EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 42, No. 1, 1-11, January 2010 Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: A brief review Youngjun Kim1, Selina Rahman Shanta2, cult to identify the composition of phosphoinositides. Li-Hua Zhou2,3 and Kwang Pyo Kim2,4 Recent advances in mass spectrometric techniques, combined with established separation methods, have 1Department of Applied Biochemistry allowed the rapid and sensitive detection and quantifi- Konkuk University cation of a variety of lipid species including phospho- Chungbuk 380-701, Korea inositides. In this mini review, we briefly introduce 2Department of Molecular Biotechnology progress in profiling of cellular phosphoinositides us- Institute of Biomedical Science and Technology ing mass spectrometry. We also summarize current Konkuk University progress of matrices development for the analysis of Seoul 143-701, Korea cellular phospholipids using matrix-assisted laser de- 3Faculty of Chemical Engineering and Light Industry sorption/ionization mass spectrometry. The phospho- Guangdong University of Technology inositides profiling and phospholipids imaging will Guangzhou 510006, China help us to understand how they function in a biological 4Corresponding author: Tel, 82-2-2049-6051; system and will provide a powerful tool for elucidating E-mail, [email protected] the mechanism of diseases such as diabetes, cancer DOI 10.3858/emm.2010.42.1.001 and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides Accepted 1 September 2009 using electrospray ionization mass spectrometry and Available Online 4 November 2009 matrix-assisted laser desorption/ionization mass Abbreviations: APCI, atmospheric pressure chemical ionization; spectrometry will suggest new insights on human dis- ATT, 6-aza-2-thiothymine; CHCA, cyano-4-hydroxycinnamic acid; eases, and on clinical application through drug devel- CHCl3, chloroform; CID, collision-induced dissociation; DHB, opment of lipid related diseases. 2,5-dihydroxybenzoic acid; eIF4E, eukaryotic translation initiation factor 4E; ESI-MS, electrospray ionization mass spectrometry; Keywords: lipids; lipid metabolism disorders; phos- FAB-MS, fast atom bombardment mass spectrometry; IMS, imaging pholipids; phosphatidylinositols; spectrometry, mass, mass spectrometry; LC, liquid chromatography; MALDI-MS, electrospray ionization; spectrometry, mass, ma- matrix-assisted laser desorption/ionization mass spectrometry; trix-assisted laser desorption-ionization MALDI-TOF-MS, matrix-assisted laser desorption and ionization/time of flight mass spectrometry; MeOH, methanol; MS, mass spectrometry; MS/MS, tandem mass spectrometry; mTOR, Introduction mammalian target of rapamycin; PA, phosphatidic acids; PAF, platelet-activating factor; PDK1, phosphoinositide-dependent Lipids are an important class of cellular compo- kinase-1; PEps, phosphatidylethanolamine plasmalogens; PH, nents and play diverse roles in human physiology. pleckstrin homology; PI3K, phosphoinositide 3-kinase; PIP, Although they are important, the study of lipids has phosphatidylinositol phosphate; PIP2, phosphatidylinositol bispho- been limited by analytical difficulties. However, sphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKB/Akt, recently improved analytical methods, such as protein kinase B or Akt; S/N, signal to noise; SA, sinapinic acid; mass spectrometry (MS) and liquid chromato- TFA, trifluoroacetic acid; TLC, thin layer chromatography graphy (LC) provide systems-level investigation in lipid research (Wenk, 2005). This systems-level analysis of lipids (lipidomics) is a growing field with different applications in drug and biomarker deve- Abstract lopment. There are various efforts to explore abnormal lipid metabolism in human diseases, Phospholipids are key components of cellular mem- such as insulin-resistant diabetes, Alzheimer's brane and signaling. Among cellular phospholipids, disease, schizophrenia, cancer, atherosclerosis, phosphoinositides, phosphorylated derivatives of and infectious diseases through lipidomics phosphatidylinositol are important as a participant in approach using mass spectrometry (Oresic et al., essential metabolic processes in animals. However, 2008). Lipidomics will contribute to drug deve- due to its low abundance in cells and tissues, it is diffi- lopment for pharmaceutical therapy of lipid related 2 Exp. Mol. Med. Vol. 42(1), 1-11, 2010 diseases through mostly presenting cellular lipid ESI-MS and phospholipid analysis using MALDI-MS profile and lipid distribution in a tissue. in this review. A brief suggestion on clinical Electrospray ionization (ESI)-MS was initially applications of phosphoinositide profiling and developed by Fenn and colleagues for analysis of phospholipid analysis using mass spectrometry is biomolecules (Fenn et al., 1989). It depends on the additionally given. formation of gaseous ions from polar, thermally labile and mostly non-volatile molecules. The "soft" electrospray ionization causes little or no fragmen- Profiling of cellular phosphoinositides tation and virtually all phospholipid species are using ESI mass spectrometry detected as molecular ion species. The identifi- cation and structural information about a certain Phosphoinositides are phospholipids with an peak is acquired by fragmentation (tandem mass inositol head groups. They have a central position spectrometry, MS/MS) (Han and Gross, 1994; Kim in the fields of cell signaling and regulation, et al., 1994). Advances in mass spectrometry (MS) including calcium homeostasis, membrane traffic- have created powerful new avenues for the king and cytoskeletal dynamics (Di Paolo and De measurement of global changes in the cellular lipid Camilli, 2006). They are able to achieve signaling inventory. effects directly by binding to cytosolic proteins or Electrospray ionization (ESI) mass spectrometry cytosolic domains of membrane proteins via their has been successfully used to analyze lipids in polar head groups (Figure 1). In this way, they can various cell types. However, for less abundant and regulate the function of proteins integral to more complicated chemical compositions, HPLC is membranes, or they can attract cytoskeletal and needed for upfront separation before ionization by signaling components to the membrane (Di Paolo ESI and detection by MS (Watson, 2006). In the field of lipidomics HPLC or TLC have been coupled with electrospray ionization (ESI)/atmospheric pressure chemical ionization (APCI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS, respectively (Wenk, 2005). These methods provide the iden- tification of lipid composition and quantification of cellular lipids, such as fatty acids, phospholipids, cholesterols, and sphingolipids. The identification of lipid composition and quan- tification of cellular lipids provide molecular signature for a lipid related pathway. One of the most obvious applications of lipidomics will include profiling lipid extracts in order to identify metabolic pathways and enzymes that are affected (Wenk, 2005). A major advantage of such lipidomics-ba- sed discovery is that, together with our relatively good understanding of many biosynthetic and metabolic pathways, it will lead to the identification of pathways and enzymes governing human diseases (Cascante et al., 2002). A recent review (Wenk, 2005) well summarized diverse aspects and fundamental principles of lipidomics. Among lipidomics technologies, phosphoinositide profiling using electrospray ionization mass spec- trometry (ESI-MS) and phospholipid analysis using matrix-assisted laser desorption/ionization mass Figure 1. A phosphatidylinositol structure. This figure is originated from spectrometry (MALDI-MS) are quite challenging http://en.wikipedia.org/wiki/File:Phosphatidylinositol.jpg. The hydroxyl due to their technical difficulties although they moieties at 3', 4', and 5' in the head group can be phosphorylated to pro- could suggest more information on lipid related duce seven phosphoinositides, phosphatidylinositol 3-phosphate (PI3P), diseases. In these fields, several attempts have phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 5-phos- phate (PI5P), phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), phos- been reported in order to beat our technical phatidylinositol (3,5)-bisphosphate (PI(3,5)P2), phosphatidylinositol limitation. We are bringing out the technical (4,5)-bisphosphate (PI(4,5)P2), and phosphatidylinositol (3,4,5)-tri- progress of cellular phosphoinositide profiling with sphosphate (PI(3,4,5)P3, PIP3). Analysis of phospholipids by MS 3 and De Camilli, 2006). high resolving power. Phosphoinositides are less abundant lipids in Several methods such as fast atom bombardment eukaryotic cell membranes. Therefore, analysis of mass spectrometry (FAB-MS), matrix-assisted phosphoinositides requires special cautions during laser desorption and ionization/time of flight mass sample treatment. According to several reports spectrometry (MALDI-TOF-MS) and ESI-MS have (Hsu and Turk, 2000; Wenk et al., 2003; Milne et al been used for the analysis of phosphoinositides. 2005; Shui et al., 2007) to minimize the MALDI-TOF-MS has been recently applied for interference of other phospholipids during analysis, phosphatidylinositols in murine brain extracts a selective two-step extraction was used (Scheme (Berry et al., 2004) and ESI-MS has been used for 1). First, the cell material was extracted with PIPs and PIP2s in lipid extracts from cultures