs
It was previously shown that DAGs form positively charged ammonium adducts that display characteristic fragment ions upon dissociation when analyzed by nano-ESI MS/MS [34]. To analyze the fragmentation behavior of DAG with the instrumentation used in this study, a commercially available DAG species with fatty acid composition 17:0/17:0 (DAG 34:0) was analyzed in the presence of ammonium acetate. Collision-induced dissociation (product ion analysis) of the ammoniated precursor (m/z 614.6) resulted in a fragment pattern typically observed for DAGs [34] (    (Figure 3a,c,e). However, we observed that the detection sensitivity of polyunsaturated DAG 38:4 was drastically reduced when compared to DAG species with a higher degree of saturation ( Figure 3a). Thus, although NL 35 scanning allows rapid identification of DAG species, accurate quantification of endogenous DAG requires an alternative approach. DAG mixtures were therefore analyzed by multiple precursor ion scanning (MPIS) [33] for DAG-specific MAG-H 2 O ions. As displayed in Figure 3b MPIS allowed the correct quantification of all endogenous DAG species, which can be quantified over a dynamic range of at least two orders of magnitude. Importantly, in contrast to quantification of DAG species by NL 35 scanning, MPIS was not significantly affected by fatty acid chain length and degree of unsaturation, i.e., number of double bonds (Supplementary Figure 1), allowing to use only one standard for quantitation, as shown before by [33,53]. Likewise, neutral loss scanning allowed accurate quantification of PIP and PIP 2 over a broad dynamic range (Figure 3d,f). The limit of quantification (LOQ) was determined to 10-15 pmol, both for DAG and PIP, and to 70 pmol for PIP 2 . This result is in line with previous observations [43] and is likely due to a lower ionization efficiency of PIP 2 .
Taken together, the mass spectrometric investigation of DAG, PIP and PIP 2 species showed that both lipid classes can be detected as ammonium adducts, facilitating their mass spectrometric analysis in positive ion mode. Collision-induced dissociation confirmed the formation of DAG-specific product ions as described [34] and revealed lipid class-specific fragments of PIP and PIP 2 that allow their rapid identification.

Identification and Quantification of DAG, PIP and PIP 2 in Human T Cells
The above described two-step extraction procedure together with lipid class-specific multiple precursor ion and neutral loss scanning was performed for the simultaneous identification and quantification of molecular species of DAG, PIP and PIP 2 in primary human CD4 + CD25 − T cells.  Table 1). The amount of total DAG (sum of all species) was quantified to 8 ± 2.5 fmol/pmol PC (n = 3). Assuming that PC accounts for ~45% of phospholipids in lymphocytes [54], DAG comprises ~0.36 mol% of phospholipids in T cells.
For the identification of PIP and PIP 2 species, neutral loss scanning was performed in acidic extracts of T cells. The neutral loss spectra NL 357 Da and NL 437 Da display endogenous PIP and PIP 2 species and the internal standards used (Figure 4b,c). Scanning for neutral losses of 357 Da enabled the identification of 11 PIP species, with PIP 38:4 being the most abundant ion (accounting for ~82 mol% of total PIP) ( Table 1). Scanning for neutral losses of 437 revealed 10 PIP 2 species, with PIP 2 38:4 as most abundant ion accounting for ~70 mol% of total PIP 2 . For absolute quantification of PIP and PIP 2 , the raw values were corrected for losses during the neutral extraction (factor of 1.72 for PIP and 1.27 for PIP 2 ). The absolute amount of PIP and PIP 2 in T cells was quantified to 4.8 ± 1.7 and 5 ± 1.8 fmol/pmol PC (n = 3), respectively. With PC accounting for ~45% of phospholipids [54], the amount of PIP and PIP 2 comprises each around 0.22 mol% of total phospholipid.

Effect of TCR Stimulation on DAG, PIP and PIP 2 in Human T Cells
To test whether the described approach is suitable to monitor transient changes in the overall levels of PIP, PIP 2 and DAG in cells, lipid extracts from T cells were analyzed before and after TCR stimulation. To this end, primary human T cells were stimulated for 5 min using agonistic anti-CD3 antibodies. Co-stimulation was achieved with anti-CD28 antibodies, and cross-linking antibodies were added to enhance stimulation. Phosphorylation of PLCγ1 and PKCθ confirmed the activation of intracellular signaling pathways upon TCR ligation (Figure 5a). Unstimulated and stimulated T cells were subjected to a two-step lipid extraction. DAG quantification was performed in neutral extracts, PIP and PIP 2 levels were quantified in acidic extracts. As depicted in Figure 5b, TCR stimulation significantly increased the level of DAG 38:4. Detection of an elevated level of DAG 38:4 is in accordance with the fact that PIP 2 38:4 is the main source for PLCγ1-generated DAG in activated T cells. In contrast to DAG, TCR stimulation did not affect the species profiles of PIP and PIP 2 (Figure 5b). Quantification of the major species PIP 2 38:4, PIP 38:4, and DAG 38:4 confirmed elevated levels of DAG 38:4, which was accompanied by a slight but significant reduction of PIP 2 38:4. The amount of PIP 38:4 did not significantly change upon T cell activation (Figure 5c). Interestingly, the increase in DAG 38:4 was not accompanied by an equivalent decrease of PIP 2 38:4, which one might expect due to PLCγ1-induced conversion of PIP 2 to DAG. This effect may indicate that upon TCR stimulation PIP 2 38:4 is rapidly re-synthesized by phosphorylation of its precursor PIP 38:4 and/or by dephosporylation of PIP 3 38:4.

Discussion
The purpose of this study was to establish a workflow for the simultaneous and rapid identification and quantification of the signaling lipids DAG, PIP and PIP 2 by nano-ESI MS/MS. These lipid classes are intimately involved in early signaling processes, as PIP 2 is rapidly converted to DAG by action of PLCγ during T cell activation [9], and PIP 2 is re-synthesized by phosphorylation of PIP [55]. Therefore, monitoring subtle changes in the abundance of these lipids is of great importance in order to understand the mechanisms of lipid-mediated signaling and potential alterations in diseases. A variety of methods for the mass spectrometric analysis of PIPs [37] and DAG [34,44,45] are currently available. All methods, however, consider both lipid classes separately with respect to their analysis. Capitalizing on different extraction properties of structurally diverse lipids [27], we were able to selectively enrich DAG and PIPs by a two-step extraction [52] allowing their simultaneous mass spectrometric profiling from one sample.
DAG can be quantitatively recovered by neutral extraction procedures, whereas PIPs are protected substantially from the neutral extraction. Different observations were made regarding the extent of PIP and PIP 2 recovery during neutral extraction. Whereas Milne et al did not detect any PIP 2 in neutral extracts of RAW 264.7 cells [38], Gray et al observed a partial extraction of PIP (26%) and PIP 2 (6%) in astrocytoma cells [52]. We tested the extraction efficiency of PIPs in HeLa cells and found even higher losses of PIP (42%) and PIP 2 (21%) during the neutral extraction. These discrepancies in the extraction behavior are most likely due to the different cell types used.
The mass spectrometric analysis of commercially available DAG, PIP and PIP 2 species showed that these lipid classes can be detected as ammonium adducts, facilitating their mass spectrometric analysis in positive ion mode. Most of the available methods for the measurement of PIPs rely on the analysis in negative ion mode [38][39][40][41]43], which should be beneficial because of the negatively charged phosphate groups. However, we found that negatively charged PIPs showed a variable ionization behavior on ESI-MS/MS systems equipped with a nanospray source (data not shown). Consequently, it was not possible to create conditions that resulted in a constant stream of either singly or doubly charged PIPs. Negatively charged PIPs were rather fluctuating between singly and doubly charged states and thus did not allow for robust analysis. In contrast, positively charged ammonium adducts resulted in a stable ionization behavior of PIP and PIP 2 . PIP 3 is an extremely low abundant lipid accounting for <0.002 mol% of phospholipids in mammalian cells [56], and we could not detect this lipid with the described approach. For the detection of PIP 3 , LC-MS based analyses using higher flow rates than nano-flow with [42] or without [40,41] headgroup modification were reported as alternative approaches with sufficient sensitivity.
The mass spectrometric analysis of DAG confirmed the formation of positively charged ammonium adducts that, upon collision-induced dissociation, display lipid class-specific fragments [34]. The neutral loss of fatty acid fragments of DAG species results in characteristic MAG-H 2 O ions that were chosen to perform DAG-specific MPIS as reported [29]. Although DAG species can be detected by scanning for neutral losses of 35 Da, quantification should be done by MPIS or neutral loss scanning of fatty acid-derived ions [34], as only these scanning procedures allow accurate quantification of DAG independent of the saturation state. In particular DAG 38:4 showed a drastically reduced NL 35 detection sensitivity. Most likely, this effect can be attributed to the polyunsaturated fatty acid 20:4, which might influence the fragmentation behavior of polyunsaturated DAG species. Further experiments are required to reveal additional information about the mass spectrometric dissociation behavior of DAG 38:4. A combination of MPIS and neutral loss scanning of fatty acid-derived ions also allows to analyze ether-and odd-numbered fatty acyl-DAGs. However, additional internal standards may be required for the absolute quantification of ether-DAGs since the mass spectrometric fragmentation behavior of ether-DAGs might differ from acyl-DAGs, such as previously observed for acyl-PE and alkenyl-PE [57].
Profiling of DAG and PIPs in T cells before and after TCR stimulation demonstrated that subtle changes in the abundances of signaling lipids can be monitored with the method described here. A 5 min stimulation of the TCR resulted in significantly increased levels of DAG 38:4, consistent with the activation of PLCγ1 and PKCθ. Furthermore, the level of PIP 2 38:4 was slightly but significantly reduced upon TCR stimulation. The increase in DAG 38:4 did not go along with a corresponding reduction of PIP 2 38:4 to the same extent. Two possibilities can be envisaged to explain this effect. First, re-synthesis of PIP 2 : This scenario is supported by slightly elevated levels of PIP 38:4, the precursor of PIP 2 biosynthesis. Indeed, the synthesis rate of PIPs is known to be increased upon TCR stimulation [55]. Second, DAG might be generated from other lipid precursors than PI(4,5)P 2 . Although it is widely accepted that TCR stimulation triggers PLCγ1 that cleaves plasma membranelocated PI(4,5)P 2 , there is evidence that activation of Ras/MAPK pathways by DAG can also occur at the Golgi complex [58]. Thus, other lipid precursors might contribute to the production of DAG, such as PC that can be converted to DAG by the action of phospholipase D and PA phosphatase, enzymes that have previously been linked to TCR signaling [59]. To identify the source of DAG in activated T cells, the analysis of PIP 2 -derived inositol-phosphate intermediates is required in future studies. In addition, it will be important to monitor other metabolites that are affected during T cell activation, such as PA and PIP 3 [9,60].

Phosphoinositide extraction from myo-[ 3 H]-inositol labeled HeLa cells:
Phosphoinositide extraction was performed as described [52]. Briefly, after transferring the cells (~ 5 × 10 6 ) to ice and removal of the medium, cells were washed two times with 10 mL cold PBS. After adding 1.2 ml cold 0.5 M TCA, the cells were immediately scraped off the dish, transferred to an Eppendorf tube and vortexed for 10 sec followed by 5 min incubation on ice. After centrifugation (13,800 × g, 2 min at 4 °C), the supernatant was discarded and the pellet washed two times with 1 mL cold 5% TCA/1 mM EDTA. For neutral extraction, the pellet was resuspended in 1 mL chloroform/methanol (1:2, v/v) and incubated for 10 min at RT. The sample was vortexed for 30 s every 3 min. After precipitation (13,800 × g, 2 min at 4 °C), the supernatant was transferred to a new Eppendorf tube. For acidic extraction, the remaining pellet was resuspended in 750 µL chloroform/methanol/37% HCl (40:80:1, v/v/v) and incubated for 15 min at RT while vortexing the sample for 30 s every 5 min. After transferring the tube to ice, 250 µL cold chloroform and 450 µL cold 0.1 M HCl was added followed by 1 min vortexing and centrifugation (6,500 × g, 2 min at 4 °C). The bottom organic phase was transferred to a new tube. Before drying, ~1 vol% of the neutral and the acidic extract was subjected to scintillation counting to determine the radioactivity. Based on the sum of radioactivities in the neutral extract (100 vol%) and the acidic extract (100 vol%), a typical myo-[ 3 H]-inositol incorporation of 0.5-1.5% was calculated compared to the starting labeling amount (150 µCi). The organic phases were dried and stored at −20 °C until further processing.
Thin-layer chromatography (TLC) and autoradiography: For TLC analysis, Silica 60 plates (Merck, Darmstadt) were covered with 1% (w/v) potassium oxalate and 2 mM EDTA in 50% methanol followed by drying for 15 min with a blow-dryer. Dried lipid extracts were dissolved in appropriate volumes (20-
Lipid extraction from primary human T cells: Two-step lipid extraction was performed similar as described [52]. Briefly, T cell lysates were either immediately processed or snap frozen and stored at −80 °C until further processing. The storage time was restricted to a maximum of 2-3 weeks to minimize sample degradation. Prior to lipid extraction, TCA lysates were precipitated (13,800 × g, 2 min at 4 °C) and pellets washed two times with 1 mL cold 5% TCA/ 1 mM EDTA. After centrifugation (13,800 × g, 2 min at 4 °C), the supernatant was discarded and lipids were extracted in Eppendorf tubes. Prior to neutral extraction, 15-50 pmol DAG 17:0/17:0 and 250 pmol of an equimolar mixture of PC standards was spiked to the pellet and lipids were extracted with 750 µL chloroform/methanol (1:2, v/v) by incubating 10 min at RT and vortexing every 3 min for 30 s. After centrifugation (13,800 × g, 2 min at 4 °C), the supernatant was carefully removed from the pellet and transferred to a new tube. The supernatant was subjected to a second extraction after the addition of 250 µL chloroform and 450 µL water. Phase separation was induced by centrifugation (6,500 × g, 2 min at 4 °C) and the lower phase was transferred to a new tube. Dried lipids were dissolved in 10 mM ammonium acetate in methanol and subjected to mass spectrometry.
Prior to acidic extraction of PIPs, 15-25 pmol PI(4)P 37:4 and 160-300 pmol PI(4,5)P 2 37:4 was spiked to the remaining pellet (which was kept on ice all the time). The pellet was resuspended in 375 µL chloroform/methanol/37% HCl (40:80:1, v/v/v), incubated at RT for 15 min and vortexed every 5 min for 30 s. After transferring the tube to ice, 125 µL cold chloroform and 225 µL cold 0.1 M HCl were added followed by 1 min vortexing. Phase separation was induced by centrifugation (6,500 × g, 2 min at 4 °C). The lower organic phase was immediately transferred to a new tube and kept on ice until further processing. For mass spectrometric analysis, an aliquot (10 µL) of the organic phase was diluted 1:2 with 50 mM ammonium acetate in chloroform/methanol (1:1, v/v).
Mass spectrometry: Before mass spectrometric analysis, lipid extracts were subjected to centrifugation (13,800 × g, 5 min at 4 °C) to remove debris. 20 µL of lipid samples were loaded into 96-well plates and sealed. Lipid infusion and ionization was achieved with the Triversa Nanomate® (Advion Biosciences) operated with the ChipSoft TM Software (Advion Biosciences) under the following settings: sample infusion volume: 10 µL, volume of air to aspirate after sample: 1 µL, air gap before chip: enabled, aspiration delay: 0 s, pre-piercing: with mandrel, spray sensing: enabled, cooling temperature: 12 °C, gas pressure: 0.5 psi. The ionization voltage was set to 1.3-1.6 kV. For infusion in 10 mM ammonium acetate in methanol (DAG analysis) vent headspace was enabled and pre-wetting was done once. For infusion in 25 mM ammonium acetate in chloroform/methanol/H 2 O (PIP and PIP 2 analysis) vent headspace was disabled and pre-wetting was performed two times.
Mass spectrometric analysis was performed with the QStar® Elite (Applied Biosystems) operated by Analyst® 2.0 and the AB Sciex QTrap® 5500 operated by Analyst® 1.5.1. Following, instrumentdependent settings were used (Table 2). Product ion analysis and MPIS [29,33] was performed with the QStar® Elite. MPIS of DAG-specific MAG-H 2 O fragments (Table 3) was carried out at a CE of 25 eV. 44 MCA spectra for every precursor ion scan were acquired. Q1-scanning was done in the profile mode with a step size of 0.2 Da. The TOF mass range was set to m/z 250-400. Trapping of ions in Q2 (peak enhancement) was applied to m/z 327.3. Scanning for neutral losses of 35 Da, 357 Da and 437 Da was achieved with the AB Sciex QTrap® 5500 at collision energies of 15 eV, 25 eV and 35 eV, respectively. Scanning was performed in the profile mode (step size 0.1 Da) at a scan rate of 200 Da/s. For every scan mode 100-500 MCA spectra were accumulated. Mass spectra were evaluated with the LipidView TM Software Version 1.1 (AB Sciex). DAG, PIP and PIP 2 amounts were normalized to PC, which was quantified in neutral extracts as described [62].

Conclusions
Here we present a mass spectrometric method to rapidly quantify signaling lipids down to the molecular species level. The method capitalizes on different extraction properties of DAGs and phosphoinositides allowing to efficiently recover by a quick two step-extraction procedure both lipid classes. Quantitative profiling is performed in the presence of internal standards by applying lipid class specific neutral loss (phosphoinositides) and multiple precursor ion scanning (DAGs). As a proof of principle, we monitor transient changes in the level of signaling lipids in human T cells before and after TCR stimulation. Quantitative profiling of signaling metabolites by nano-ESI MS/MS thus provides a valuable tool for future lipidomics studies in order to unravel mechanisms of lipid-based signaling events in health and disease.