Methods for analyzing phosphoinositides using mass spectrometry

Biochimica et Biophysica Acta 1811 (2011) 758–762

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Review

Methods for analyzing phosphoinositides using mass spectrometry☆ Michael J.O. Wakelam a,⁎, Jonathan Clark b a b

The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom Babraham Biosciences Technologies Ltd, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom

a r t i c l e

i n f o

Article history: Received 14 June 2011 Received in revised form 7 September 2011 Accepted 9 September 2011 Available online 23 September 2011 Keywords: Mass spectrometry Phosphoinositide HPLC Acyl chain PtdIns3,4,5P3

a b s t r a c t The polyphosphoinositides are key signaling lipids whose levels are tightly regulated within cells. As with other cellular lipids multiple species exist with distinct acyl chain makeups. There are methods which analyze the phosphoinositides as their deacylated derivatives which cannot address these distinct forms. Lipidomic analysis of the polyphosphoinositides has been hampered by difficulties with extraction and problems associated with binding of the lipids to surfaces. This review outlines the available MS methodologies, highlighting the difficulties associated with each. However, at present, no single methodology is available that can successfully and reproducibly quantitate each inositol phospholipid. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Advances in separation technology and particularly in mass spectrometry have revolutionized the analysis and quantification of cellular and tissue lipids. As outlined elsewhere in this issue, methodological advances have made such analyses routine, however one area that has not progressed to the same degree has been the phosphoinositides. This article summarizes the issues that have made progress in phosphoinositide analysis a challenge and highlights the recent steps forward which have made the determination of some of the members of the phosphoinositide family of lipids achievable. In addition to phosphatidylinositol (PtdIns), there are seven identified phosphoinositides in mammalian cells: phosphatidylinositol-3phosphate (PtdIns3P), phosphatidylinositol-4-phosphate (PtdIns4P), phosphatidylinositol-5-phosphate (PtdIns5P), phosphatidylinositol3,4-bisphosphate (PtdIns3,4P2), phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2), phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns3,4,5P3). Fig. 1 shows the structure of PtdIns3,4,5P3 and the inositol ring numbering system for phosphorylation. Critically, with the exception of PtdIns, each of the inositol containing phospholipids has a distinct messenger function; essentially this is determined by their ability to selectively bind to specific modules or domains within a range of target proteins [1]. This binding is reasoned in some cases to induce catalytic activation in certain enzymes, PtdIns4,5P2 binding to and activating ☆ This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry. ⁎ Corresponding author. Tel.: + 44 1223 496202; fax: + 44 1223 496212. E-mail address: [email protected] (M.J.O. Wakelam). 1388-1981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2011.09.004

phospholipase D being a good example, but in most cases the lipid binding-mediated signaling appears to be brought about through localization of the signaling protein to an appropriate site in a membrane, e.g. PDK-1. Probably the best characterized of the binding modules are the PH (plexstrin homology) domains. More than 100 PH domain-containing proteins have been identified in mammalian cells, see Ref. [1] for additional detail. While many PH domains can bind PtdIns3,4,5P3 others can bind PtdIns3,4P2 or PtdIns4,5P2 and indeed not all are specific for one lipid. PtdIns3,5P2 does not interact with PH domains rather it has been demonstrated to bind PROPPIN domains [2]. PtdIns3,4P2 and PtdIns3P can both bind to PX domains [3] while PtdIns3P also binds to FYVE domains [4]. PtdIns4P regulates the activity of a number of cellular processes, though there is less clarity regarding the existence of a specific binding domain, while PtdIns5P has been suggested to bind to PHD domains (PlantHomeoDomain) in nuclear proteins [5]. In addition, as will be discussed below, there is an input from the lipid's acyl chain structure into signaling specificity [6]. Despite the clear importance of the phosphoinositides in signaling biology and also their relevance in, for example, the putative lipid composition of membrane lipid rafts [7], there has been limited progress in determining the cellular amounts and structures of these lipids. Many of the methods utilized to date have continued to make use of the procedures established thirty years ago involving radiolabelling of cells with [ 32P]Pi or [ 3H]inositol, followed by chemical deacylation of the cellular lipids and separation of the generated head-groups or glycerophosphates by ion exchange HPLC. There are a number of issues with these procedures, not least that it requires the ability to radiolabel the lipids in the sample to isotopic equilibrium and it fails to permit definition of the acyl chain structure.

M.J.O. Wakelam, J. Clark / Biochimica et Biophysica Acta 1811 (2011) 758–762

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OH HO

OH 3 4

HO

2 5

1 6 OH

OH O OH (HO)2OP

O O

P OH

(HO)2OP

OH

O O

O O

PO(OH)2 Fig. 1. Polyphosphoinositide structure. The structure of a polyphosphoinositide is dictated by the inositol ring numbering system (upper figure). This permits definition of polyphosphoinositide structures such as that shown — 18:0 20:4 PtdIns3,4,5P3 whose full name is 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′, 5′-trisphosphate).

While also failing to address the lipid backbone, more quantitative measures have been made by adopting an enzyme assay-linked approach, e.g. Divecha's method for determining PtdIns5P concentration [8]. In an attempt to address these limitations a number of methods have been introduced to measure and characterize the phosphoinositides using mass spectrometric (MS) methods. There are a number issues that complicate phosphoinositide analysis by MS including the very low cellular concentrations of the different lipids relative to other phospholipids, the likelihood that most of the phosphoinositides are protein bound in cells, the highly charged head group which affects the phase behavior distinct from other amphipathic lipids and their consequent solubility. The high charge associated with the phosphoinositides also promotes their binding to surfaces frequently used in lipid extraction and separation such as borosilicate glass and stainless steel. The distinct phosphorylations of the inositol headgroup also make the chromatographic separation of the positional isomers a challenge, indeed there does not exist at present a published method capable of separating the intact lipids, as distinct from the glycerophosphoinositides or inositol phosphates, prior to MS analysis. 2. Methods to analyze the phosphoinositides This article will not in detail consider the many methodologies that have been reported able to determine phosphoinositides following deacylation. These do not provide the necessary complete structural analysis and the complete chromatographic separation of each of the deacylated lipids even using ion-pair chromatography prior to MS remains challenging, not least because most ion-pairing reagents are not MS-compatible [9]. The first approach to the use of ESI-MS to examine phosphoinositides in cell extracts was provided by Wenk [10]. While this work was able to identify and semi-quantitate a number of PtdIns, PtdInsP and PtdInsP2 molecular species from mouse brain and S. cerevisiae using negative ion ESI-MS, it was not able to determine PtdInsP3 nor the positional isomers of PtdInsP and PtdInsP2. Examination of the reported phosphoinositide species detected also suggests that not all were extracted, or identified when the data is compared to subsequent reports. Importantly, Wenk and colleagues recognized that the addition of organic buffers, in this case triethylammonium acetate or piperidine, was necessary to enhance signal intensity allowing detection of the phosphoinositide molecular ions. This was a key step that has also been adopted by subsequently reported methods. The utility of the methodology was demonstrated by its use to demonstrate

elevated PtdInsP2 levels in fibroblasts from Low syndrome patients and phosphoinositide changes in S. cerevisiae deficient in Sac1p, Vps34p, or Pik1p. In addition to the recognition of the need to enhance signal intensity, phosphoinositide analysis by MS is further compromised by ion suppression, potentiating the problems associated with very low levels and charge. Two approaches have been adopted to address this: selective extraction or chromatographic separation prior to MS. Milne et al. [11] adopted the former approach pre-extracting RAW 264.7 cell and murine peritoneal macrophage in neutral solvents before utilizing an acidic solvent step to extract the PtdInsP2 and PtdInsP3 species. Milne et al. reported that they did not extract any of the PtdInsP2 and PtdInsP3 species into the neutral solvent, however it is unclear, but unlikely, that none of the PtdIns or PtdInsP species was unextracted. Reduction in the complexity of the extract in this way allowed Milne et al. to detect a large number of intact phosphoinositides in infused samples, in the presence of piperidine, by ESItandem MS. Using this procedure Milne et al. observed increased amounts of PtdInsP, PtdInsP2 and PtdInsP3 species in both cell types stimulated with LPA, C5a or zymosan [11]. Collision-induced dissociation (CID) of the MS peaks was used to unambiguously identify the lipid structures. This showed that there was no acyl chain structure of PtdInsP2 or PtdInsP3 species that was not observed in PtdIns, though in two cases (32:0 and 32:1) this did not hold for PtdInsP. Despite this, the data demonstrates that PtdInsP2 and PtdInsP3 are probably generated in these cells by kinases and phosphatases rather than acyltransferases. Unexpectedly, however, Milne et al. detected the agonist-stimulated generation of 7 distinct PtdInsP3 species of which one was saturated, three monounsaturated, one diunsaturated and only one species had the expected 3- or 4-double bonds [11]. Ogiso and Taguchi [12] and Pettitt et al. [13] adopted a different approach, choosing to chromatographically concentrate the phosphoinositides prior to ESI-MS. Because of the extremely low concentrations of the lipids of interest it is necessary that any LC system adopted is connected online. Thus it is not possible to dry down samples and resolvate prior to MS, consequently the solvents adopted must be MS-compatible and for such protocols it may be necessary to make post-column additions to ensure ionization. Both these reports also considered problems associated with extraction and ion suppression brought about by contaminating lipids. Ogiso and Taguchi separated the phosphoinositides by reverse phase chromatography using a C8 column prior to MS analysis using an LTQ Orbitrap. However, Ogiso and Taguchi addressed the complication brought about by ion suppression by pre-fractionating the

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lipid extracts on a DEAE-Cellulose column. This methodology allows the successive elution of neutral, basic and acidic lipids and indeed was introduced nearly 50 years ago to separate brain phosphoinositides [14]. Once the acidic phospholipids were eluted from the ion exchange column, they were dried down prior to resolvation and loading onto a C8 reverse phase column which fractionated the inositol lipids prior to MS. This methodology, while being somewhat cumbersome allowed Ogiso and Taguchi to separate and identify a range of PtdInsP, PtdInsP2 species, but surprisingly only a single (36:2) species of PIP3 was detected. This limited detection questions either the sensitivity of the methodology, or more likely, the limited recovery of the higher phosphorylated inositol lipids through this rather involved procedure, particularly when the issues involved in the binding of phosphoinositides to multiple surfaces is considered. The detection limit of added standard PtdIns3,4,5P3 in the MS was about 1 pmol and the analysis suggested that EGF stimulated a twofold increase in PtdIns3,4,5P3 in A431 cells from a basal level of 0.3 pmol/6 × 10 6 cells. Pettitt et al. [13] adopted a different approach using normal phase HPLC upon a silica column prior to MS analysis. The methodology was adapted from the work of Gunnarsson et al. [15] who demonstrated separation of PtdIns4P from PtdInsP2 and made use of previous studies that had demonstrated that other neutral and phospholipids could be separated from the phosphoinositides on this column by use of an appropriate gradient [16]. The separation reported in this publication remains the only reported method that is able to separate the majority of intact phosphoinositides in a biological extract. Nevertheless, even this procedure was unable to separate chromatographically PtdIns3P from PtdIns4P and the separation of PtdIns5P from the other two PtdInsPs was incomplete. In contrast, each of the PtdInsP2 forms and PtdInsP3 were separated. Following this separation the phosphoinositides were detected by MS/MS. Additionally, MS 3 was adopted to distinguish between the chromatographically unseparable PtdIns3P and PtdIns4P. The fragmentation of PtdIns3P and PtdIns5P was identical even using MS 3, but the chromatographic separation achieved for these two allowed the complete analysis of all phosphoinositides using this methodology. Using this methodology Pettitt et al. analyzed the phosphoinositides in thrombin-stimulated human platelets and salt-stressed S. cerevisiae. While it was possible in both cell types to detect a number of PtdInsP and PtdInsP2 species and stimulated changes in concentrations, only 18:0/20:4 PtdIns3,4,5P3 was

detected in platelets again suggesting incomplete recovery or detection. Detection limits were approximately 1–10 pmol and the analysis showed that thrombin stimulated an increase in PtdIns3,4,5P3 from undetectable levels to approximately 70 pmol/10 9 platelets. While this methodology looks highly promising for analyzing phosphoinositides and its use has been reported by the Larijani group in their analysis of nuclear envelope assembly [17], there are issues particularly with recovery and reproducibility. Extraction of phosphoinositides is extremely difficult. Being highly charged these lipids are protein bound in cells and tissues and thus require acidification of extracts, to protonate the phosphate groups, in order to facilitate extraction and increase solubility in organic solvents. Unfortunately, some phosphoinositide hydrolysis occurs in acidified solvents and sustained acidification can also promote phosphate migration around the inositol ring. Once extracted the recovery of the phosphoinositides is a significant problem as they bind to glass and common HPLC surfaces such as stainless steel. Pettitt et al. attempted to alleviate these problems by silanization of all glassware and replacement of steel, where possible, with inert materials [13], nevertheless this cannot be complete and undoubtedly has contributed to the variable utility of this methodology. This is particularly an issue with those lipids present in the lowest amounts, i.e. the PtdIns3,4,5P3 which is the lipid of particular interest from a signaling perspective. A number of binding and enzymatic assays have been suggested as solutions to the difficulty of measuring PtdIns3,4,5P3 but a MS approach remains desirable, not least because it can address the acyl structural differences. Recently Clark et al. [18] have presented a novel approach to this problem. Recognizing the issues associated with the inositol ring phosphates binding inappropriately, or being subject to hydrolysis, Clark et al. have developed a derivatization method whereby the phosphate groups are methylated using trimethylsilyl diazomethane. The derivatized cell or tissue lipid extracts are then chromatographically separated using a C4 reverse phase column HPLC prior to MS infusion. A key aspect of this methodology is that detection and measurement of the derivatized phosphoinositides require multiple reaction monitoring and neutral loss of the methylated inositol phosphate headgroup. Fig. 2 outlines the steps of this methodology whereby each phosphate is methylated and the methylated inositol phosphate headgroup is released by neutral loss with the diacylglycerol unit being detected in the mass spectrometer.

O OH (MeO)2OP

O O

P

O O

O

OMe (MeO)2OP

O

OH PO(OMe)2

OH (MeO)2OP

O O

P

OH

OMe (MeO)2OP

OH PO(OMe)2 O O +

H 2C

O O

Fig. 2. Derivatization and fragmentation of PtdIns3,4,5P3. The figure shows the neutral loss of the derivatized phosphoinositol head of the PtdIns3,4,5P3 species shown in Fig. 1, to leave the charged diacylglycerol fragment within the mass spectrometer. The diacylglycerol unit gives rise to the signal at the mass spectrometer detector which facilitates its identification.

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and 18:0/20:2, as was found by Milne et al. [11] similar species were found in PtdInsP2 and PtdInsP. Intriguingly the pattern of stimulated PtdIns3,4,5P3 species that was generated in wild type and PTEN −/− cells was subtly different [18] implying different regulation of and functions for individual molecular species, perhaps through interaction with distinct PH domain containing proteins.

Fig. 3 shows neutral loss scans of derivatized PtdInsP2 (Fig. 3a) and PtdIns3,4,5P3 (Fig. 3b) species. The figures show the parental ions that lost the head group through neutral loss — 490 amu (PtdInsP2), 598 amu (PtdIns3,4,5P3) allowing identification of the acyl chain composition. Using this method Clark et al. have successfully determined PtdInsP, PtdInsP2 and PtdInsP3 levels in cell and tissue extracts. Because the individual PtdInsP and PtdInsP2 isoforms are not separated by the reverse phase chromatography step, this methodology cannot determine changes in the levels of these messengers. Nevertheless, by including relevant standards the method provides for semi-quantitative, sensitive and reproducible determination of PtdIns3,4,5P3 with the same for total PtdInsP2. Clark et al. [18] report use of the method with cultured cells, freshly isolated neutrophils and mouse and human tissues. In analyzing PtdIns3,4,5P3 in human adipose tissue samples it was apparent that the high levels of triglycerides affected the separation and detection of the derivatized lipids leading to poor recoveries and ion suppression. Thus Clark et al. adopted the extraction procedure to pre-deplete neutral lipids from the samples prior to acidic solvent extraction of the phosphoinositides with a detection limit of approximately 1 pmol. Unlike Pettitt et al. [13], but similar to Milne et al. [11], Clark et al. [18] observed multiple PtdIns3,4,5P3 species in cells and tissues. The species identified were 18:0/20:4, 18:0/18:2, 18:0/18:1, 18:0/20:3

3. Remaining challenges in phosphoinositide analysis Clark et al.'s methodology [18] is a significant step forward since it addresses a number of the key problems in phosphoinositide analysis — extraction, stability and recovery, while at the same time allowing for molecular species analysis. Nevertheless, it doesn't provide a complete solution because it does not allow the analysis and quantification of the individual PtdInsP and PtdInsP2 species. Pettitt et al. [13] devised a chromatographic separation for the PtdInsP2 forms, but this was unable to fully resolve the PtdInsP species. Unfortunately, this methodology is not directly applicable as an add-on to Clark et al.'s methodology because the methylation of the phosphate groups changes the chromatographic behavior of the phosphoinositides. Consequently, there remains a requirement to develop an appropriate chromatographic separation methodology, whether this will involve normal phase, reverse phase or chiral chromatography is unclear. As an alternative a

a 3.0e4

/counts per second

C18:0 C20:4 m/z 1117.9

C18:0 C20:3 m/z 1119.9 C18:0 C18:2 m/z 1093.8

1070

1090

1120

1100

1150

m/z, Da

b 5.6e4

/counts per second

C16:0 C17:0 PtdIns3,4,5P3 Internal standard m/z 1163.6

C18:0 C18:1 m/z 1203.8

C18:0 C20:4 m/z 1225.7 C18:0 C20:3 m/z 1227.8

C18:0 C18:2 m/z 1201.8

1110

1150

1200

12201230

1290

m/z, Da Fig. 3. Neutral loss scan data. Loss of 490 amu (PtdInsP2) in a, or 598 amu (PtdIns3,4,5P3) in b allows identification of the acyl chain composition. In the figures the parent ions are shown whose structure is deduced from the neutral loss data.

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means of improving reproducible recovery of phosphoinositides from cells and tissues and reducing the loss of the highly charged lipids during separation would allow more extensive use of the Pettitt et al. methodology. Irrespective of the methodology adopted, there remains a need for appropriate standards. A commonly adopted approach by analysts is to include forms of lipids not thought to be present in mammalian samples, in particular lipids with odd chain length acyl chains. There are a number of issues with this approach, first there is increasing evidence that odd chain acyl groups are indeed found in mammalian samples, for example in macrophage cell lines [19]. Second, the degree of ionization is not always constant within a class of lipid and thus the use of single standards may give an unreliable assessment of the concentration of each lipid species. Attempts to circumvent this problem have involved utilizing multiple standards for instance short, medium and long chain forms of each particular lipid class. The most accurate method, however, involves the inclusion of a deuterated form of each species. Clearly this is not feasible when there are many species within each class but the most appropriate compromise will involve the use of a range of deuterated standards for each class that includes the most commonly detected species. For PtdIns3,4,5P3 and PtdIns4,5P2 this will therefore include 18:0/20:4, 18:0/20:3, 18:0/18:2, 18:0/18:1, 18:0/20:3 and 18:0/20:2. Cost considerations will dictate what is possible and in many cases these will require de novo synthesis as they are not presently commercially available. Nevertheless, this will be the only methodology that will allow quantitative, rather than semi-quantitative analysis of the individual messenger lipids. Acknowledgements Work in the author's laboratory is supported by grants from the BBSRC, MRC and EU FP7 Lipidomic Net. References [1] M.A. Lemmon, Membrane recognition by phospholipid-binding domains, Nat. Rev. Mol. Cell Biol. 9 (2008) 99–111. [2] S.K. Dove, K. Dong, T. Kobayashi, F.K. Williams, R.H. Michell, Phosphatidylinositol 3,5-bisphosphate and Fab1p/PIKfyve underPPIn endo-lysosome function, Biochem. J. 419 (2009) 1–13. [3] M.L. Cheever, T.K. Sato, T. deBeer, T.G. Kutateladze, S.D. Emr, M. Overduin, Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat. Cell Biol. 3 (2001) 613–618.

[4] J.M. Gaullier, E. Ronning, D.J. Gillooly, H. Stenmark, Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes, J. Biol. Chem. 275 (2000) 24595–24600. [5] O. Gozani, P. Karuman, D.R. Jones, D. Ivanov, J. Cha, A.A. Lugovskoy, C.L. Baird, H. Zhu, S.J. Field, S.L. Lessnick, J. Villasensor, B. Mehrotra, J. Chen, V.R. Rao, J.S. Brugge, C.G. Ferguson, B. Payrastre, D.G. Myszka, L.C. Cantley, G. Wagner, N. Divecha, G.D. Prestwich, J. Yuan, The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor, Cell 114 (2003) 99–111. [6] T.R. Pettitt, M.J.O. Wakelam, Bombesin stimulates distinct time-dependent changes in the sn-1,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts as analysed by 3,5-dinitrobenzoyl derivitisation and HPLC separation, Biochem. J. 289 (1993) 487–495. [7] K. Simons, M.J. Gerl, Revitalizing membrane rafts: new tools and insights, Nat. Rev. Mol. Cell Biol. 11 (2010) 688–699. [8] D.R. Jones, Y. Bultsma, W.J. Keune, N. Divecha, Methods for the determination of the mass of nuclear PtdIns5P, PtdIns5P and PtdIns(4,5)P2, Methods Mol. Biol. 462 (2009) 75–88. [9] S. Kiefer, J. Rogger, A. Melone, A.C. Mertz, A. Koryakina, M. Hamburger, P. Kuenzi, Separation and detection of all phosphoinositide isomers by ESI-MS, J. Pharm. Biomed. Anal. 53 (2010) 552–558. [10] M.R. Wenk, L. Lucast, G. Di Paola, A.J. Romanelli, S.F. Suchy, R.L. Nussbaum, G.W. Cline, G.I. Shulman, W. McMurray, P. De Camilli, Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry, Nat. Biotechnol. 21 (2003) 813–817. [11] S.B. Milne, P.T. Ivanova, D. DeCamp, R.C. Hsueh, H.A. Brown, A targeted mass spectrometric analysis of phosphatidylinositol phosphate species, J. Lipid Res. 46 (2005) 1796–1802. [12] H. Ogiso, R. Taguchi, Reversed-phase LC/MS method for polyphosphoinositide analyses: changes in molecular species levels during epidermal growth factor activation in A431 cells, Anal. Chem. 80 (2008) 9226–9232. [13] T.R. Pettitt, S.K. Dove, A. Lubben, S.D.J. Calaminus, M.J.O. Wakelam, Analysis of intact phosphoinositides in biological samples, J. Lipid Res. 47 (2006) 1588–1596. [14] H.S. Hendrickson, C.E. Ballou, Ion exchange chromatography of intact brain phosphoinositides on diethylaminoethyl cellulose by gradient salt elution in a mixed solvent system, J. Biol. Chem. 239 (1964) 1369–1373. [15] T. Gunnarsson, L. Ekblad, A. Karlsson, P. Michelsen, G. Odham, B. Jergil, Separation of polyphosphoinositides using normal phase high performance liquid chromatography and evaporative light scattering detection or electrospray mass spectrometry, Anal. Biochem. 254 (1997) 293–296. [16] T.R. Pettitt, M. McDermott, K.M. Saqib, N. Shimwell, M.J.O. Wakelam, Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells, Biochem. J. 360 (2001) 707–715. [17] R.D. Byrne, M. Garnier-Lhomme, K. Han, M. Dowicki, N. Michael, N. Totty, V. Zhendre, A. Cho, T.R. Pettitt, M.J. Wakelam, D.L. Poccia, B. Larijani, PLC[gamma] is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly, Cell. Signal. 19 (2007) 913–922. [18] J. Clark, K.E. Anderson, V. Juvin, T.S. Smith, F. Karpe, M.J.O. Wakelam, L.R. Stephens, P. T. Hawkins, Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry, Nat. Methods 8 (2011) 267–272. [19] P.T. Ivanova, S.B. Milne, H.A. Brown, Identification of atypical ether-linked glycerophospholipid species in macrophages by mass spectrometry, J. Lipid Res. 51 (2010) 1581–1590.