Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS

Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS Alexei Belenky CASSS, Meritage Resort, Napa 2008

Outline Role of lipid analysis in drug discovery and disease diagnostics Analytical tools and methods for lipid analysis and extraction from tissues Quantitation and normalization of the results Multidimensional approaches for Lipidomics

Lipids a free fatty acid a triglyceride cholesterol a phospholipid Lipids are broadly defined as any fat-soluble (lipophilic), naturally-occurring molecule, such as fats, oils, cholesterol, sterols, mono-, di-, tri- glycerides, phospholipids, sphingolipids and others. (From Wikipedia) The main biological functions of lipids include energy storage, acting as structural components of cell membranes, and participating as important signaling molecules. Because of the combinatorial nature of sphingolipids biosynthesis, the sphingolipidome is comprised of thousands of related and biologically connected species

Sphingolipid Pathway Map Sphingolipidomics similarly to genomics or proteomics helps to understand the disease progression, identify targets, mechanism of drug action and toxicity

Gaucher Disease Lysosomal storage disorder caused by deficiency of the enzyme β-glucosidase, leading to an accumulation of its substrate glucosylceramide. Fatty material can collect in the spleen, liver, kidney, braine and bone marrow. occurring in approximately 1 in 50,000 live births Structure of a Glucosylceramide Girl with Gaucher Disease..

Glycosphingolipid Pathways and Target Diseases Gangliosides Globosides G M1 G B4 Tay-Sachs Disease G M2 Type 2 Diabetes G M3 LacCer G B3 Fabry Disease Sandhoff Disease Enzyme Replacement Therapy glucosylceramide Gaucher Disease ceramide + glucose GluCer synthase Substrate Reduction Therapy

Analytical Challenges Separation analytes are a small part of a huge family of endogenous lipids Absence of pure synthetic standards Analyte concentration is ng/ml Solution to the problem: SELECTIVITY, SELECTIVITY, SELECTIVITY!

Work Flow for Sphingolipids Analysis Cells/Tissue/plasma/serum Cells/Tissue/plasma/serum 96/384-well plate format QQQ-MS Extraction HPLC UPLC API 4000 API 5000 LC separation MRM Int. (arb.) 0.0 DHSPH SPH 3.09 4.47 3.50 2 min 8.01 Higher Cer throughput GluCer 10.17 SM 9.35 S1P MRM-TIC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 15 min

Extraction and Analysis of Tissue Samples Kidney samples ( 100 mg/ml) are homogenized and extracted with a modified Folch method: Extraction buffer : 0.47/ 1.7/1.7 of water / methanol / chloroform; ~ 5mg/ml tissue HPLC-MS/MS of glucosylceramide isoforms, GL1 std 0.5ug/ml Intensity, cps 4.4e5 4.0e5 3.6e5 3.2e5 2.8e5 2.4e5 2.0e5 1.6e5 1.2e5 8.0e4 4.0e4 0.0 XIC of +MRM (6 pairs) C16 C18 C 24 1 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2 7.6 8.0 Time, min C22 C 24 C 23 HPLC (UPLC) Column: Waters Xbridge Phenyl 3.0 x 100 mm 3.5 um column. (Alternative: Phenomenex Luna C8,) The mobile phases : 0.2% (v/v) FA and 5 mm AmF in water (A) 0.2% FA (v/v) and 5 mm AmF in 1:1 meth/acetonitrile (B) The flow rate was 0.6 ml/min. The column was heated to 60 C. The gradient: 70% B to 100% B in 5 min, hold at 100% B for 3 min, reset ESI- MS, API-4000 (API-5000) ESI-MS conditions were: needle voltage, 3.5 kv. drying gas temperature, 400 C, collision gas density (CAD), 7. All MRM transitions included m/z 264.2 or 184 for SM as the product ions.

MRM Transitions for Analysis of Sphingolipids in Tissues Intensity, cps Intensity, cps 1799 1600 1400 1200 1000 800 600 400 200 0 80 70 60 50 40 30 20 10 GalCer std. 11.7 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 Time, min 264.29 Sphingoid backbone -H2O 728.65 548.60 -Gal 530.58 710.65 0100 150 200 250 300 350 400 450 500 550 600 650 700 750 m/z, amu 800 fatty acid chain O Ceramide R=H Glycosphingolipids R= carbohydrate O + Sphyngomyelin R = O-P-OCH 2 CH 2 N(CH 3 ) 3 OH m/z 184 Alfred H.Merrill Jr, et.al. Methods 36(2005) 207-224

LC-MRM Analysis of GL1 Isoforms Extracted from Different Amount of Tissue Tissues are extracted and reconstituted in the same volume MRM signal peak area 3.50E+05 3.00E+05 2.50E+05 2.00E+05 1.50E+05 1.00E+05 5.00E+04 0.00E+00 0 50 100 150 200 250 Tissue, mg GL1 C16 GL1 C24.1 GL1 C22 GL1 C24 GL1 C23 GL1 C18 C16 C 24 1 C18 Time, min 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2 7.6 8.0 C22 C 24 C 23? Extraction or Matrix?

When is LC QQQ Analysis Quantitative? when matrix effect does not exist when isotopically labeled IS is available.. and if not? The alternative is - method of standard additions, which requires a well characterized standard.

LC-CAD/TOF Characterization Analysis of Standards of a Sphingomyelin using CAD-TOF Standard Method: 20 15 10 5 8.0e4 6.0e4 4.0e4 HPLC HPLC Column Charged Aerosol Detector, CAD 9 1 Eluent Split Mass Spectrometer TIC of +TOF MS: 600 to 850 amu from SM 214ug/m stnd Luna C8 Max. 9.2e4 cps. CAD 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min Since the abundance of isoforms in the standard is not known, a universal detector (CAD) was used to quantitate all the isophorms present in the standard. 2.0e4 CAD, mv CAD of SM 214 ug/m stnd, Luna C8 MW 701.65 SM C16:1 MW 675.63, C14 MW 703.64,C16 MW 689.64 SM C15 MW 717.677, C17 MW 731.68,C18 MW 757.72,C20:1 MW 811.78, C24:2 Intensity, mv Intensity, cps Sphingomyelin standard, 214ug/ml 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min MW 759.73, C20 MS - Q STAR 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min MW 799.78, C23:1 MW 787.76, C22 MW 813.77, C24:1 MW 801.80, C23 MW 815.79, C24 MW 84187, C26:1 MS - Q STAR CAD

Slopes for Different Mouse Models Kidney Samples, GL1 C24:1 Isoform Analysis Peak area 3.50E+05 3.00E+05 2.50E+05 2.00E+05 y = 9292.9x + 59476 R 2 = 0.9983 y = 9293x y = 6167.3x + 82791 R 2 = y 0.9997 = 6167x y = 6990x WT 45d Jck 45d Jck 64d WT 45d Jck 45d Jck 64d 1.50E+05 1.00E+05 5.00E+04 y = 6989.6x + 45330 R 2 = 0.9997 0 5 10 15 20 25 30 GL1 conc., ng/ml

Normalization of Results Not all the tissues are equal

Difference in Tissue Morphology Makes Normalization by Weight Inadequate normal adult kidney cystic adult kidney Conventional method of normalization is using total phosphate. We have developed a fast, sensitive and reliable method for total phosphate analysis based on inductively coupled plasma spectrometry (ICP).

Total Phosphate Levels in Tissues are Associated with Disease State 3000.0 healthy 2500.0 ICP count 2000.0 1500.0 cystic 1000.0 500.0 0.0 202 203 205 1223 1504 2027 mouse,# Normal Kidney ADPKD Kidney

Normalization by Total P Revealed Increased GL1 Levels in Cystic Mice: Distribution of GL1 isoforms in kidney tissues for cystic and normal mice 1.80E+05 1.60E+05 1.40E+05 1.20E+05 1.00E+05 8.00E+04 6.00E+04 4.00E+04 2.00E+04 Control Mice Cystic Mice GL1 C16 GL1 C18 GL1 C22 GL1 C24 1 GL1 C24 0 2.50E+02 2.00E+02 Same data after normalization Control Mice Cystic Mice 1.50E+02 1.00E+02 5.00E+01 0.00E+00 GL1C16 GL1 C18 GL1 C22 GL1 C24 1 GL1 24 0

Correlation of Endogenous SM C16 and Total P Has Been Identified Comparison of Total Phosphate and SM C16 levels; data normalized to the same scale Phosphate SM C16/100 Correlation between Total P and levels of endogenous SM C16 in mice kidney 2.00E+05 1.60E+05 y = 77.128x - 17644 Animal #184 Animal #183 Animal #182 Animal #2274 Animal #5780 Animal #1222 Total phosphate by ICP 1.20E+05 8.00E+04 4.00E+04 0.00E+00 R2= 0.9944 0.00E+00 5.00E+02 1.00E+03 1.50E+03 2.00E+03 2.50E+03 SM C16 level by LC-MS/MS Alternative approach: normalization using selected endogenous markers

Why Phospholipids can serve as relevant normalization markers? Napa Phospholipids are cell building materials and can be used for estimation of the number and the nature of the extracted cells

Phospholipids 141 184+ 184+ palmitate Separate by HILIC and detect using precursor scan:+184 & neutral loss: 141

HILIC Mode of Separation Water Acetonitrile/Water (90:10) Separation is based on partitioning between adsorbed polar component of mobile phase (water) and hydrophobic mobile phase (acetonitrile). This mode can be used to separate sphingolipids by their polar groups.

A Standard and a Mouse Kidney Homogenate Sample, Analyzed by HILIC Intensity, cps 4.8e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e5 0.0 1.29 PC PM 789.0 SM PC 2.83 standard 3.23 PE PE 1 2 3 4 5 6 7 Time, min 760.9 Intensity, cps 6.5e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e6 0.0 1.21 1.29 PC PC SM SM 2.83 3.19 kidney sample PE PE 1 2 3 4 5 6 7 Time, min 760.7 735.0 758.8 782.9 810.8 787.0 680 700 720 740 760 780 800 820 840 m/z, amu +184 precursors of PC from standard 660 680 700 720 740 760 780 800 820 840 m/z, amu +184 precursors of PC from sample Mobil Phase A: 96:2:2 (ACN:MeOH:Acetic acid:water, 5mM Ammonium Acetate) Mobil Phase B: 98:1:1 (MeOH:Acetic Acid:Water, 5mM Ammonium Acetate) Column: Phenomenex Luna 50 x 2mm, 3 micron HILIC; gradient : 0 to 15%B

Correlation Between PC Level Measured by LC-MS/MS and Total Phosphate Measured by ICP Acute jck Mice Model Study 16 14 12 PC 10level by MS/MS P level by ICP ug/ml 10 8 6 4 2 0 0 2 4 6 8 sample# Treated Untreated

Correlation Between PC Level Measured by LC-MS/MS and Total Phosphate Measured by ICP 0.45 0.4 0.35 Acute jck mice kidney study Time Course y = 2.087x + 0.0863 R 2 = 0.890 ICP, total P, mm 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 LC-MS/MS, PC mm

GL1 Analysis from Cell Pellets Extracts in Triplicate Data normalized by cell count 0.450 0.400 0.350 0.300 0.250 0.200 0.150 0.100 0.050 0.000 control 100nM-C9 300nM-C9 1uM-C9 Normalization by cell count is hard because of cell clamping 90.00 Data normalized by PC level 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 12hr 24hr 48hr 0.00 control 100nM-C9 300nM-C9 1uM-C9 Normalization by PC level helps because it reflects true cell count. As a result RSD is much improved and effect of treatment can be clearly seen.

Comments PC levels correlate well with the total phosphate levels Normalization by PC significantly reduces sample preparation work required by conventional total phosphate analysis

What have we accomplished so far? We have a selective LC MS/MS method to analyze Sphingolipids We are able to quantitate multiple lipids and their isoforms We know how to normalize data

Characterization of 7 Animal Models by Sphingplipids Analysis MRM raw data data after quantitation and normalization SM C18 GL1 C16 GL1 C57Blk6 (wt) 45 days C57Blk6 (wt) 64 days Jck (disease model ) 45 days Jck (disease model ) 64 days CD1(wt) Pcy (disease model ) 4-15 wk Pcy (disease model )15-30 wk

Quantitation of Glycosphingolipids Using 2D-LC-MS/MS Method

Time, min Reversed Phase Chromatography Separates GLs According to the Lengths of Their Fatty Acid Chains and Not By the Sugar Moieties Luna C8, 3u, 2x100mm Mobile Phase A= 95%ACN, 5%MeOH, 5mM NH4OOC, 0.2% FA Mobile Phase B= MeOH, 5 mm NH4OOC, 0.2% FA 250ul/min 50% B isocratic,. Q-Star TOF, EIC 1.91 Max. 2117.8 cps. 2.66 3.03 GM 3 20ug/ml : C16, C22, C24 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 2.85 Max. 1158.6 cps. 1.98 2.51 3.26 GL 2 2ug/ml: C16, C20, C22, C24 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Max. 2523.5 cps. Max. 2523 cps 3.20 3.73 2.80 2.45 GL1 1ug/ml: C18, C20, C22, C24 1.2 1.4 1. 6 Max. 630.1 cps. 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 2.97 4.77 4.04 CER 1ug/ml : C18, C22, C24 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2

HILIC Chromatography Separates GLs According to their Sugar Moieties and Not by the Fatty Acid Chains Atlantis HILIC Silica, 2.1 x 150mm, 3um GL1 Mobile Phase A = 95%ACN, 4.8%MeOH, 5mM NH 4 F, 0.2% FA Mobile Phase B = MeOH, 4.8 mm NH 4 F, 0.2% FA 250 ul/min, 55C Q-Star TOF, EIC 2% B to 10%B in 2 min,10%b to 40%B in 0.1min ; hold 2 min, re-equilibrate 6min at 2%B, 250 ul/min. Max. 593.7 cps. CER GM3 GL2 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Time, min

Analysis of GM3 - GL1 Mixture Using both Columns Connected in Sequence Atlantis HILIC Silica, 2.1 x 150mm, 3um Luna C8, 3u, 2x50mm Summed EIC of +TOF Sample 3 (GL1 & GM3 at 20ug/ml ) 3.71 4.53 4.97 Max. 1.4e4 cps. 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 Time, min GL1 C16, C22, C24. 9.21 Max. 1916.0 cps. 8.82 9.44 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 Time, min GM3 C16, C22,C24 Gradient used in the 1st dimension works as a 100% organic isocratic mode in the 2nd (RP) dimension.

Analysis of CER GM3 Mixture Using Two Columns Connected in Sequence Atlantis HILIC Silica, 2.1 x 150mm, 3um Luna C8, 3u, 2x50mm Summed EIC of +TOF Sample 3 (CER & GM3 at 20ug/ml) 4.10 4.34 5.98 Max. 1.2e4 cps. 4.74 5.30 9.01 9.42 8.77 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 CER C16, 18, 22, 24 9.17 8.77 9.40 Max. 1744.0 cps. 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 GM3 C16, 22, 24 Time, min

Comments: Direct hyphenation of normal and reversed phase separations on a single HPLC system was demonstrated for the 2D separation of GLs The presented on-line 2D separation system is fully compatible with ESI- MS/MS analysis Dramatic increase of the separation window for gangliosides was achieved in this 2D setup, which allowed for monitoring of GM3, GL2, GL1 and CER transitions in the same LC-MS/MS run.

Conclusions: We have demonstrated a new set of tools for quantitative targeted lipidomics These tools were successfully applied for analysis of many tissue types, including kidney, liver, muscle, adipose and blood serum New normalization approach simplified comparison of different animal models and disease states

Acknowledgments: Analytical R&D Aharon Cohen Bing Wang Eva Budman Clifford Phaneuf Alla Kloss Lingyun Li Kim Alving John Bailey Biology: Tom Natoli

Distribution of Glucosylceramide Isoforms for a Mice Kidney Sample and a Matreya Standard 3.00E+06 2.50E+06 Sample mice kidney Standard 2.00E+06 MRM, Are 1.50E+06 1.00E+06 5.00E+05 0.00E+00 C16 GL1 C18 GL1 C22 GL1 C23 GL1 C24:1GL1 C24 GL1

In this sequential setup GLs are separated first by their sugar moieties on the HILIC column (1 st dimension = normal phase separation), and then additional separation by the length of the fatty acid chain is achieved on the Luna column (2 nd dimension = reversed phase separation). Gradient used in the 1 st dimension works as a 100% organic isocratic mode in the 2 nd (RP) dimension. The last feature allows to use this 2D methodology in on-line flow through mode. Dramatic increase of the separation window for gangliosides was achieved in this 2D setup, which allowed for monitoring of GM3, GL2, GL1 and CER transitions in the same LC-MS/MS run.

Fragmentation of GM3 in the ESI-MS Source +TOF MS: 7.735 to 8.669 min from Sample 1 (GL1 and GM3 at 20 ug/ml) Cer LC - QSTAR Results Max. 245.6 counts. Cer Cer Cer GL1 GL2 GM3 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 m/z, amu Multiple GLs with shorter sugar chains are produced as result of fragmentation in ESI, which reduces selectivity of the MRM detection. Therefore separation of different GLs prior to MS/MS analysis is crucial.

Method for Phospholipid Analysis using HILIC Chromatography: Instrument UPLC-API 4000 LC method: Mobil Phase A: 96:2:1:1 (ACN:MeOH:Acetic acid:water, 5mM Ammonium Acetate) Mobil Phase B: 98:1:1 (MeOH:Acetic Acid:Water, 5mM Ammonium Acetate) Column: Phenomenex Luna 50 x 2mm 3 micron HILIC MS/MS method: Period 1 Scan type: precursor Polarity: positive Precursor of: 184 amu Period 2 Scan type: neutral loss Polarity: positive Neutral loss of: 141 amu Gradient profile corrected for system volume 20 % B PC SM PE 15 10 Phosphatidylcholine Sphingomyelin Phosphatidylethanolamine PC SM PE 5 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 minutes