Yeast Lipid Analysis and Quantification by Mass Spectrometry

C H A P T E R

F I F T E E N

Yeast Lipid Analysis and Quantification by Mass Spectrometry Xue Li Guan,* Isabelle Riezman,‡ Markus R. Wenk,*,† and Howard Riezman‡ Contents 1. Introduction 2. Methods 2.1. Sample preparation 2.2. Normalization for starting amount of material 2.3. Lipid extraction (for glycerophospholipids and sphingolipids) 2.4. Sterol isolation 2.5. Lipid analysis Acknowledgments References

370 373 373 375 375 376 377 389 389

Abstract The systematic and quantitative analysis of the different lipid species within a cell or an organism has recently become possible and the general approach has been termed ‘‘lipidomics.’’ Traditional methods of identification and quantification of lipid species were laborious processes and it was necessary to use a wide variety of techniques to analyse the different lipid species, especially concerning the assigning of particular acyl chain lengths, hydroxylations, and desaturations to the diverse lipid species. While it is still not possible to quantitatively analyze all lipid species in one fell swoop, great progress has been made with the intensive use of quantitative mass spectrometry approaches. It is now relatively simple to quantify most of the lipid species, including all of the major ones, in a yeast cell. Different degrees of sophistication of mass spectrometric analysis exist and the available techniques and instrumentation are evolving rapidly. Therefore, we have decided to present robust, simple methods to quantify the major yeast lipids by mass spectrometry that should be accessible to anyone who has access to a standard mass * Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore { Department of Biochemistry, University of Geneva, Switzerland {

Methods in Enzymology, Volume 470 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)70015-X

#

2010 Elsevier Inc. All rights reserved.

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spectrometry equipment. The methods to identify and quantify yeast glycerophospholipids and sphingolipids involve electrospray ionization mass spectrometry using fragmentation to characterize the lipid species. A simplified gas chromatographic method is used to quantify the major sterols that occur in wild-type yeast cells and ergosterol biosynthesis mutants.

1. Introduction Studies in yeast, notably Saccharomyces cerevisiae, have played a particularly important role in the advancement of our knowledge in biology. An indispensable aspect of yeast research, in addition to genetics and molecular biology, is lipid biochemistry and analysis. Lipids make up a bulk of cellular membrane and have gained immense interest due to their emerging new cellular functions other than their structural roles. Three major classes of membrane lipids in all eukaryotic organisms include glycerophospholipids, sphingolipids, and sterols and each class is structurally diverse, arising from both nonpolar chains and head group substitutions (Fig. 15.1). The glycerophospholipids are diversified by their head group and fatty acyl compositions and the major head groups are conserved among all eukaryotes. Lipid composition varies between organelles (Schneiter et al., 1999). On the other hand, the sphingolipids of S. cerevisiae are distinct and relatively simple compared to mammals. There A O

O

P O –O O R

O O O

B OH OH R

sn-1

O

sn-2

R-hydrogen (GPA); Inositol (GPIns); Ethanolamine (GPEtn); Choline (GPCho); Serine (GPSer); Glycerol (GPGro)

Sphingoid base

HN O

OH

Fatty acid

R-hydrogen (phytoceramide); Inositol phosphate (IPC); Mannosyl inositol phosphate (MIPC); Mannosyl diinositol phosphate (M(IP)2C)

C

HO

Figure 15.1 Structures of major membrane lipids in S. cerevisiae: (A) glycerophospholipids, (B) sphingolipids, (C) ergosterol. R refers to possible head group modifications.

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are three classes of inositol-containing sphingolipids; inositol-phosphorylceramide (IPC), mannose-inositol-phosphorylceramide (MIPC), and mannose(inositol-P)2-ceramide (MIP2C), localized mainly to the plasma membrane where they constitute 20–30% of all the lipids (Patton and Lester, 1991). Their biosynthesis begins in the endoplasmic reticulum (ER) and is completed in the Golgi apparatus (Dickson et al., 2006). Another distinct characteristic of the lipid composition of a normal yeast cell is that instead of cholesterol, it contains approximately 75% ergosterol (ergosta-5,7,22-trienol) and a variety of other sterols, most of which are intermediates in the sterol biosynthesis pathway (Munn et al., 1999). Most of the ergosterol is found in the plasma membrane and many of the other sterol molecules are enriched in the ER where sterol synthesis occurs (Zinser et al., 1993). Sterols can be esterified and stored in lipid bodies from where they can be rapidly mobilized (Wagner et al., 2009). Although the major lipids and the biosynthetic machineries in S. cerevisiae have been extensively characterized (Daum et al., 1998; Ejsing et al., 2009; Guan and Wenk, 2006), novel chemical entities may have escaped discovery in previous analyses and warrants development of high resolution and sensitive methodologies for detection and characterization. Furthermore, the molecular signature of individual lipids is increasingly recognized to encode for highly specific, though not necessarily unique, functions. This has been further supported by recent revelations that the intricate interactions between specific lipids and proteins are important for cellular functions (Guan et al., 2009; Valachovic et al., 2004). High resolution analysis of lipids therefore is critical for enhancing our understanding of the biological roles of lipids. Traditional methods of lipid analysis include metabolic labeling, thinlayer chromatography (TLC) and gas chromatography (GC) (Wenk, 2005). Metabolic labeling using lipid precursors (such as radiolabeled inositol, ethanolamine, or fatty acyls) have been widely used to (selectively) label certain classes of lipids, which will typically be separated by TLC and visualized by autoradiography. However, these techniques only deliver mass levels of lipids under conditions of steady-state incorporation of the label. Moreover, generally, TLC separation is of low resolution and does not provide molecular information of the diversity of fatty acyl compositions, which would require subsequent analysis, often by GC. Nonetheless, although these methods suffer from limited sensitivity, selectivity, and resolution, they are still commonly used today due to the relative ease and low investment cost in instrumentation. In the last 15–20 years, the field of lipid research has been spurred by the development of mass spectrometry (MS), and in particular soft ionization such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). These methods have allowed rapid and sensitive profiling, characterization as well as quantification of lipids in complex lipid mixtures (Han et al., 2008; Shui et al., 2007). However, despite

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continuous advancement in the technology, such as achieving enhanced sensitivity by microfluidics-based ionization (Han et al., 2008) and high resolution by MS such as Fourier transform ion cyclotron resonance (FTICR) or Orbitrap MS (Schwudke et al., 2007), there is no one method to probe the entire lipidome of a cell. S. cerevisiae has a relatively simple lipidome compared to other eukaryotic cells, yet it is only recently that a fairly comprehensive analysis of its lipidome has been described, and such a detailed catalogue is still currently unavailable for the relatively more complex lipidomes of cell types in other organisms. Ejsing and coworkers described the absolute quantification of 250 lipid molecular species in S. cerevisiae and estimated that the coverage of the lipids analyzed encompasses 95% of the yeast lipidome (Ejsing et al., 2009). Failure in the complete annotation and measurement of an entire cellular lipidome is attributed to the complex chemistry between different classes of lipids as well as the dynamic range of their concentration, which limits their extractability and ionization using a single method. In other words, depending on the lipid(s) of interest, employing multiple extraction protocols, as well as different analytical tools are still required to examine the lipidome for any given biological sample. In this chapter, we describe the extraction and mass spectrometric analysis of various classes of lipids, including glycerophospholipids, sphingolipids, and sterols from yeast, specifically S. cerevisiae. Important considerations for isolation of lipids from the cellular milieu include recovery and potential activation of lipases during the extraction procedure. While protocols based on Folch and Bligh and Dyer methods (Bligh and Dyer, 1959; Folch et al., 1957) are seemingly efficient for isolating lipids from mammalian cells and tissues, these methods are effective for isolating glycerophospholipids and sphingolipids from broken yeast cells but not from intact cells (Hanson and Lester, 1980). Furthermore, a technical challenge is recovery of the entire cellular lipidome due to the diverse chemistry of lipids in cells. Polarity of lipids differ depending on the lipid backbone as well as the head group modification and therefore the selection of organic solvents is critical. For instance, phosphorylation of sphingolipids renders them highly polar, and thus they may escape into the aqueous milieu during phase separation using Folch or Bligh and Dyer’s methods. Here, we describe a modified protocol for isolation of lipids from intact yeast cells based on Angus and Lester’s method (Angus and Lester, 1972). This involves the use of a slightly alkaline mixture of ethanol–diethyl ether–water–pyridine–ammonium hydroxide at elevated temperatures, which leads to effective extraction of inositolcontaining lipids and glycerophosphatidylcholine from intact S. cerevisiae and Neurospora crassa (Hanson and Lester, 1980). However, for analysis of subcellular organelles, the modified Bligh and Dyer method may be applicable. An additional isolation method will be added to analyze total sterols, including an alkaline hydrolysis and extraction with petroleum ether.

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Various MS-based methods will be described for the analysis of the different classes of lipids, including (1) the direct analysis of major yeast glycerophospholipids and sphingolipids in complex lipid mixtures using ESI-MS and tandem mass spectrometry (MS/MS) and (2) analysis of sterols and metabolites by GC–MS (Fig. 15.2). Lipid analysis of yeast using ESI-MS is not a novel technique and dates back to the 1990s (Hechtberger et al., 1994; Schneiter et al., 1999) but comprehensive characterization and quantification of multiple classes of lipids in an ‘‘omic-centric’’ fashion has only been reported in recent years (Ejsing et al., 2006, 2009; Guan and Wenk, 2006). For successful MS analysis, often, prior knowledge of the chemistry and the fragmentation patterns of lipids of interest is required. Numerous works have been reported, particularly for the analysis of glycerophospholipids and sphingolipids in mammalian cells and tissues (Brugger et al., 1997; Han and Gross, 2005; Sullards, 2000; Taguchi et al., 2005) and inference can be drawn for lipid analysis in yeast. Moreover, often instruments have accompanying software that allow automated acquisition (termed datadependent acquisition) (Schwudke et al., 2006). Despite the wealth of information available and the ease of automation, we nonetheless aim to provide a detailed description of the operation to allow the reader to reproduce the techniques in probing yeast lipidomes as it should be noted that the lipid inventory depends on culture conditions (Tuller et al., 1999) and further differs between yeast species ( Jungnickel et al., 2005), for example, Schizosaccharomyces pombe versus S. cerevisiae (Shui et al., under revision). Previous methods have been published to analyze yeast sterols by GC with or without MS (van den Hazel et al., 1999; Xu and Nes, 1988; Zinser et al., 1991). These methods usually involve silylation of the isolated sterol sample before GC, which improves separation, but which also has some disadvantages. In this chapter, we will present a simple protocol for the semiquantitative analysis of free and total yeast sterols by GC–MS. The term semiquantitative is used because appropriate standards are not commercially available for the large variety of sterols present in wild-type (WT) and mutant yeast cells and therefore, their exact quantities cannot be measured by GC–MS.

2. Methods 2.1. Sample preparation Cells (10 OD600 units) are harvested by addition of trichloroacetic acid (TCA) to the media to a final concentration of 5% (w/v) and the cells are put onto ice. The cooled cells are pelleted by centrifugation at 3000
g for 5 min and the pellets are washed once with ice-cold 5% TCA, followed by

Single stage MS

General lipid extraction (glycerophospholipids and sphingolipids) Biological sample (yeast cell pellet)

PIS Direct infusion

General lipid profile (GPL and SPL)

Characterization

ESI-MS and MS/MS NL; PREIS

Lipid classspecific profile

MRM

Sterol isolation

Quantitative analysis of individual lipid species

Solid phase extraction for separation of sterols from other lipids (optional) GC-MS

Figure 15.2 General strategy for the extraction and analysis of major membrane lipids from yeast. Glycerophospholipids, sphingolipids, and sterols are extracted according to respective procedures. The former are analyzed by ESI-MS using various scan modes including single stage MS, product ion scan (PIS), precursor ion scan (PREIS), neutral loss (NL) scan, and multiple-reaction monitoring (MRM) for profiling, species identification, and quantification. Sterols are analyzed by GC–MS.

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another wash with ice-cold deionized water. The cells are transferred to screw cap tubes and they can be snap-frozen in liquid nitrogen and stored at 80  C or immediately used for extraction. Except for Teflon, all plastic should be avoided in subsequent procedures involving organic solvents because they leach contaminants into the solution. Furthermore, handling of organic solvents should be carried out in a fume hood.

2.2. Normalization for starting amount of material Two parameters can be used for normalizing the amount of lipids: (1) Dry weight: In this case, cells are freeze-dried and weighed before extraction. (2) Protein amount: An equivalent OD of cells used for lipid extraction is used for protein determination using standard assays.

2.3. Lipid extraction (for glycerophospholipids and sphingolipids) Cells (10 OD600 units) are resuspended in 1 ml of extraction solvent containing 95% ethanol, water, diethyl ether, pyridine, and 4.2 N ammonium hydroxide in the ratio of 15:15:5:1:0.18 by volume. A cocktail of lipid standards (Table 15.1) and 100mL of glass beads are added. It should be noted that lipid materials removed from storage should be allowed to reach room temperature before opening for use. In addition, the standards are stored as concentrated stock (mM to M) for stability, and are diluted in ethanol prior to use (Moore et al., 2007). We routinely sonicate the lipids to ensure complete solubilization. The sample is vortexed vigorously for several minutes and incubated at 60  C for 20 min. Cell debris are pelleted by centrifugation at 10,000
g for 10 min and the supernatant is collected. One milliliter of extraction solvent Table 15.1 List of synthetic standards for quantitative analysis of major yeast lipids

Standard

Source

MRM transition Ionization mode (Q1/Q3)

Didodecanoyl GPEtn Didodecanoyl GPSer Dioctanoyl GPIns d18:1/19:0 Ceramide Dodecanoyl, tridecanoyl GPA Didecanoyl GPGro Dinonadecanoyl GPCho Cholesterol

Avanti Avanti Echelon Matreya Avanti Avanti Avanti Avanti

Negative Negative Negative Positive Negative Negative Positive Negative

578.6/196.1 622.6/535.5 585.5/241.1 580.7/264.2 549.5/153.1 553.6/153.1 818.7/184.1 386.3/368.3

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is added to the pellet, which is vortexed again, and the extraction is repeated. The supernatant is pooled together and dried under a stream of nitrogen or in a Centrivap (Labconco Corporation, Kansas City, MO). In the event of using a Centrivap, the temperature should be raised gradually to 60  C to avoid boiling. For complex lipids including glycerophospholipids, sphingolipids, and neutral lipids, the extract is desalted using butanol and water. The lipid film is resuspended in 300 ml of water-saturated butanol and 150 ml of water is added. The mixture is vortexed vigorously, followed by centrifugation at 10,000
g for 2 min. The top butanol phase is recovered and the aqueous phase is reextracted again with 300 ml of water-saturated butanol. The butanolic phase is pooled together and dried under a stream of nitrogen or with a Centrivap (50–60  C). The lipid film is resuspended in 400 ml of chloroform–methanol (1:1, v/v) for MS analysis. 2.3.1. Alkaline methanolysis (optional) For analysis of ceramides and complex sphingolipids, an optional step of mild alkaline hydrolysis is recommended to reduce ion suppression by other lipids such as glycerophospholipids during MS analysis. This treatment hydrolyzes acyl bonds (and hence the majority of glycerophospholipids) but leaves amide linkages largely intact (Brockerhoff, 1963). Two different methods are available. In the first method, the lipid film from the pyridine extraction is reconstituted in 400 ml of a mixture of chloroform, methanol, and water in the ratio of 16:16:5 by volume. Four hundred microliters of 0.2 N of methanolic NaOH is added and the mixture is incubated for 1 h at 37  C on a thermoshaker with mild shaking. Eighty microliters of 1 N acetic acid is then added to neutralize the mixture, followed by the addition of 400 ml of 0.5 M EDTA and 400 ml of chloroform. The sample is vortexed vigorously for 1 min, followed by centrifugation at 10,000
g for 2 min. The lower organic phase is recovered and the aqueous phase is reextracted with 600 ml of chloroform. The organic phase is pooled together and dried under a stream of nitrogen or in the Centrivap (Guan and Wenk, 2006). In the second method, the lipid film is reconstituted in 400 ml of monomethylamine reagent (methanol:H2O:n-butanol:methylamine ¼ 4:3:1:5 by volume) and incubated for 1 h at 53  C (Cheng et al., 2001). The mixture is then dried under a stream of nitrogen or in a Centrivap (50–60  C). The lipid extract is then desalted using butanol and water as described above.

2.4. Sterol isolation In order to quantify free, nonesterified sterols, the procedure used for the isolation of glycerophospholipids and sphingolipids cannot be used because it results in partial hydrolysis of sterol esters. Therefore, cells are extracted with a chloroform/methanol procedure. Cells (10 OD600 units) are

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harvested as above. Cholesterol (2 nmol) and 100 ml glass beads are added to the cell pellet, which is resuspended in 50 ml water and 50 ml methanol is added. After vortexing, 100 ml of chloroform is added and the sample is vortexed for 6 min followed by centrifugation at 100
g for 5 min. The supernatant is transferred to a new tube and the pellet is vortexed again with 100 ml of chloroform/methanol (2:1). After centrifugation the supernatants are combined. Thirty-four microliters of 0.034% MgCl2 is added, vortexed, and centrifuged at 800
g for 5 min. The aqueous phase is removed and 34 ml of 2 M KCl/methanol (4:1) is added to the lower phase. After vortexing and centrifugation the aqueous phase is removed. This procedure is repeated two times with 34 ml of artificial upper phase (chloroform: methanol:water, 3:48:47). The organic phase is then removed without taking the protein interface and dried. Sterol esters are present in this preparation, but are difficult to quantify by GC–MS. To determine the amounts of total cellular sterols (esterified and nonesterified) the following protocol is used. Two nanomoles of cholesterol are added to the cell pellet as internal standard. Cells are resuspended with 300 ml 60% KOH to which is added 300 ml methanol containing 0.5% pyrogallol and 450 ml methanol in a screw cap glass tube. The tube is placed at 85  C and once hot, the cap is tightly closed. After 2-h of incubation with occasional mixing, the sample is returned to room temperature and the sterols are extracted three times with 1 mL of petroleum ether. The combined petroleum ether phases are dried and analyzed by GC–MS. 2.4.1. Solid phase extraction for separation of sterols from other lipids (optional) A column of ChromabondÒ SiOH (Macherey-Nagel, Germany) (0.1 g) is washed with two times 1 ml chloroform. Total lipid extract from 50 OD600 units of cells is resuspended in 0.25 ml chloroform by vortexing and sonication. The extract is then applied to the column and eluted with two times 0.65 ml chloroform. The flow through and chloroform elutions are combined and dried (sterol extract). The column is then eluted with three times 0.5 ml methanol. The combined methanol elutions are dried and can be used as a total lipid extract as above.

2.5. Lipid analysis 2.5.1. Direct analysis of glycerophospholipids and sphingolipids in complex lipid mixtures by ESI-MS and ESI-MS/MS Glycerophospholipids, ceramides, and inositol sphingolipids are analyzed by direct infusion using ESI-MS and MS/MS (Fig. 15.3). The samples from the butanolic extraction or after alkaline hydrolysis are reconstituted in 400 ml of chloroform–methanol (1:1, v/v). Samples are centrifuged at maximum speed for 10 min to remove any residues. A high-performance

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MS1

34:1 GPIns 835

A

MS2 CID

Single stage MS

m/z 400 - 1200 32:1 GPIns 807 28:0 GPIns 753

34:2 GPEtn 714 500

B

O

HO HO HO

O

P − O 241

600

700

800 m/z

281

255

36:1 GPIns 863 t18:0/26:0 IPC-C 952 900

1100

1000 MS1

MS2

O OH

CID m/z 835

O O P O O

−

OH

153

100

200

34:1 GPIns 835

391

300

400

553 500 600 m/z

700

Tandem mass spectrometry

800

900

34:1 GPIns 835

C 32:1 GPIns 807

500

600

700

m/z 241

t18:0/26:0 IPC-C 952 800 m/z

900

1000

1100

MS1

D

MS2 CID

30,000

m/z 241

m/z 833

25,000 Intensity

MS2 CID

m/z 400 - 1200

26:0 GPIns 725

16:0 GPIns 571

MS1

Data integration

20,000 15,000 10,000 5000 0

0

30

60

90

0

60

120

180

240

300

Time (s) pmoles/mL 34:2 GPIns

Figure 15.3 Analysis of yeast lipids by electrospray ionization–mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). (A) Single stage ESI-MS in the negative ion mode. The majority of glycerophospholipids and sphingolipids are detected in the mass range of 400–1200. The ions can be tentatively assigned by their mass-to-charge (m/z) ratio. Characterization of ions can be achieved by collisioninduced dissociation (CID) and MS/MS. (B) MS/MS spectra of ions with m/z 835. (C) Precursor ion scans for lipids containing inositol phosphate head group (m/z 241). Samples can be spiked with internal standards, which are typically not found naturally in the samples under investigation, to allow for semiquantitative profiling. (D) Overlay of chromatogram (left panel) and standard curve (right panel) obtained from

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liquid chromatography system (Agilent Technologies, Santa Clara, CA) is coupled to a 4000 Qtrap triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) with an ESI source. The mass spectrometer is operated in both the positive and negative ion modes, depending on the propensity of the class of lipids of interest to ionize (Table 15.2). The spray voltage in the positive ion mode is 5.5 and 4.5 kV in the negative mode. The MS is operated with a curtain gas of 20 psi, source temperature of 250  C and both ion source gas 1 and 2 set to 30 psi. For all MS/MS experiments, each individual ion dissociation pathway has to be optimized with regard to collision energy and declustering potential to minimize variations in relative ion abundance due to differences in rates of dissociation (Table 15.3 and Fig. 15.5). Samples are introduced into the mass spectrometer by loop injections with chloroform–methanol (1:1 v/v) as a mobile phase at a flow rate of 200 ml/min. Typically, 25 ml of samples is injected for analysis. 2.5.1.1. Single stage (nontargeted) profiling The mass spectrometer is operated in enhanced MS mode to obtain the profiles of the total lipid extract. Measurement of lipids by ESI-MS is based on the ability of each class of lipids to acquire positive or negative charges when in solution during ionization and the structures of these lipids entail their inherent ionization property. Table 15.2 summarizes the charge states of major glycerophospholipids and sphingolipids. An example of a single stage MS profile, in the negative ion mode, of an extract obtained from S. cerevisiae grown in rich media (yeast–peptone–dextrose) to logarithmic phase is shown in Fig. 15.3A. Typically, the scan range is between m/z 400 and 1200, which includes the detection of various classes of lipids including glycerophosphatidylethanolamine (GPEtn), glycerophosphatidylserine (GPSer), glycerophosphatidic acid (GPA), glycerophosphatidylglycerol (GPGro), glycerophosphatidylinositol (GPIns), ceramide, and inositol sphingolipids. The profiling of complex lipid mixtures using single stage MS may serve as an initial screen when different conditions or strains are to be compared. The comparison of profiles can be achieved by multivariate analysis or simply generating a differential profile which displays differences in ion response between the two conditions of interest and softwares for analysis, alignment, and comparison of full scan MS, are increasingly available (De Vos et al., 2007; Song et al., 2009; Wong et al., 2005). Such a nontargeted survey may serve as a starting point to reveal perturbations in the lipid quantification of varying concentrations of a commercially available 34:2 GPIns by multiple-reaction monitoring (MRM). Selective quantification can be attained with a reasonably good linearity. Note that 34:2 GPIns is a minor ion in the complex lipid mixture and MRM offers a selective and sensitive method for quantification.

380

Table 15.2 Precursor ions, MS/MS scan modes and associated parameters for analysis of major glycerophospholipids and sphingolipids in S. cerevisiae

Lipid

Precursor ion

Glycerophospholipid GPA [M  GPGro [M  [M þ GPCho [M þ GPEtn [M 

H] H] NH4]þ H]þ H]

PREIS 153 PREIS 153 NL 189 PREIS 184 PREIS 196

þ   þ   þ

H]þ H] H] H]þ H] H] NH4]þ

NL 141 NL 87 PREIS 153 NL 185 PREIS 241 PREIS 153 NL 277

GPSer

GPIns

[M [M [M [M [M [M [M

Mass range (m/z)

Declustering potential (V)

Collision energy (V)

Glycerophosphate derivative Glycerophosphate derivative Phosphoglycerol Phosphocholine Glycerophosphoethanolamine derivative Phosphoethanolamine Serine Glycerophosphate derivative Phosphoserine Cyclic inositol phosphate Glycerophosphate derivative Phosphoinositol

370–800 400–800

 75  75

 40 to  60  40 to  60

400–900 400–800

70  75

45–65  40 to  65

400–800 400–800

 75  75

 25  40 to  60

450–900 450–900

 90  75

 45 to  60  45 to  65

Double dehydration product of d18:0 sphingoid base Double dehydration product of t18:0 sphingoid base Double dehydration product of d20:0 sphingoid base Double dehydration product of t20:0 sphingoid base

600–800

80

40–50

80

40–50

100

40–50

100

40–50

MS/MS modes Fragment

Sphingolipid Phytoceramide [M þ H]þ

PREIS 266

[M þ H]þ

PREIS 282

[M þ H]þ

PREIS 294

[M þ H]þ

PREIS 310

IPC

MIPC

M(IP)2C

[M  H] [M þ H]þ

PREIS 241 PREIS 266

[M þ H]þ

PREIS 282

[M þ H]þ

PREIS 294

[M þ H]þ

PREIS 310

[M  H] [M þ H]þ

PREIS 421 PREIS 266

[M þ H]þ

PREIS 282

[M þ H]þ

PREIS 294

[M þ H]þ

PREIS 310

[M  2H]2

PREI 241

Cyclic inositol phosphate Double dehydration product of d18:0 sphingoid base Double dehydration product of t18:0 sphingoid base Double dehydration product of d20:0 sphingoid base Double dehydration product of t20:0 sphingoid base Cyclic inositol phosphate Double dehydration product of d18:0 sphingoid base Double dehydration product of t18:0 sphingoid base Double dehydration product of d20:0 sphingoid base Double dehydration product of t20:0 sphingoid base Cyclic inositol phosphate

800–1000

950–1200

600–750

 120 100

 60 to  70 60–70

100

60–70

100

60–70

100

60–70

 160 100

 60 to  70 75–80

100

75–80

100

75–80

100

75–80

 180

 65 to  75

381

Table 15.3 Precursor/product ion m/z’s for multiple-reaction monitoring of major species of S. cerevisiae glyerophospholipids GPA

GPGro

GPEtn

GPSer

GPIns

GPCho

Molecular species

Q1 [M  H]

Q3

Q1 [M  H]

Q3

Q1 [M þ H]þ

Q3

Q1 [M  H]

Q3

Q1 [M  H]

Q3

Q1 [M þ H]þ

Q3

14:0 (Lyso) 16:1 (Lyso) 16:0 (Lyso) 18:1 (Lyso) 18:0 (Lyso) 26:1 26:0 28:1 28:0 30:1 30:0 32:2 32:1 32:0 34:2 34:1 34:0 36:2 36:1 36:0

379.4 407.4 409.4 435.4 437.4 561.6 563.6 589.6 591.6 617.6 619.6 643.7 645.7 647.7 671.7 673.7 675.7 699.7 701.7 703.7

153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1

453.4 481.4 483.4 509.4 511.4 635.6 637.6 663.7 665.7 691.7 693.7 717.7 719.7 721.7 745.7 747.7 749.7 773.7 775.7 777.7

153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1 153.1

426.4 452.4 454.4 480.4 482.4 606.7 608.7 634.7 636.7 662.7 664.7 688.7 690.7 692.7 716.7 718.7 720.7 744.7 746.7 748.7

285.5 311.6 313.5 339.6 341.5 465.7 467.7 493.7 495.7 521.7 523.7 547.7 549.7 551.7 575.7 577.7 579.7 603.7 605.7 607.7

466.4 494.4 496.4 522.4 524.4 648.7 650.7 676.7 678.7 704.7 706.7 730.7 732.7 734.7 758.7 760.7 762.7 786.7 788.7 790.7

379.4 407.4 409.4 435.4 437.4 561.7 563.7 589.7 591.7 617.7 619.7 643.7 645.7 647.7 671.7 673.7 675.7 699.7 701.7 703.7

543.4 569.4 571.4 597.4 599.4 723.7 725.7 751.7 753.7 779.7 781.7 805.7 807.7 809.7 833.7 835.7 837.7 861.7 863.7 865.7

241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1 241.1

468.4 494.4 496.4 522.4 524.4 648.6 650.6 676.7 678.7 704.7 706.7 730.7 732.7 734.7 758.7 760.7 762.7 786.7 788.7 790.7

184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1 184.1

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profiles which cannot be easily anticipated (Guan et al., 2006). For instance, Fig. 15.4 represents profiles obtained from a total lipid and sphingolipid extract obtained from WT cells and cells deficient in the acyltransferase, Slc1p. Note in panel A that the signal of a prominent inositol-phosphorylceramide (IPC, m/z 952, asterisk) is substantially increased after alkaline hydrolysis.

835

A

Phytoceramide

807

IPC

MIPC

952 *

714

WT 710 686 742

427

863 400

600

800 m/z

B

952 * 1000

746 1200

500

600

700

968 1114

936

800 900 1000 1100 1200 m/z

807

835

714

710 952

686 742 427

746 569

400

800

1000

1200

500

0.5

569

686 770 807

924

0 −0.5

600

700

800

968 1114

900 1000 1100 1200

Δslc1

2 .0

1 Differential profile

Differential profile

C

600

924

427 545

632

952

−1 −1.5 400

725 600

682 726

762

924 968

0 936

−1.0

952

−2.0

753 800 m/z

1.0

1000

1200

500

600

700

800 900 1000 1100 1200 m/z

Figure 15.4 Glycerophospholipid and sphingolipid profiling of yeast mutants. (A) Typical phospholipid (left panels) and sphingolipid profiles (right panels) of wild-type (WT) yeast and (B) deletion mutants of the slc1 gene. Mass spectra from at least four independent samples were averaged for each condition (n ¼ 4). (C) Differential lipid profile, which are ratios of single stage mass spectrometry scans plotted as log 10 ratios, are used to compare differences in glycerophospholipid and sphingolipid composition between Dslc1 and WT. Note that this approach does not require knowledge of the underlying lipid species for a given ion of interest. Instead, it serves as an ‘‘unbiased’’ screening tool for discovery of lipids which are present in different amounts between two conditions. Reproduced and modified from Guan and Wenk (2006).

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The spectra are aligned and the differences between ion intensity between the two conditions (Dslc1 vs. wt) are computed to generate the differential profiles as shown in Fig. 15.4C. The major differences lie in ions at m/z 725 and 753, which, based on the mass, can tentatively be assigned to GPIns with a total fatty acyl carbon number of 26 and 0 double bonds (26:0-GPIns) as well as 28:0-GPIns. The precise identity of ions can be confirmed by MS/MS. A nontargeted profiling approach offers the advantage of discovering a previously uncharacterized lipid moiety or an unexpected lipid mediator under a given condition. However, the likelihood of missing a relevant lipid cannot be ruled out because of the incomplete detection of all lipid species due to the dynamic range and complex chemistries of lipids in a mixture, which is beyond the capacity of existing instrumentations. For instance, sterol is not detected under this condition and requires an alternative method (see Section 2.5.2). 2.5.1.2. Characterization by tandem mass spectrometry To characterize an ion of interest, the mass spectrometer is set to product ion scan (PIS) mode, in which the first quadrupole is set to transmit a selected precursor ion, for instance, m/z 835 in negative ion mode. These ions enter the second quadrupole, often referred as the collision cell, where they are fragmented by collisioninduced dissociation (CID) and the fragments are resolved by the third quadrupole which scans over a mass range, typically starting from m/z 70 to the m/z of the precursor ion selected. In this instance, CID of m/z 835 with collision energy of 55 V and declustering potential of 100 V yields fragments with m/z 153, 241, 255, and 281, which are dehydrated glycerophosphate ions, inositol phosphate ions, and decarboxylate ions of palmitic acid (16:0), and oleic acid (18:1), respectively (Fig. 15.3B). This identifies the ion to be a 34:1 GPIns. Public databases to facilitate interpretation of MS/MS data for a range of lipids are available, including LipidSearch (Taguchi et al., 2007), LipidMaps MS tool (Fahy et al., 2007), and LipidQA (Song et al., 2007). 2.5.1.3. Targeted profiling and semiquantitative analysis of lipids by class using precursor ion and neutral ion scans For selective detection and semiquantification of specific class of lipids, the mass spectrometer is operated in precursor ion (PREIS) or neutral loss (NL) scan. Table 15.2 summarizes the PREIS and NL scan modes for major glycerophospholipids and sphingolipids in S. cerevisiae. In the PREIS scan mode, the first quadrupole scans over a selected mass range and the ions sequentially enter the collision cell where collision energy is applied to induce fragmentation. The third quadrupole is set to transmit a selected single product ion, which is typically the diagnostic fragment of a specific class of lipid. For instance, a PREIS of m/z 241 in the negative mode scans ions with a phosphoinositol group which results in a mass spectrum that contains all precursor ions that decompose to produce the fragment with m/z 241 and in yeast, this

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385

generates a profile of GPIns, IPC, as well as doubly charged ions of M(IP)2C (Fig. 15.3C). It should however be noted that a 15:0 fatty acyl shares the same m/z as the dehydrated phosphoinositol fragment and such data should be handled with care. Odd chain fatty acids are uncommon in S. cerevisiae cultured in rich media, and therefore the fragment of m/z 241 is considered specific for phosphoinositol-containing lipids. An NL scan is used to profile several classes of glycerophospholipids when the charge of the head group does not localize to the lipid head group after fragmentation. In this mode, the first and third quadrupoles are linked and scanned at the same speed over the same mass range with a constant mass difference, for instance 87 amu in negative mode for the loss of serine, between the two analyzers. Because of the mass offset at any time, the third quadrupole will transmit product ions with a fixed lower m/z value than the mass selected precursor ions transmitted by the first quadrupole. The resultant mass spectrum contains all the precursor ions that lose a neutral species of selected mass, in the case of an NL of 87 amu, a glycerophosphatidylserine profile is generated. 2.5.1.4. Quantitative analysis by multiple-reaction monitoring Multiplereaction monitoring (MRM) is a highly selective and sensitive method for quantification of specific lipids of interest. Quantitative analysis requires the use of internal standards, which are typically stable-isotope incorporated lipids or unnatural lipids which are usually chemically synthesized (Table 15.1). In MRM experiments, the first quadrupole is set to pass the precursor ion of interest to the collision cell where it undergoes CID. The third quadruple, Q3, is set to pass the structure-specific product ion characteristic of the lipid of interest. Again, these transitions require user-defined input of the parent ion (Q1) and product ion (Q3). Figure 15.5 and Table 15.3 summarize the MRM transitions for several major yeast sphingolipids and glycerophospholipids for quantification of these lipids in our previous work (Guan et al., 2009; Guan and Wenk, 2006). A chromatogram is generated (Fig. 15.3D) and the area under each curve is integrated for each lipids measured. Lipid concentrations are calculated relative to the relevant internal standards which have been spiked in based on the amount of starting material and is expressed as moles (M)/mg dry weight or M/mg protein. It should be noted that the absolute quantification of inositol-containing lipids was limited by the availability of pertinent standards but such is not the case with the production of nonnatural lipids by genetic manipulation of yeast strains (Ejsing et al., 2009).

2.5.2. Sterol analysis by GC–MS Extracts are analyzed by GC–MS as follows. Samples are injected into a VARIAN CP-3800 gas chromatograph equipped with a Factor Four Capillary Column VF-5ms 15 m
0.32 mm i.d. DF ¼ 0.10 and analyzed by a

Serine + palmitoyl-CoA

Endoplasmic reticulum

Lcb1p/Lcb2p/Tsc3p

Serine palmitoyltransferase 3-ketodihydrosphingosine (KDS)

Tsc10p

OH

3-KDS reductase

HO NH2 OH OH

Sur2p

Dihydrosphingosine hydroxylase

HO NH2 OH OH

Lag1p/Lac1p

Ceramide synthase

HO HN O

Scs7p

OH OH

Ceramide hydroxylase

HO HN O OH

Golgi

Aur1p

OH OH O HO OH HO O P O HO – OH O HN O OH

Csh1p/Csg1p/Csg2p OH HO OH O OH O P O O HO – HO HO HN OH O O OH HO O OH Ipt1p HO HO HO

OH

–

O HO O O PHO OH O

OH OH O HO OH O O PO HO – O OH HN O OH OH O

IPC synthase

Mannosyltransferase

Inositolphosphotransferase

Species Sphingoid base Dihydrosphingosine d18:0 d20:0 Phytosphingosine t18:0 t20:0 Phytoceramide (sphingoid base/fatty acid-type) d18:0/18:0-A t18:0/18:0-B t18:0/24:0-C t18:0/26:0-B t18:0/26:0-C t18:0/26:0-D t20:0/26:0-B t20:0/26:0-C t20:0/26:0-D Inositol phosphorylceramide (IPC) t18:0/24:0-C t18:0/26:0-B t18:0/26:0-C t18:0/26:0-D t20:0/26:0-B t20:0/26:0-C Mannosyl inositol Phosphorylceramide (MIPC) t18:0/26:0-B t18:0/26:0-C t20:0/26:0-B t20:0/26:0-C t20:0/26:0-D Mannosyl diinositol Phosphorylceramide (M(IP)2C) t18:0/26:0-B t18:0/26:0-C Internal standard d18:1/19:0 ceramide d18:1/8:0 glucosylceramide

Precursor/ production m/z

DP (eV)CE (eV)

302.2/284.2 330.2/312.3 318.3/282.3 346.3/310.3

568.6/284.3 584.6/282.3 684.7/282.3 696.7/282.3 712.7/282.3 728.7/282.3 724.7/310.3 740.8/310.3 756.8/310.3

60 80 80 80 80 80 100 100 100

40 40 45 45 45 50 45 45 50

926.7/282.3 938.7/282.3 954.7/282.3 970.7/282.3 966.8/310.3 982.8/310.3

80 80 100 100 80 80

60 65 65 70 70 70

1100.8/282.3 1116.8/282.3 1128.8/310.3 1144.8/310.3 1160.8/310.3

100 100 100 100 100

75 80 80 80 80

670.2/241.0 678.2/241.0

–180 –180

–75 –75

580.6/282.3 588.6/282.3

45 45

35 45

Figure 15.5 Summary of sphingolipid pathway of S. cerevisiae and precursor/product ion m/z’s and associated parameters for MRM detection of individual molecular species of sphingoid bases and complex ceramides. Reproduced and modified from Guan and Wenk (2006). Abbreviations: DP, declustering potential; CE, collision energy.

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Varian 320 MS triple quadrupole with electron energy set to 70 eV at 250  C. Samples are applied with the column oven at 45  C, held for 4 min, then raised to 195  C (20  C/min). Sterols are eluted with a linear gradient from 195 to 230  C (4  C/min), followed by raising to 320  C (10  C/min). Finally, the column temperature is raised to 350  C (6  C/min) to elute sterol esters. This protocol allows sufficient separation of sterols as judged by elution of standards and extracts from ergosterol biosynthesis mutants of known sterol composition. An example of a chromatogram of a sample of WT yeast is shown (Fig. 15.6) and the profiles of a crude extract and one purified over SPE are compared. Standard curves can be constructed by

CE total ions

CE 396

CE 386

SPE total ions

SPE 396

SPE 386

10

15 Minutes 20

25

Figure 15.6 Use of SPE separation for GC–MS analysis of sterols. Total lipid extracts (top three panels) or the chloroform eluate from an SPE column of the same extract were analyzed by GC–MS as described. The total ion intensity or intensities of the 396 (ergosterol) or 386 (cholesterol standard) are shown. Separation by SPE eliminates the peaks appearing at around 25 min, which are products of glycerophospholipids and other substances and slightly improves the resolution in the region where sterols are found (15–20 min).

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WT total ions

A

WT 396

erg2 total ions

B

erg2 396

16 100%

17

A

75% 50% 25%

18 Minutes

19 363.3 1.992e+7

143.0 1.538e+7

81.0 7.320e+6 97.0 1.861e+6

253.2 1.279e+7 211.1 171.0 1.038e+7 8.048e+6 130.9 271.2 5.875e+6 173.1 213.1 3.575e+6 2.400e+6 3.515e+6

396.3 1.257e+7 364.4 5.770e+6

0%

397.5 3.615e+6

Ergosterol CAS No. 57-87-4, C28H44O, MW 396 363.0 811 396.0 695

100% 75% 253.0 143.0 501 432 211.0 119.0 271.0 171.0 287 255 246 147.0 214 195.0 237.0 123.0 146 269.0 114 87 88 65

81.0 473 83.0 296 25% 77.0 103 0% 50%

100 100%

20

200

337.0 332 364.0 238 338.0 128 365.0 67 300

363.3 9.886e+6

B 157.0 6.065e+6

75% 50%

211.1 171.0 3.277e+6 2.498e+6

118.9 2.161e+6

25%

253.2 4.156e+6

HO 397.0 219

400

m/z

364.4 2.744e+6 396.3 2.051e+6

254.3 953929

0%

Ergosta-5, 8, 22-trien-3-ol, (3.beta., 22E) CAS No.50657-31-3, C28H44O, MW 396

100%

363.0 999

75% 50% 25%

81.0 280 79.0 120

143.0 370 119.0 147.0 171.0 180 180 150

0% 100

150

364.0 330

253.0 211.0 300 200 237.0 269.0 100 60 200

250

337.0 100 300

365.0 70

350

396.0 450 HO

397.0 170 400

450 m/z

Figure 15.7 GC–MS analysis of wild-type (WT) and erg2 mutant strain. The portion of the GC–MS profile of the WT and erg2 mutant strains where sterols elute is shown. The major ion at 396 is ergosta-5,7,22-trienol in WT (A), as seen by its fragmentation pattern (panel A) compared to the pattern found in the NIST library. Exiting the GC column 0.5 min earlier is the major ion at 396 in the erg2 mutant, ergosta-5,8,22-trienol (B), whose fragmentation pattern is very similar, but not identical to ergosterol (panels B), also similar to the profile from the NIST library. Fragmentation occurs in the source.

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extracting data for the relevant ions for known amounts of cholesterol (386) and ergosterol (396). Compounds are identified by their retention times (compared to standards) and fragmentation patterns, which are compared to the NIST library or previously characterized sterols from WT and mutant yeast cells. Most stereoisomers can be separated by this method. In general, stereoisomers with a double bond at position 8 elute before the corresponding isomer with a double bond at position 7 (Fig. 15.7). It is important to pay attention to the ion used for quantification because the major ions may not be of similar intensities for different sterols. For example, the 396 ion for ergosterol is quite a bit more intense than the same ion for ergosta-5,8,22-trienol. To compare these sterols it is best to use a unique, but more representative ion (see Fig. 15.7, e.g., 363) or total ion counts, if sterols are sufficiently well separated. We have recently published data on the major sterols found in five isogenic ergosterol biosynthesis mutants (Guan et al., 2009) and these strains can be used as sources to determine the retention times of yeast sterols that are not commercially available.

ACKNOWLEDGMENTS Research in the laboratory of Markus Wenk is supported in part by the Singapore National Research Foundation under CRP Award No. 2007-04, the Biomedical Research Council of Singapore (R-183-000-211-305), the National Medical Research Council (R-183-000224-213), and the Swiss SystemsX.ch initiative, evaluated by the Swiss National Science Foundation. Howard Riezman is funded by the Swiss National Science Foundation, the Human Frontiers Science Program Organization, the Swiss SystemsX.ch initiative, evaluated by the Swiss National Science Foundation, and the University of Geneva. We thank Karl Abele at Varian AG, Basel, for help in setting up sterol analysis.

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