Article pubs.acs.org/ac
Comprehensive and Quantitative Analysis of Polyphosphoinositide Species by Shotgun Lipidomics Revealed Their Alterations in db/db Mouse Brain Chunyan Wang,† Juan Pablo Palavicini,† Miao Wang,† Linyuan Chen,† Kui Yang,‡ Peter A. Crawford,† and Xianlin Han*,† †
Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, Florida 32827, United States ‡ Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, United States S Supporting Information *
ABSTRACT: Polyphosphoinositides (PPI) play crucial roles in cellular signaling and functions. However, comprehensively determining the changed levels of these species during different cellular processes has faced difficulties. Herein, we applied a novel methylation pattern recognition and simulation approach, and we exploited newly derived fragmentation patterns of methylated PPI species for comprehensive analysis of PPI species including phosphate position(s) and fatty acyl chains capable of circumpassing previous limitations. The developed method was applied for quantitative analysis of PPI species present in diabetic mouse cortex and liver, and it allowed us to unravel the marked reduction of PPI levels in brain cortices of db/db mice for the first time. Taken together, we developed a powerful and high-throughput method for comprehensive analysis of PPI species, which should greatly contribute to the elucidation of PPI biology under different disease states.
P
With the development of lipidomics, a variety of electrospray ionization-mass spectrometry (ESI-MS)-based methods for the analysis of PPI species have been developed.13−18 Wenk et al. analyzed PPI by MS/MS in the precursor-ion scan (PIS) mode targeted to inositol phosphate fragment ions after an enrichment of these lipids with an affinity SPE column.13 The method was labor intensive and in relatively low sensitivity. Milne et al. determined PPI species after three-step extraction.14 The method did not absolutely quantify PPI levels. Haag et al. developed a method based on the neutral loss scans (NLS) of ammoniated inositol phosphate from their ammoniated molecular species after direct infusion.17 The method did not differentiate isomers from different PIP and PIP2 classes. Clark et al. derivatized PPI species from extracts with trimethylsilyldiazomethane (TMS-diazomethane) and analyzed methylated PIP3 species by LC-MS/MS.15 The same group also extended the method to analyze other PPI classes.18 The approach significantly enhanced the analysis sensitivity and showed promising results for the analysis of PPI species. However, this approach is relatively time-consuming, because it requires running NLS of methylated inositol phosphate head groups first in order to identify the presence of particular PPI species and accurately quantify them by multiple-reaction-monitoring (MRM)-based MS. Moreover, the method was unable to totally
olyphosphoinositide (PPI) species are a category of cellular membrane lipids that are dynamically phosphorylated/dephosphorylated from phosphatidylinositol (PI) and its phospho-derivatives at the positions 3, 4, and/or 5 of the inositol ring through different kinases and phosphatases to generate seven distinct PPI classes, including PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3.1−4 They are present in nearly all cellular membranes, but in low abundance, and predominantly reside on the cytoplasmic side. Previous studies have shown that PPI species are classspecifically synthesized and located at different subcellular membranes.5 Therefore, some PPI classes may be used as organelle markers. PPI species or their hydrolysis products play pivotal roles in numerous cellular processes, including membrane trafficking, cell growth, survival, and motility.2,3,5−7 Aberrant PPI signaling is associated with numerous human diseases, such as cancer, neurological disorders, diabetes, and cardiovascular dysfunction.1,2,6,8,9 Although the importance of PPI species in biological processes has been well-recognized, determining their precise structures and accurate cellular levels remains a major challenge. This is largely due to their low abundance and high polarity, which makes their recovery from biological samples more difficult relative to the majority of other membrane lipids. Classic methods including TLC, HPLC, receptor displacement assay, and radioactive labeling10−12 are usually laborious and do not provide information about fatty acyl (FA) composition. © 2016 American Chemical Society
Received: July 31, 2016 Accepted: November 16, 2016 Published: November 16, 2016 12137
DOI: 10.1021/acs.analchem.6b02947 Anal. Chem. 2016, 88, 12137−12144
Article
Analytical Chemistry
specified) was added into the eppendorf tube (2 mL) containing a lipid extract in approximately 1 mL of organic phase as described above. The tube was capped, gently shaken shortly (one or two strokes), and reopened immediately to release fume in the fume hood for a few seconds. Then the tube was recapped and further shaken for 20 min or as indicated at room temperature. The reaction was quenched by adding 5 μL of glacial acetic acid. Recovery of the derivatives was performed by adding 500 μL of MTBE/MeOH/H2O (20:6:5, v/v/v) to the reaction vessel and followed by vortexing for 1 min and centrifuging at 1500 rpm for 2 min at 4 °C. The upper organic phase (∼800 μL) was collected into an Eppendorf tube. The wash step was repeated once as described above. The final upper phase (∼750 μL) was collected into a new 6 mL conical glass tube, dried under a N2 stream, and resuspended in 80− 100 μL chloroform/methanol (1:1, v/v) for MS analysis. MS Analysis of Derivatized PPI Species. Methylated PPI species (at the concentration specified in the legends of figures under different experimental conditions) were diluted 20−50fold using a mixture of chloroform/methanol/isopropanol (1:2:4, v/v/v) containing of 2000-fold diluted saturated LiCl methanol solution or 5 mM ammonium acetate, respectively. Mass spectra were acquired as described in the Supporting Information. Experimental sequences were programmed and controlled as described.20,27 Quantification of individual PIP, PIP2, or PIP3 ion was then conducted by summarizing the ion intensities of all ions of each species containing different numbers of methyl groups relative to the summarized ion intensity of the corresponding IS after correction for 13C isotope effects.28,29 Phosphate positional isomers of individual PIP or PIP2 ion were resolved through simulation of the methylation pattern of the ion with corresponding methylation patterns of individual isomer standards similar to the simulation method described.30 Data Processing and Analysis. All mass spectral data were automatically acquired by a customized sequence subroutine operated under Xcalibur software.20 Data processing were conducted as described20 based on the principles of shotgun lipidomics such as selective ionization, low concentration of lipid solution, an appropriate number of internal standards, two-step quantification, and correction for differential kinetics of fragmentation.28,31 All data are presented as the means ± SEM of five separate animals. Statistical significance was determined by a two-tailed Student t-test relative to control, where *p < 0.05, **p < 0.01, and ***p < 0.001.
resolve and quantify all PPI species carrying different fatty acyl chains and phosphate positional isomers. Recently, the methylation method has been exploited by shotgun lipidomics for relative quantification of comparable samples with and without stable isotope methyl labeling.16,22 This approach is limited to relative comparison of two groups of samples in the current setting without identification of PPI isomers. Collectively, absolute quantification of methylated PPI species including isomers with different phosphate positions and FA chains in a high-throughput manner for broad applications remains elusive. To resolve these obstacles for comprehensive analysis of PPI species, we exploited the lithium adducts of methylated PPI species in the positive-ion mode and the demethylated ions from their chlorine adducts in the negative-ion mode, respectively, to achieve a comprehensive analysis of individual PPI isomers in the presence of internal standards (IS). Moreover, the developed method was applied for identification and absolute quantification of PPI isomers present in the liver and brain cortex of diabetic mice. We revealed significant reduction of PPI species in the brain for the first time. The application clearly demonstrated the power of the method for analysis of PPI species. We believe that the developed method should greatly facilitate the elucidation of PPI biology under disease states.
■
MATERIALS AND METHODS Materials. The standard PPI species utilized were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). TMS-diazomethane (2.0 M in hexane) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). All solvents used for lipid extraction and sample preparation were obtained from Burdick and Jackson (Muskegon, MI). Other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Animals. Homozygous diabetic (db/db) mice and nondiabetic controls of the C57BL/6 strain (male, 4 months of age, 5 animals per group) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed with 12-h light/dark cycles at 20−22 °C, and were fed with standard rodent chow diet and water ad libitum. Animals were euthanized by asphyxiation with CO2 followed by decapitation. All tissue samples were prepared as described23 and stored at −80 °C until lipid extraction and analysis. All animal procedures performed in the study were approved by the Institutional Animal Care and Use Committee at the Sanford Burnham Prebys Medical Discovery Institute. Preparation of Lipid Extracts. Mouse liver, cortex, or spinal cord tissue (∼3.5 mg) was homogenized in diluted PBS (0.1×). The protein content of individual homogenate was determined with a BCA assay kit (Pierce, Rockford, IL). Lipid extraction was performed with three different extraction methods: (1) a two-step extraction method with addition of IS in the second step,16,17,24 (2) a two-step extraction method with addition of IS in the first step (a modified methyl-tert-butyl ether (MTBE) extraction method25 under acidic conditions (MTBE/MeOH/2 N HCl (200:60:13, v/v/v) was used in the second step), and (3) a two-step extraction method with addition of IS in the first step (a modified Folch extraction method26 under acidic condition was used in the second step). Detailed procedures of the extraction methods 2 and 3 were provided in the Supporting Information. Derivatization of Polyphosphoinositides with TMSDiazomethane. TMS-diazomethane in hexane (2 M, 50 μL or
■
RESULTS Determining Methylation Patterns and Ionization Ratios of Phosphate Positional Isomers. Previous studies used an identical methylation pattern of PPI isomeric classes for quantification.16,18 When we broadly examined the methylation conditions including reaction duration (1 to 20 min), reaction temperature (0 to 70 °C), and TMS-diazomethane concentration (200- to 10 000-fold to total lipid concentration), we found that methylation of these lipids was class-specific and independent of the reaction conditions examined (e.g., Figure S1). However, extreme reaction conditions such as long reaction time (>24 h) and very high TMS-diazomethane concentration (over a million of folds to lipids) led to a shift of the methylation pattern to contain higher methyl moieties (data not shown). We further found that these types of methylation patterns did not vary with examined individual PPI species 12138
DOI: 10.1021/acs.analchem.6b02947 Anal. Chem. 2016, 88, 12137−12144
Article
Analytical Chemistry
Figure 1. Determination of methylation patterns of PIP2 classes and the ratios of ion intensities between different PIP2 classes. Methylation of PIP2 equimolar mixtures (0.3 pmol/μL each) was conducted in the presence of trimethylsilyl-diazomethane as described under the method section. Mass spectral analysis of the methylated PIP2 mixtures in the full MS mode as indicated was performed in the presence of a small amount of LiCl in the positive ion mode on a triple quadrupole mass spectrometer (Quantiva, Thermo Fisher Scientific) as described under the methods section. (n,m)/ (s,t) in panels A to F indicates the phosphate positions of di18:1 and 17:0−20:4 PIP2 species, respectively, which were used to make up the equimolar mixtures for acquiring the mass spectra. xMe in the spectra indicates the ion of PIP2 species containing “x” methyl groups. Normalized ratios of ion intensities of different PIP2 classes relative to that of PI(3,4)P2(Me)5 were determined based on the peak intensities displayed in the mass spectra (panels A−F) and tabulated in Table 1.
containing different acyl chains (Figure 1). We used different combinations of all commercially available PIP2 species and demonstrated that methylation of five sites was predominant in the classes of PI(3,4)P2 and PI(4,5)P2, while methylation of six groups in PI(3,5)P2 was predominant (Figures 1 and S2). However, the total ion counts summarizing all the methylated ions of a species were virtually identical to each other between the classes (Figure 1), indicating that the difference in ionization efficiency among differently methylated ions of a PIP2 species was minimal. Therefore, we normalized all the methylated PIP2 ions to the ion corresponding to Me5PI(3,4)P2/Li+ ion which showed a most intense ion peak and determined the methylated patterns of PIP2 classes (Table 1). These patterns were used for simulation of the mixtures of isomer standards and endogenous unknowns utilizing the methods described for simulation of other lipid classes.30 Simulation of the mixtures of standards at a broad range of ratios through this pattern recognition approach demonstrated an excellent accuracy and dynamic range. The class-specific patterns for PIP classes were similarly determined with commercially available species (Figure S2 and Table 1). Of note, the methylation patterns of PI(4)P and PI(5)P were virtually identical. Thus, only their mixtures could be
Table 1. Normalized Methylation Patterns of Polyphosphoinositide Classesa PIP
(3)
(4)
(5)
PIP2
(3,4)
(4,5)
(3,5)
3Me 4Me 5Me
1.00 0.76 0.18
0.74 0.76 0.43
0.76 0.77 0.41
5Me 6Me 7Me
1.00 0.57 0.08
0.88 0.60 0.18
0.73 0.74 0.20
a
The data were determined from the mass spectral analyses of differently paired, equimolar mixtures of PIP or PIP2 isomers after methylation (Me), as demonstrated in Figures 1 and S2. The data tabulated were normalized to the intensities of ions corresponding to Me3PI(3)P/Li+ and Me5PI(3,4)P2/Li+, respectively, which were arbitrarily assigned as 1.
determined from biological samples by the method. It should be recognized that the PI(4)P content in the majority of these samples is predominant relative to PI(5)P.32 Characterizing Methylated PPI Species in Both Positive- and Negative-Ion Modes. Previous studies employed protonated ions of methylated PPI species for their identification and quantification.15,16,18 Because methylated PPI species are charge-neutral, they can be ionized as adducts of small cations or anions available in the matrix in the positive- or negative-ion mode, respectively, depending on the affinity and 12139
DOI: 10.1021/acs.analchem.6b02947 Anal. Chem. 2016, 88, 12137−12144
Article
Analytical Chemistry
Figure 2. Representative determination of linear dynamic range of the method with optimized collision energy for quantification of PIP2 species. A fixed amount of lipid extract from mouse spinal cord was added with different amount of 17:0−20:4 PI(4,5)P2 as an internal standard (IS) (0.46 nmol/mg protein, panel A; 1.05 nmol/mg protein, panel B; 2.27 nmol/mg protein, panel C; 4.3 nmol/mg protein, panel D; 8.99 nmol/mg protein, panel E; 16.34 nmol/mg protein, panel F; and 27.66 nmol/mg protein, panel G) for quantification of total PIP2 species present in mouse spinal cord extracts. Linear regression (panel H) of peak intensity ratios of spinal cord PIP2 species and IS vs their molar ratio was performed, and the linear result was plotted as its logarithm format to demonstrate the true linearity of the data as previously described.39
availability of these small ions.21,22 We initially conducted a comparison among proton, ammonium, sodium, and lithium adducts of these methylated species in both ionization and fragmentation after collision-induced dissociation (CID). Protonated ions of methylated PPI species were readily formed under acidic conditions or in the presence of relatively low concentration of ammonium salt with some complications (Figure S3). It should be recognized that both proton and ammonium adducts were formed if an ammonium salt was used as a modifier, which thus diversified the molecular ions of these lipids, made the MS analysis complicated, and led to a relatively low sensitivity in detection of these methylated species (Figure S3). Furthermore, sodiated ions of the methylated PPI species were also detected occasionally under the conditions (Figure S3). In contrast to the case of ammonium or proton adduction, the mass spectra of lithium adducts of methylated PPI species showed intense solely lithiated peaks, which made the mass spectra simple (Figure S3). This observation led us to further explore lithiated PPI species for their identification and quantification. Through comparison of ion peak intensities between individual species of a PPI class or between molecular species containing identical fatty acyl chains but different position(s) of phosphate(s) (i.e., phosphate position isomers), we demonstrated that the effects of acyl chains and/or phosphate positions on ionization efficiency as lithium adducts after correction for 13C isotope effects28,29 were minimal in comparison to the effects of
different methylation patterns of phosphate position isomers (Figures 1 and S2). In addition to the ionization efficiency, we also determined and compared the fragmentation patterns yielded from proton, ammonium, sodium, and lithium adducts of methylated PPI species after CID. Both protonated and ammoniated PPI species gave rise to a product ion corresponding to the neutral loss of methylated polyphosphoinositol.16 Therefore, in those previous studies, either neutral losses of these fragments prior to setting up and performing MRM assays15,18 or multiple PIS corresponding to diglyceride-like ions16 were performed. Different from those adducts, lithiated species of a PPI class containing same number of methyl groups yielded an identical fragment ion corresponding to lithiated polymethyl phosphoinositol (Figures S4 and S5). This fragment ion was different in the number of methyl groups on the inositol ring (Figures S4 and S5). The molecular structure of methylated PPI species and their fragmentation scheme were illustrated in Figure S6 using PIP2 as an example. Therefore, PIS analysis of these different methylated species in the positive-ion mode can be used to explore identification and quantification of individual species (see below). It should be mentioned that the sodium adducts of methylated PPI species displayed similar fragmentation patterns to their lithium adducts (data not shown). The methylated PPI species have never been characterized in the negative-ion mode. We found that these species in the negative-ion mode could readily form a molecular ion 12140
DOI: 10.1021/acs.analchem.6b02947 Anal. Chem. 2016, 88, 12137−12144
Article
Analytical Chemistry
Figure 3. Two-dimensional mass spectral analysis and comparison of PIP2 species present in brain cortices of wild-type and db/db mice. Cortical lipids of wild-type (panel A) and db/db (panel B) mice at 4 months of age were prepared in the presence of 17:0−20:4 PI(4,5)P2 (0.6 nmol/mg protein) as an internal standard (IS) as described in the Materials and Methods section. Two-dimensional ESI MS analyses were performed by precursor-ion scanning (PIS) of all methylated PIP2 species (i.e., PIS497.1 for Me5PIP2, PIS511.1 for Me6PIP2, PIS525.1 for Me7PIP2, and PIS539.1 for Me8PIP2) in the positive-ion mode in the presence of LiCl as described under the method section. For quantification, all the mass spectra were displayed after they were normalized to the ion peak intensities of IS based on its methylation pattern listed in Table 1. The broken lines highlight the methylation patterns of the PIP2 ions. These patterns were used for simulation of phosphate positional isomers as described in the text.
Determining the Optimal Conditions for Quantification of PPI Species. MS/MS analysis (including MRM) of individual species of a lipid class is a process that depends on the structure of individual lipid species.21,28 This has led scientists to use at least two IS for general quantification of individual species of a class when a MS/MS approach was exploited.28,34,35 Ramping collision energy (CE) might be used to minimize the differential effects of the collision process on different molecular species of a class, which usually is timeconsuming. Alternatively, careful selection of the representative CE to balance the differential effects could be employed by using authentic species of the class if they are commercially available. This latter method was frequently employed in our studies.36−38 In the case of those standard species rarely commercially available, an alternative method by using biological samples with one IS to determine the appropriate CE for balance of differential fragmentation effects could be exploited as described.23 The detailed information on determined CE for quantification of each PPI class by PIS and others was summarized in Table S1. We also determined the linearity of quantification of PPI species with the optimized CE in the method because the
corresponding to the loss of a methyl group (likely due to the facile loss of methyl chloride, which usually occurs in chlorinated phosphatidylcholines33) as well as relatively less intense chlorinated ions. Product-ion MS analysis of these demethylated ions yielded complicated fragmentation patterns due to the differential positions of phosphate(s) (Figure S7), which made the assignment of individual fragment ions difficult. However, it was readily recognized that abundant fragment ions corresponding to FA constituent(s) and an ion at m/z 125 (corresponding to dimethylphosphate in the case of PIP2 species (Figure S6)) resulting from methylated phosphate derivative were present (Figure S7). These features were exploited for identification of FA constituents of individual PPI species, as analyzed by shotgun lipidomics19,20 and demonstrated with the 2D MS analysis of PPI species present in mouse spinal cord (Figure S8). Taken together, on the basis of the characterized features of MS/MS of methylated PPI species in both positive- and negative-ion modes in combination with the methylation patterns of different PPI classes, comprehensive identification of individual PPI species including phosphorylation isomers and FA chains was achieved. 12141
DOI: 10.1021/acs.analchem.6b02947 Anal. Chem. 2016, 88, 12137−12144
Article
Analytical Chemistry
Figure 4. Illustration of the determined levels of molecular species of PIP and PIP2 classes present in mouse cortex. Lipid levels were determined in comparison to the selected internal standards by using precursor-ion scan of lithium adducts after methylation of PPI species as described in the Materials and Methods section and text. The mass levels of individual PI(3)P and PI(4)P+PI(5)P species (left panel), and PI(4,5)P2, PI(3,4)P2, and PI(3,5)P2 species (right panel) were then simulated on the basis of their methylation patterns as described in the text. The data presented represent the mean ± SEM of individual species from five separate animals.
We also examined the possible losses of PPI species using the two-step extraction method as previously described.16−18,24 We rationalized that if the majority of the less-polar lipids including single phosphate-containing anionic phospholipids are extracted efficiently in the first-step exaction, PIP and even PIP2 species very likely are also lost to a certain degree in this step. In fact, we showed that substantial amounts of both PIP and PIP2 species were extracted in the first step (Figure S10A and D). Discarding the extracts from this step led to the losses of an ∼70% of PIP and 50% of PIP2 content in comparison to those in the presence of IS in the first step and extracted with either the modified Folch or MTBE method25,26 in the second step (Compared Figure S10B to C, and E to F). The latter methods did not result in any significant differences of the profiles relative to the selected IS, indicating that the extraction efficiency of these methods were comparable under experimental conditions. These results indicate that a significant error could be introduced in the extraction procedure if IS for quantification of PPI species is not added at the earliest extraction steps. Moreover, the modified MTBE extraction method was relatively simpler (top layer extraction) and less labor intense (fewer steps). The modified MTBE extraction method was thus employed in the current biological applications. It should be pointed out that our standard(s) used for method development (i.e., di18:1 and 17:0−20:4) well-covered the range of endogenous PPI species. Any secondary effects of different PPI molecular species on extraction efficiency, if any, should be minimal. In fact, the mass range of PPI species (as known from PI species) was very narrow. Therefore, we concluded that the secondary effects of different fatty acyl chains on extraction efficiency, if existing, should be negligible
effects of phosphate-position isomers on ionization efficiency were minimal (Figures 1 and S2). For example, different amounts of 17:0−20:4 PI(4,5)P2 (from 10 to 500 pmol/mg protein) along with other PPI IS species in a premixed solution were added to the fixed amount of mouse spinal cord homogenates during lipid extraction. The mixtures were analyzed by using the settings listed in Table S1, and the acquired spectra were illustrated in Figure 2. The linearity of peak intensity ratios of individual PPI species and their corresponding standards after 13C deisotoping and summary of different numbers of methylated ion peaks vs their corresponding molar ratios in the mixtures was analyzed by linear log plots (i.e., log[IX/IIS − b] = log[CX/CIS] + c, which were derived from (IX/IIS) = a(CX/CIS) + b as discussed previously39 (Figure 2E). An essentially identical linear correlation was effectively obtained for all the PIP2 species, further indicating that the ionization response factors obtained from this method were independent of molecular species. This set of experiments was also used to further validate the reproducibility of the method through simulation of different methylation patterns of phosphate isomers, in addition to using standard mixtures. Moreover, the results demonstrated a broad linear dynamic range of over 3 orders and the low limit of quantification at less than 0.5 fmol/μL (Table S1). Similar experiments for other PPI classes were also performed to determine the linear dynamic ranges, low limits of detection and quantification, and so on (Figure S9 and Table S1). The relative standard deviations of sample analysis were
