Table 11.1.1.

Current Methods for Measuring Phosphoinositide (PI) Lipids or Soluble Inositol Phosphates (IP) in Cells or Extracts

Assay	Advantages	Disadvantages	Used for HTS	References
Metabolic radiolabeling followed by anion exchange chromatographic, TLC or HPLC analysis of intact IPs or deacylated PIs	Quantitative. HPLC separation can allow analysis of all PI and IP isomers and is sensitive (picomolar range). High-throughput anion-exchange chromatography on 96-well plates available.	Requires radioactivity and time-consuming metabolic labeling. May not appropriately distinguish between PI/IP mass and phosphorylation. TLC has limited resolution. Anion exchange chromatography and TLC do not sufficiently resolve PI/IP isomers. HPLC is logistically challenging and time consuming, requires expensive equipment.	Yes	Irvine (1986, 1990); Irvine et al. (1986); Imboden and Pattison (1987); Takazawa et al. (1990); Wreggett et al. (1990); Jenkinson (1995); Singh and Jiang (1995); Chengalvala et al. (1999); Kuksis (2003); Pouillon et al. (2003); Benjamin et al. (2004); Wen et al. (2004); Rusten and Stenmark (2006); Skippen et al. (2006); Stevenson-Paulik et al. (2006); Berrie et al. (2007); Guillou et al. (2007b); Otto et al. (2007); Sergeant and McPhail (2007)
MDD-HPLC	Quantitative. Allows analysis of many PI and IP isomers. Very sensitive (<50 pmol).	HPLC is logistically challenging and time consuming, and requires expensive equipment		Guse et al. (1995a,b); Singh and Jiang (1995); Adelt et al. (1999); Casals et al. (2002); Kuksis (2003); Lorke et al. (2004); Lin et al. (2009)
HPLC with suppressed conductivity detection	Quantitative. Non-radioactive. Can separate most isomers.	Less sensitive than radioactivity based detection and MS. HPLC is logistically challenging and time consuming, requires expensive equipment.		Nasuhoglu et al. (2002); Rusten and Stenmark (2006)
Liquid chromatography - mass spectrometry (LC-MS) or HPLC-MS	Non-radioactive. No post-column derivatization required. Sensitive (100 fmol to 100 pmol scale). Can analyze multiple PIs in a mixture, including some regioisomers. Can yield information about the PI fatty acid components.	Requires sophisticated, expensive equipment and specific expertise. Requires specialized MS equipment such as a fast ion trap mass spectrometer for isomer identification. Unclear if suitable for higher order PIs/IPs.		Singh and Jiang (1995); Pettitt et al. (2006); Vats et al. (2008)
Electrospray-ionization tandem mass spectrometry (ESI-MS-MS)	Sensitive and quantitative. Non-radioactive.	Does not distinguish between isomers. May require prior PI/IP isomer separation by one of the other techniques. Requires expensive equipment. Sophisticated.		Wenk et al. (2003); Rusten and Stenmark (2006); Berrie et al. (2007)
High-performance thin-layer chromatography (HPTLC) with molybdate staining	Simple, rapid, sensitive (100 to 200 pmol), non-radioactive, can monitor several PIs and IPs	Low resolution, requires HPLC confirmation		Hatzack and Rasmussen (1999); Kuksis (2003)
Radioreceptor/competitive radioligand displacement assays, radio-immunoassays (RIA); scintillation proximity (SPA) assays	Homogeneous, sensitive (high femtomolar to low picomolar range), simple, fast. Commercial kits available. More accurate IP mass measurements than via metabolic labeling. Can quantify specific IPs/PIs in tissue or cell extracts with little purification. Allow kinetic analyses of PI/IP levels in cell extracts or in vitro, measuring PI/IP-kinase or -phosphatase activities	Radioactive. Limited to single analytes such as PIP3, IP3, or IP4. In extracts, may read out combined activities of PIPs and corresponding IPs.	Yes	Amersham, Perkin Elmer; Challiss et al. (1988); Donie and Reiser (1989); Park et al. (1997); van der Kaay et al. (1997, 1998); Chang et al. (2002); Brandish et al. (2003); Kuksis (2003); Liu et al. (2003); Wen et al. (2004); Zheng et al. (2004); Sergeant and McPhail (2007); Mueller et al. (2008)
Luminescent oxygen channeling (LOCI), amplified luminescent proximity homogeneous assay (AlphaScreen), fluorescence polarization (FP) or fluorescence resonance energy transfer (FRET) assays	Homogeneous, non-radioactive, sensitive. Quantitative. Commercial kits available. Can quantify specific IPs/PIs in tissue or cell extracts with little purification. Allow kinetic analyses of PI/IP levels in cell extracts or in vitro, measuring PI/IP-kinase or -phosphatase activities.	Limited to single analytes such as PIP3, IP3, or IP4. In extracts, may read out combined activities of PIPs and corresponding IPs. LOCI/AlphaScreen sensitive to reactive oxygen scavengers.	Yes	Discoverex, Amersham, Echelon, Perkin Elmer; Ullman et al. (1996); Drees et al. (2003); Gray et al. (2003); Sato et al. (2003); Cicchetti et al. (2004); Prestwich (2004, 2005); Boldyreff et al. (2008)
Enzyme-linked immunosorbent assays (ELISA)	Quantitative. Commercial kits available.	Limited to single analytes such as PIP3, IP3, or IP4. In extracts, may read out combined activities of PIPs and corresponding IPs.	(Yes)	Rusten and Stenmark (2006)
PI extraction followed by spotting on nitrocellulose filters and antibody or PI-binding protein lipid overlay detection	Non-radioactive. Simple.	Low sensitivity, limited to few analytes. Nonquantitative.		Guillou et al. (2007b); Johnson et al. (2008); Rusten and Stenmark (2006)
Ectopic expression of fluorophor-tagged PI or IP binding protein domain fusion proteins in cells followed by immunofluorescent, FRET or electron microscopic analysis. Examples: PLCδ1 PH domain-EGFP fusion protein for PI(4,5)P2 detection, GRP1, Btk or ARNO PH domain-EGFP fusion proteins for PI(3,4,5)P3, PKCε C1 domain-EGFP fusion protein for diacylglycerol.	Allows in situ imaging of PI/IP levels in live or fixed cells. In life cells, kinetic analyses are possible. Probes for multiple different PIs and IPs available. Domain point mutants can provide specificity controls.	Semiquantitative. Requires transient cell transfection or stably transfected cell line. Probe overexpression may interfere with normal cell functions. FRET requires rigorous controls. Probes may bind to several different PIs and IPs. Limited to specific PI isomers. The probe for a given PI may also bind to and be modulated by its soluble IP analog, I(1,3,4,5)P4 in the case of PI(3,4,5)P3, for example (Huang et al., 2007). Thus, the assay reads out the combined action of both rather than absolute levels of one specific analyte. Physiologically, however, this is likely more relevant than individual analyte levels. Thus, this “integrated detection” can be advantageous in biological systems.	Yes, requires high-throughput/content imaging technology.	Oatey et al. (1999); Zeidman et al. (1999); Holz et al. (2000); Balla and Varnai (2002); Parmryd et al. (2003); Sato et al. (2003); Cicchetti et al. (2004); Rusten and Stenmark (2006); Guillou et al. (2007a); Sakaguchi et al. (2009)
Direct fixed cell labeling with recombinant PI/IP binding protein domains or antibodies	Does not require transfection. Does not interfere with cell function. Non-radioactive.	Limited to fixed cells, where PI and in particular IP preservation is difficult. May not detect protein-bound IPs/PIs.		Rusten and Stenmark (2006)
Enzymatic detection	Sensitive, non-radioactive, quantitative	Requires prior IP separation by HPLC or other techniques. Indirect multi-step process, requiring IP de-phosphorylation to myo-inositol which is then oxidized, followed by enzymatic measurement of oxidation products, or by measuring of the released inorganic phosphate. Salt interferes with IP dephosphorylation. Dephosphorylation rates differ for different IPs. IP5/IP6 are very poorly dephosphorylated. Thus, enzymological detection is not ideal for analyses of multiple different IPs/PIs and may be limited by elution reagent or buffer conditions.		Singh and Jiang (1995); Kuksis (2003)