using skyline for targeted lipidomics user's group meeting june 9, 2013 hari nair, phd andy...
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Using Skyline for Targeted Lipidomics
User's Group MeetingJune 9, 2013
Hari Nair, PhDAndy Hoofnagle, MD PhDUniversity of Washington
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HDL is the Good Cholesterol
The Framingham Heart Study
Castelli, Can J Cardiol, 1988
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HDL Cholesterol is Not the Whole Story
Torcetrapib (ILLUMINATE)60% increase in HDL cholesterol40% increase in poor outcomes
Dalcetrapib (dal-OUTCOMES)35% increase in HDL cholesterolNo improvement in outcomes
Niacin (AIM-HIGH)30% increase in HDL cholesterolTwice as many strokes
Three HDL-C raising studies terminated early
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HDL Does More than Just Carry LipidsFunctional Assays of HDL
Particles remove lipids from lipid-laden macrophagesHDL activity in vitro is predictive of coronary artery disease
Khera, NEJM, 2011
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The HDL Proteome is Vast
SAA1
PON1ApoA-I
SAA2
ApoHApoA-IV
C3
C4A
C4B
VTN
ApoA-IIApoL-1
ApoDSAA4
ApoC-IV
ApoC-III
ApoC-IIApoC-ILCATCETP
PLTPApoM
ApoF
ApoE
HRP SERA1
KNG1
ORM2
AGT
AHSG AMP
SERF2SERF1
Clusterin
PON3
C9
Lipid Metabolism
ITIH4TTR RBP4 TF FGA
HPX
Proteinase Inhibitor
Complement Regulation
Acute Phase Response
Vaisar, JCI, 2007
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Thrombotic Occlusion of Coronary Artery
Enzymatic Weakening of the Fibrous Cap
Platelet and Coagulation Activation at the Lipid Core
Acute Coronary Syndrome and Sudden DeathProteolysis
Complement Activation
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Targeted Proteomics for HDLInternal Standard Protein
apoA-I apoB apoC-II
apoC-III apoE apoJ
Hoofnagle, ClinChem, 2012
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Paraoxonase 3 is Depleted in Patients withType 1 Diabetes and Artery Calcification
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Endothelial Cell FunctionImplicating HDL Lipids
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Phosphotidylcholine
Ceramide
Sphingomyelin
Glucosylceramide
Lactosylceramide
Thinking Beyond CholesterolPhospholipids and sphingolipids
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Phosphotidylcholine
Ceramide
Sphingomyelin
Glucosylceramide
Lactosylceramide
184
264
Common Fragment Ions
Common Head Group Fragment (m/z)
Common BackboneFragment (m/z)
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Time (minutes)
Inte
nsity
(cp
s x1
04 )Typical Chromatogram
4 6 8 10
2.0
1.5
1.0
0.5
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Inte
nsity
(cp
s x1
04 )Biological Complexity
Time (minutes)4 6 8 10
2.0
1.5
1.0
0.5
SM20
PC
dehydro SM20
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Step 1: Design Data Processing Method
Assign transitions
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Set RT and Integration window
Step 2: Design Data Processing Method
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Step 3: Design Data Processing Method
Assign smoothing parameters
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Manually pick peak
Step 4: Design Data Processing Method
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Method 1 Method 2 Method 3
Step 5: Process the Data
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An Excel spreadsheet with a single transition per tab
Step 5: Export the Data
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Step 6: Manual Integration & Re-process
Identify samples with wrong retention time, etc.Fix processing parameters, reprocessThere are 100 tabs here!
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Step 7: Compile Data: A Single Spreadsheet
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Workflow ComplexityThree different LC-MS runsCreate a processing method for each transitionUsing a previously acquired data file:
Assign RT, peak width, smoothing parameters.Process data using this processing methodAdjust processing parameters as neededExport data:
One worksheet in Excel per transitionRandom order of worksheets in ExcelCompile data into a single file
Perform QC:Manually ensure proper integration (retention time, peak shape)Export dataPerform QC
Lather, Rinse, Repeat...
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Possible Solution: Skyline
Easy to manipulate large data sets
Chromatographic alignment
Automated peak integration
Simple report configuration
Simple data export
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Possible Solution: Skyline
Tricking Skyline
Set one amino acid to be the fragment for the class of lipids
Set another amino acid to add up to the right precursor mass
Pretend the two amino acids are a peptide
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Ceramides (CER, GluCer, LacCer)
Phosphatidylcholine (PC)
Sphingomyelins (SM)
M+H+
Modified GlycineChemical Formula
Inventing Amino Acids: The Constant Part
264
184
C15H24
C10H4
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Inventing Amino Acids: The Variable Part
C18 Ceramide (d18:1/18:0)
C20 Ceramide (d18:1/20:0)
C22 Ceramide (d18:1/22:0)
M+H+
Modified AlanineChemical Formula
548
604
C13H13
C17H21
576 C20H13
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Step 1: Invent Modified Amino Acids
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Step 2: Invent New "Proteins" and "Peptides"
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Step 3: Modify the Amino Acids on the "Peptides"
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Step 4: Import Data
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Step 5: Manually Pick Peaks if Needed
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Step 6: Export the Data
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A Deliverable: One Tab on One Spreadsheet
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An Experiment: The Data First
How long would you be willing to spend to get this amount of data?Vendor Software: 15 hours, Skyline: 4 hours
Actively collaborating with the author of the software: Priceless
Lipid in HDL extract Within Day CV Between Day CV Lipid in HDL extract Within Day CV Between Day CVCer(d18:1/12:0) 9% 9% PC(18:0/20:3) 12% 15%Cer(d18:1/24:0) 12% 11% PC(18:0/22:5) 11% 16%
Glucosylceramide (d18:1/12:0) 8% 9% PC(18:0/22:6) 10% 15%PC(10:0/16:0) 12% 30% PC(18:1/22:6) 8% 14%PC(10:0/18:1) 10% 19% PC(20:5/16:0) 13% 15%PC(12:0/17:2) 11% 35% SM(d18:0/16:0) 9% 14%PC(12:0/18:1) 9% 14% SM(d18:0/18:0) 10% 17%PC(12:0/18:2) 10% 14% SM(d18:0/20:0) 11% 15%PC(13:0/20:3) 11% 14% SM(d18:0/22:0) 9% 15%PC(14:0/18:1) 11% 13% SM(d18:1/12:0) 8% 14%PC(14:0/18:2) 9% 14% SM(d18:1/12:0)-18 11% 22%PC(14:0/18:3) 13% 11% SM(d18:1/16:0) 10% 13%PC(15:0/20:5) 11% 14% SM(d18:1/16:1) 11% 15%PC(15:0/22:6) 11% 15% SM(d18:1/18:1) 11% 13%PC(16:0/18:1) 11% 15% SM(d18:1/20:0) 11% 14%PC(16:0/18:2) 10% 13% SM(d18:1/22:0) 11% 14%PC(16:0/20:3) 10% 14% SM(d18:1/22:1) 10% 14%PC(16:0/20:4) 10% 13% SM(d18:1/24:0) 10% 16%PC(16:0/22:4) 11% 15% SM(d18:1/24:1) 8% 15%PC(16:0/22:5) 10% 14% SM(d18:2/18:1) 13% 14%PC(18:0/11:1) 14% 17% SM(d18:2/22:1) 11% 13%PC(18:0/18:2) 11% 15% SM(d18:2/24:1) 10% 17%PC(18:0/20:2) 10% 17%