Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

Review

Advances in phosphoinositide proﬁling by mass spectrometry
Tsumugi Iwata, Yuki Ishino, Junken Aoki, Nozomu Kono*
Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, the University of Tokyo,
7‒3‒1 Hongo, Bunkyo-ku, Tokyo 113‒0033, Japan
Abstract Phosphoinositides (PIPs), which are phosphorylated forms of phosphatidylinositol (PI), are minor but essential
components of membrane phospholipids. There are seven PIP classes diﬀering in phosphorylation at the 3, 4, and 5 positions of the inositol ring of PI. Each PIP class plays crucial roles in various cellular events, such as signal transduction andmembrane traﬃcking, while dysregulation of PIP metabolism is associated with the development of diseases such as cancer. Recent studies suggest the importance of the composition of the two fatty acyl chains in PIPs as well as the phosphorylation status of the inositol ring of PIPs. Therefore, there is an increasing demand for analysis of PIPs by mass spectrometry
which can provide information on the fatty acyl chains of PIPs. Recently, mass spectrometric methods that can comprehensively analyze molecular species of all PIP regioisomers have been developed. In this short review, we describe past andpresent mass spectrometric analyses of PIPs as well as potential future improvements in the method.
Key words: phosphoinositides; regioisomer; methylation; supercritical ﬂuid chromatography; chiral column chromatography

Introduction
Phosphoinositides (PIPs), which are phosphorylated

agy induction. PI(3,4)P2 and PI(3,4,5)P3 are produced at theplasma membrane in response to stimuli. Although the pre-forms of phosphatidylinositol (PI), are minor but essential

cise localization of PI(5)P is unclear, it has been found incomponents of membrane phospholipids in eukaryotes.
Phosphorylation of PI at the 3-, 4-, and 5- hydroxyls of thediverse compartments, including the plasma membrane,
3)
early endosomes, and the nucleus . These unique subcellu-

inositol head group generates seven PIP classes, namely,

lar localizations are formed by the local actions of PIP

PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, andkinases and phosphatases.

PI(3,4,5)P3 (Fig. 1A). Each of the seven PIPs has a uniquesubcellular distribution with predominant localization in

The unique subcellular localizations of PIPs allow themto act as signposts for each organelle, regulating the subcel-

subsets of membranes: PI(4,5)P2 localizes to the plasma
membrane, PI(4)P to the Golgi apparatus and plasma mem-

lular localization and function of various proteins involvedin cell migration, adhesion, signal transduction, and mem-brane, PI(3,5)P2 to late endosomes and lysosomes, andbrane traﬃcking . In addition, PI(4,5)P2 is hydrolyzed byphospholipase C upon stimulation to produce inosi-

2)

1, 2)

(Fig. 1B). PI(3)P is
also produced in the endoplasmic reticulum during autophPI(3)P to early and late endosomes

tol-1,4,5-triphosphate and diacylglycerol, both of which act
4)

as second messengers . In the past decade, it has become*Corresponding author

clear that PI(4)P plays an essential role in intracellular lipid
2)transport . On the other hand, dysregulation of PIP metabo-Nozomu Kono

Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, the University of Tokyo, 7‒3‒1 Hongo,

lism is reportedly associated with the development of dis4)

Bunkyo-ku, Tokyo 113‒0033, Japan
Tel: ＋81‒3‒5841‒1076, Fax: ＋81‒3‒3818‒3173

eases . For example, genes PIK3CA, PTEN, and INPP4B,

Received: July 31, 2022. Accepted: September 18, 2022.

cancers .

which encode enzymes involved in the metabolism ofPI(3,4,5)P3, were found to be frequently mutated in many

E-mail: nozomu@mol.f.u-tokyo.ac.jp

5)

PIPs are membrane lipids whose amounts are very small,

Epub October 18, 2022.

making their measurement very diﬃcult, and substantial

DOI: 10.24508/mms.2022.11.004
93Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

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Fig. 1.

Metabolism and subcellular localization of phosphoinositides.
A. Chemical structure of PI and PIPs. For PIPs, only the polar heads are shown. Addition and removal of phosphate groups at the-hydroxy, 4′-hydroxy and 5′-hydroxy groups of PI by phosphoinositide kinases and phosphatases (solid arrows) creates seven3′
distinct PIP species. B. Subcellular localization of PIPs. The spatiotemporally controlled activity of phosphoinositide kinases andphosphatases creates a distinctive enrichment of PIPs across the cellular compartments.

Proﬁling Intact PIPs by ESI-MS

eﬀorts have been made to measure them. Classically, cellu3
lar PIPs have been measured by labeling cells with [ H]ino-

Michelsen et al. applied ESI-MS for analyzing phosphatidylinositol-monophosphate (PIP1) and phosphatidylinosi-

32

sitol or [ P]phosphate and separating deacylated PIPs
according to their polar head using high-performance liquid

tol-bisphosphate (PIP2) and measured 1-stearoyl-2-arachidonyl (18:0‒20:4) species of PIP1 and PIP2 in bovine brain

chromatography (HPLC) . Non-radiolabeled PIPs can bemeasured by utilizing PIP kinase or PIP-binding pro6)

7‒9)

15)

extracts . They showed that about 200 pmol of PIP1 and. Although these methods can quantify the total

PIP2 could be quantiﬁed by single ion monitoring in theamount of each PIP class, they do not provide information
on fatty acyl chains.

negative ion mode. Hsu et al. intensively studied thedetailed mechanism of fragmentation of phosphatidylinosi-

Recently, it has become clear that the composition of
fatty acyl chains of PIPs is associated with protein func-

tol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] ions under collision-activated dissoci-teinsneed for PIP proﬁling that includes information on fatty

ation (CAD) with ESI-tandem MS (ESI-MS/MS), showingthat ESI-MS/MS is a powerful tool for structural characteri-acyl chains. Electrospray ionization mass spectrometry(ESI-MS) is a powerful and commonly used method forzation of PIPs . Proﬁling of PIP molecular species in biological samples by ESI-MS/MS was ﬁrst reported by Wenk

10,11)tions

12)and cancer progression , thus increasing the16)

13,14)

17). In the past 20
years, several analytical techniques for PIPs by ESI-MSet al. . They found that the addition of piperidine to thehave been developed, enabling highly sensitive and versa-

PIP2 ions in the negative ion mode. They detected elevatedtile measurements of PIP molecular species. More recently,
analytical methods that distinguish between positional iso-levels of PIP2 species in human ﬁbroblasts from patients

quantifying individual lipid species

lipid samples markedly enhance the detection of PIP1 andmers of PIPs have been developed. In this short review, wewith Lowe syndrome, which is caused by mutations in
OCRL1, which encodes a phosphoinositide 5-phospha-

describe past and present mass spectrometric analyses oftase . They also showed that deﬁciency of enzymes

PIPs as well as potential future improvements in the

involved in PIP metabolism in Saccharomyces cerevisiaeaﬀects not only the levels of PIPs but also PIP proﬁles, sug-

18)

method.

gesting that PIP metabolizing enzymes are selective for cer94

Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

Table 1.

Advances in the analysis of PIPs by mass spectrometry

Authors

Year

Advances/Novelty

ref.

Michelsen et al.

1995

Application of ESI-MS to PIP1 and PIP2 analysis.

15

Hsu et al.

2000

The detailed mechanism of fragmentation of PI(4)P and PI(4,5)P2 ions under CAD with ESI-MS/MS.

16

Wenk et al.

2003

Measurement of PIP1 and PIP2 acyl vriants in biological samples by ESI-MS/MS.
Enhancement of PIPs signal intensities by piperidine.

17

Milne et al.

2005

Measurement of PI(3,4,5)P3 molecular species in biological samples
Development of 2-step extraction method.

19

Pettitt et al.

2006

Measurement of PIP molecular species and PIP2 regioisomers by normal phase LC-MS.
Development of buﬀered citrate extraction method.
Use of ethylamine as a mobile phase modiﬁer.
3Distinct fragmentation pattern of PI(4)P from other PIP1s at MS .

20

Ogiso et al.

2008

Measurement of PIP molecular species by reverse phase LC-MS
Use of DEAE column to obtain a PIP-rich fraction.

21

Clark et al.

2011

Development of PIP derivatization method with TMS-diazomethane.
Measurement of methylated PIPs by LC-MS/MS.

22

Kim et al.

2017

Development of LC-MS/MS method for structural identiﬁcation of sn-1 and sn-2 fatty acyl chains of PIPs.
Enhancement of PIPs signal intensities by ammonium ion adduction.

23

Wang et al.

2016

Development of a method for quantifying each PIP2 regioisomer and PI(3)P by simulation using theinformation of methylation patterns.

24

Bui et al.

2018

Development of a technique for measuring non-derivatized PIP2 regioisomers by RPLC-MS with theaddition of the ion-pairing reagents.

25

Melek et al.

2017

Development of a method to distinguish between 18:0/20:4 PI(4,5)P2 and PI(3,4)P2 in biological samples by shortening the acyl groups of PIPs by ozone cleavage.

26

Li et al.

2021

Development of a method to separate all methylated PIP1 and PIP2 regioisomers by HPLC-MS using acellulose-based chiral column
Use of SWATH to obtain a comprehensive PIP proﬁle.

27

Morioka et al.

2022

Development of a method for simultaneously quantifying molecular species of all seven PIP classes by
HPLC-ESI-MS/MS using a cellulose-based chiral column.

28

Shimanaka et al.

2022

Development of a method for simultaneously quantifying molecular species of all seven PIP classes by
SFC-ESI-MS/MS using a β -cyclodextrin column.

29

20)

tain acyl chains or that organelles have a speciﬁcity of PIP
proﬁles. Milne et al. reported proﬁling of PI(3,4,5)P3

method to resolve PIP regioisomers . They thoroughly
investigated each step from the extraction to the ﬁnal analy-

together with PIP1 and PIP2 in macrophages . They used a
selective two-step extraction, in which cells were treatedsis of PIPs and made several improvements. Especially,
they developed a highly-eﬃcient buﬀered citrate extraction

with neutral solvents to extract unwanted neutral phospholipids, and then PIPs were extracted from the resulting pel-methodology to minimize acid-induced phosphoinositidedegradation and a sensitive LC-MS method using ethyl-

let with acidic solvents. Their analysis revealed that diﬀer-

amine as a mobile phase modiﬁer. They showed that levels

ent proﬁles of PIP3 were produced in macrophagesof PIP1 were 2.5 times higher than those of PI in humanstimulated by diﬀerent agonists, suggesting the activation
mechanism of PI-3 kinase and/or PI(4,5)P2 species that may

platelets. The detection limits were 250 fmol for PIP1, 1
pmol for PIP2, and 5 pmol for PIP3. Reversed-phase (RP)19)

serve as substrates are diﬀerent for diﬀerent agonists. Wenk
et al. and Milne et al. used direct infusion mass spectrome-

LC is excellent for separating phospholipid molecular species and detecting minor components. Ogiso et al. devel-

try, in which ion suppression eﬀects may aﬀect the measurements. Pettitt et al. developed a normal phase LC-MS

oped a sensitive method to determine PIP1, PIP2, and PIP3
21)
proﬁles by RPLC-MS . They used a (diethylamino)ethyl
95

Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

Fig. 2.

Methylation of the phosphate groups of PIPs by TMS-diazomethane.

Treatment of PI(3,4,5)P3 with TMS-diazomethane yields heptamethylated PI(3,4,5)P3 [PI(3,4,5)P3 (Me)7]. In the case of PI(4)P andPI(4,5)P2, trimethylated PI(4)P [PI(4)P (Me)3]and pentamethylated PI(4,5)P2 [PI(4,5)P2 (Me)5]are mainly produced, respectively.
The methylated PIPs are readily detected as positive ions and collision-activated dissociation (CAD) of a methylated PIP ion yields
diacylglycerol (DAG) ion and monoacylglycerol (MAG) ion.

(DEAE)-cellulose column to obtain a PIP-rich fraction fromthe lipid extracts and an alkaline-resistant C8 column. They

the addition of three methyl groups to PIP1, ﬁve to PIP2,

measured 17 PIP1 and PIP2 species in epidermal growth
factor (EGF)-stimulated A431 cells. They also showed thatgroups improves the recovery of PIPs and renders theand seven to PIP3 (Fig. 2). Methylation of the phosphatehighly polar PIPs less charged and more stable. Methylated
PIPs can be detected as positive ions by ESI-MS, and thethe level of 36:2 PIP3 species increased upon EGF stimulation. The detection limit of their method was 25 fmol foreach PIP. These and other advances in the mass spectrome-

CAD of methylated PIP ions leads to neutral loss of thetry of PIPs (described below) are summarized in (Table 1).

charged diacylglycerol fragments. Clark et al. used a C4
reversed-phase HPLC column to concentrate methylated

methylated inositol phosphate headgroup, producing

Highly-sensitive PIP Analysis by Using a
Derivatization Technique

PIPs and detected them by neutral loss scan mode or multiple reaction monitoring (MRM) mode using diacylglycerol
ions as Q3 masses. Clark et al. quantiﬁed PI(3,4,5)P3 spe-

As mentioned above, the analytical method for PIPs by
ESI-MS, including sample preparation, has been greatly

cies in fMLP-stimulated human neutrophils, glucose-stimulated human adipose tissues, and insulin-stimulated mouse

improved. However, there remain problems in PIP recovery

liver. They also showed that 18:0/20:4 PI(3,4,5)P3 accumu-

and stability, which are associated with the inositol ring
phosphate group being adsorbed on glass and metal sur-lated selectively upon EGF stimulation in MCF10a breast

20,21)

faces and undergoing hydrolysis. To address thesecancer cells, although its precursor 18:0/20:4 PI(4,5)P2 was
the least abundant species of PI(4,5)P2. Kim et al. devel-

problems, Clark et al. developed a derivatization method inwhich the phosphate group is methylated with trimethylsi-

oped an LC-MS/MS method for structural identiﬁcation of23)
sn-1 and sn-2 fatty acyl chains of PIPs . They noted that

lyl-diazomethane (Fig. 2). Derivatization of PIPs with
TMS-diazomethane can be performed directly in the acidi-

sn-1 monoacylglycerol ions are generated by the CAD ofﬁed chloroform-methanol phase from cellular or tissue

methylated PIPs and performed precursor ion scans of

extracts and takes only 10 min. The derivatization results inmonoacylglycerol ions as well as neutral loss scans of the

22)

96

Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 202225)

ethylenediamine tetraacetic acid (EDTA) . Melek et al.

inositol head groups in positive ion mode. By comparing
LC retention times between the two scan modes, they suc-developed a method to distinguish between 18:0/20:4

ceeded in determining sn-1 and sn-2 fatty acyl chains of PIP

PI(4,5)P2 and PI(3,4)P2 in biological samples by shortening

species. They also found that ammonium adduction
enhanced signal intensities of PIPs by about twofold. Using

the acyl groups of PIPs by ozone cleavage . Thus, whilevarious eﬀorts have been made to measure each PIP regio-

their method, they quantitatively proﬁled PIP species ininsulin-treated AML12 hepatocytes and mouse livers withisomer separately, there is much interest in the developmentof a simple and versatile method to separate all PIP regio-

structural information of the sn-1 and sn-2 fatty acid com-isomers by chromatography and measure them simultane-

positions.

ously.

26)

Very recently, three independent groups, including ours,

Regioisomer-speciﬁc Analysis of PIP Molecular Species

have reported a method for the simultaneous determinationof all PIP regioisomers by chromatography-tandem mass27‒29). Li et al. successfully separated all methylated PIP1 and PIP2 regioisomers by HPLC using a cellu-

As mentioned above, PIP1 and PIP2 each have three

spectrometry

regioisomers, and each regioisomer is involved in distinct
quantiﬁed separately to investigate its functions. However,

lose-based chiral column . They also utilized SWATH(sequential window acquisition of all theoretical mass spec-

the masses of the PIP regioisomers are exactly the same,tra) technology, a data-independent acquisition technique,making it impossible to distinguish the regioisomers byto obtain a comprehensive PIP proﬁle. Using their method,mass spectrometry.

they reported the full PIP proﬁles of human plasma, Pichia

Pettit et al. reported a method for measuring non-derivatized PIP regioisomers by using normal-phase LC and ion

pastoris, and HeLa cells, including proﬁle changes upon27)

cellular functions. Therefore, each regioisomer must betreatment with PI3K inhibitor wortmannin. Morioka et al.

20)

trap mass spectrometry . They showed that three PIP2
regioisomers could be separated by normal phase LC using

also reported a method for simultaneously quantifying
molecular species of all seven PIP classes by HPLC-a silica column. Although the three PIP1 regioisomers could

ESI-MS/MS using a cellulose-based chiral column . Theirnot be separated by the LC system, PI(5)P eluted slightly

method revealed (1) a severe skewing in acyl chains inPI(3,4)P2 and PI(3,4,5)P3 in Pten-deﬁcient prostate cancer

28)

ahead of PI(3)P and PI(4)P. Moreover, they found that thetissues, (2) extracellular mobilization of PIPs upon expression of oncogenic PIK3CA, and (3) a unique proﬁle for exo-

3

fragmentation pattern of PI(4)P at MS was diﬀerent fromthe fragmentation patterns of the other PIP1 regioisomers,
which allowed them to calculate the ratio of PI(4)P to other
PIP1 isomers present. Thus, the combination of the separa-

somal PIPs. Although both two methods use cellulose-based chiral columns, the order of elution of the PIP1

tion of PI(5)P by normal phase LC and the diagnosis of theregioisomers is diﬀerent: PIP1 regioisomers elute in thepresence ratio of PI(4)P and PI(3)P by ion trap MS enabledorder PI(3)P, PI(5)P, and PI(4)P in the method of Li et al.,the measurement of each PIP1 regioisomer. Using thisbut in the order PI(3)P, PI(4)P, and PI(5)P in the method of

method, they showed that PI(3,5)P2 is increased in yeast

Morioka et al. PIP2 regioisomers elute in the order PI(3,5)

cells upon hyperosmotic stress. Wang et al. found that PIP2
regioisomers and PI(3)P exhibit unique methylation pat-

P2, PI(3,4)P2, and PI(4,5)P2 for both methods.

terns and developed a method for quantifying each PIP2

oped a method for separating methylated PIP regioisomers
29)
by supercritical ﬂuid chromatography (SFC) . A supercrit-

As an alternative chromatographic approach, we devel-

regioisomer and PI(3)P by simulation using the information
24)of methylation patterns . Since the methylation patterns ofPI(4)P and PI(5)P were virtually identical, only their mix-

ical ﬂuid is a ﬂuid that is maintained at a temperature andpressure above its critical point. It has viscosity and diﬀu-tures could be determined from biological samples by thissivity close to gas and has solubility close to liquid.
Because of these characteristics, SFC enables fast and eﬃ-

method. They showed changes in the PIP proﬁle in thebrain cortices of db/db mice. Bui et al. reported a techniquefor measuring non-derivatized PIP2 regioisomers by using

cient separation. In addition, SFC and HPLC have diﬀerent

C18 reverse phase chromatography with the addition of theion-pairing reagents diisopropylethanolamine (DiiPEA) andthat cannot be separated by HPLC. We found that SFC witha β -cyclodextrin column successfully separated methylated

separation behaviors, allowing SFC to separate compounds

97Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

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in the absolute quantiﬁcation of PIP molecular species inbiological samples. Because PI(4)P and PI(4,5)P2 are far
more abundant than other PIPs in biological samples, minor
PIP1s [PI(5)P and PI(3)P] and minor PIP2s [PI(3,4)P2, andPI(3,5)P2] ended up on the shoulders of the large PI(4)Pand PI(4,5)P2 peaks, respectively, preventing their accuratequantiﬁcation of minor PIPs. In fact, the amounts of minor
PIPs [PI(5)P, PI(3)P, PI(3,4)P2, and PI(3,5)P2] in the biolog-

ical samples measured by HPLC-ESI-MS/MS or SFCESI-MS/MS were much higher than those measured by
Fig. 3.

27,29). Although this may beradioisotope-labeling methods
partly due to the eﬀect of culture conditions during radio-

Separation of PIP1 and PIP2 regioisomers by SFC.

Synthetic 1-heptadecanoyl-2-arachidonyl (17:0/20:4) PIP
standards were methylated with TMS-diazomethane andanalyzed by SFC-ESI-MS/MS. MRM chromatograms of
17:0/20:4-PIP1 (A) and 17:0/20:4-PIP2 (B) are shown.

isotope labeling on PIP metabolism, we cannot rule out thepossibility that peaks of minor PIP1 and PIP2 classes are
inﬂated by those of PI(4)P and PI(4,5)P2. Further improve-ments in separating PIP regioisomers would allow the abso-

PIP1 and PIP2 regioisomers (Fig. 3). The SFC-based methodlute quantiﬁcation of PIP molecular species in biologicalproduces a diﬀerent elution proﬁle for the PIP regioisomers
than the two HPLC-based methods: in the SFC-based

samples.

method, the PIP1 regioisomers elute in the order PI(4)P,niques have made it possible to image various phospholipid
30,31).
molecular species in tissues at the single-cell level

Recent advances in phospholipid mass imaging tech-PI(5)P, PI(3)P, and the PIP2 regioisomers elute in the orderPI(4,5)P2, PI(3,5)P2, PI(3,4)P2. In the HPLC-based method,

32,33)

However, there are few reports on MS imaging of PIPs

the PIP1 and PIP2 regioisomers elute in a narrow range ofretention times, whereas, in the SFC-based method, theand none on MS imaging of PIP regioisomers. For MS

PIP1 and PIP2 regioisomers elute in a broader range, result-regioisomers inside the mass spectrometer. One powerfulapproach for isomer-speciﬁc imaging of PIPs would be ion

imaging of PIP regioisomers, it is necessary to separate PIP

ing in a better separation of regioisomers. The SFC system
coupled with ESI-MS/MS makes it possible to measure themobility spectrometry, which is a gas-phase separation tech-molecular species of all seven PIP classes in the biologicalnique in which ion mobility is aﬀected by the molecularsamples. Proﬁling PIP molecular species in mouse tissues
by this method revealed that PIPs with two saturated fatty

shapes of ions . Ion mobility spectrometry will also help
the absolute quantiﬁcation of PIP regioisomers by LC/SFC-

acids, which were hardly detected in most of the tissues or

ESI-MS/MS. By incorporating new methodologies such as

cultured cells, were abundant in the testis. We also foundion mobility spectrometry, the analysis of PIPs by MS willthat less unsaturated PI(3)Ps such as 34:1, 34:2, 36:1, and36:2 PI(3)P, which are probably derived from newly synthe-

overcome the current challenges and further contribute to

sized PI, selectively increased during autophagy induction.

roles of PIPs.

34)

our understanding of the physiological and pathologicalThus, chiral column chromatography has made it possible

Acknowledgments

to measure the molecular species of all seven PIP classes inThis work was supported by Grants-in-Aid for Scientiﬁca single analysis.

Research on Innovative Areas (21H00228 to NK); by
AMED-CREST, AMED (JP 21gm1210013 to NK).

Future Perspectives
PIP analysis has now reached a major milestone with

Conﬂict of Interest

methods that can measure the molecular species of all

NK is a co-inventor of a Japan patent 7017704 with theseven PIP classes. Analyzing full PIP proﬁles in biologicalsamples should lead to a better understanding of the physio-

title “Biological membrane phosphoinositide separation

logical and pathological roles of PIPs at the molecular spe-method”. All other authors declare no competing interests.

cies level. However, there remains room for improvement98Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

143: 888‒895, 2010.

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