non targeted lipidomic analysis by direct infusion mass ...non‐targeted lipidomic analysis by...
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Non‐targeted Lipidomic Analysis by Direct Infusion Mass Spectrometry
Jianzhong Chen, PhDAssistant ProfessorSchool of Optometry
UAB
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Lipidome: A subset of Metabolome
2https://en.wikipedia.org/wiki/Lipidomics
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Eight Categories of Lipids
3Fahy E. et. al., J. Lipid Res. 2005, 839-861
Fatty Acyls Glycerolipids
Glycerophospholipids Sphingolipids
Sterol lipids Prenol lipids
Saccharolipids
Polyketides
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Mass Spectrometric Analysis of Lipids
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Lipids of Interest Mass Spectrometric Analysis
Targeted
one or several specific lipid species
multiple reaction monitoring (MRM)
one specific lipid class/subclass
• precursor ion scan
• Neutral loss scan
Non‐targeted all lipid classes
• SWATH (Sequential Window Acquisition of all Theoretical fragment‐ion spectra)
• MS/MS (Identification) combined with high resolution MS (Quantification)
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Non‐targeted Lipidomic Analysis• Advantages
• Comprehensive
• Rapid
• Big picture
• Challenges
• Relatively low sensitivity
• Full scan, neutral lipids, more severe interference peaks
• Complexed data analysis
• More severe interference peaks, multiple adduct forms, peak overlapping
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Interference Peaks in Mass Spectrometric Analysis
• Contaminants from plastics (additive, polymer)
• Multiple adducts formation: H+, NH4+, Na+, K+
• Non‐covalent adduct formation
• homo/hetero lipid dimers;
• between lipid and impurity
• In‐source dissociation
• Solvent degradation: CHCl3 HCl
• Carryover: previous runs, glassware, calibrants
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Common Contaminants in Samples• Plasticizers:
phthalates
esters of aliphatic dicarboxylic acids
• Polymers:
• Detergents:
• Ingredients in cosmetics and hair conditioners
44.03 DaPEG
58.04 DaPPG
74.02 Dasilicone rubber
44.03 Da
Distearyldimethylammonium chloride m/z 550.63
Triton X‐100
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Contamination from Detergent
Detergent Triton
44 Da
44 Da44 Da
44 Da
44 Da
44 Da44 Da
44.03 Da
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Contamination from Silicone Rubber in LC/MS
74.02 Da
74
74
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Approaches for Non‐targeted Lipidomic Analysis
• Minimize interference from common contaminants
• Do not use plastics
• Rinse thoroughly
• Tuning instruments to maximize signal of lipids of interest
• Electrospray probe position
• Desolvation temperature
• Flow rate
• MS profile
• Other parameters
• heating gas, nebulization gas, cone voltage/decluster voltage
• Convert multiple adduct forms to one dominant form
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Multiple adduct formation
Chen JZ, et. al, IOVS, 2013, 54: 5730‐5753. 11
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Bis(2‐ethylhexyl) adipate 101 detergent101 detergent
Dibutyl phthalate
101 detergent
Non‐targeted Mass Spectrometry Analysis of Lipids Before optimization
After optimization
Analyte peak
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MS/MSall Analysis of All Lipid Peaks in a Sample
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Q1 Q2
Fast Q1 precursor selection step-wise through mass range
CID Fragmentation Direct infusion, flow injection, and lipid-class targeted LC techniques
Collection of High resolution MS/MS
https://www.uab.edu/proteomics/metabolomics/symposium/symoposium_dec2012.php
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SWATH: Full MS/MS Archive of Every Compound in a Sample
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TICMS/MSall
MS250ms
MS/MS
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Pseudo Precursor Ion Scanning Extracted from MS/MSall Analysis
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Pseudo Precursor Ion Scanning Extracted from MS/MSall Analysis
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• Minimizing contaminants/interference peaks is particularly important for non‐targeted lipidomic analysis.
• It is important to confirm the identity of lipids by MS/MS before quantifying lipids by MS.
• SWATH appears to be a promising method for non‐targeted lipidomic analysis.
Summary
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References on Contaminants
1. Keller BO, Jie Suib, Alex B. Youngc, Randy M. Whittal, Interferences and contaminants encounterd in modern mass spectrometry, Analytica Chimica Acta, 2008, 627: 71‐81
2. Ende M, Spiteller G, Contaminants in mass spectrometry, Mass Spectrometry Review, 1982, 1: 29‐62
3. http://www.waters.com/webassets/cms/support/docs/715001307d_cntrl_cntm.pdf
4. http://www.abrf.org/index.cfm/list.msg/66994
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Useful websites for lipid analysis1. http://lipidlibrary.aocs.org/
2. http://www.cyberlipid.org/
3. http://www.lipidmaps.org/
4. http://lipidlibrary.aocs.org/news/links.html