Non targeted Lipidomic Analysis by Direct Infusion Mass Spectrometry. Jianzhong Chen, PhD Assistant Professor School of Optometry UAB
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1 Non targeted Lipidomic Analysis by Direct Infusion Mass Spectrometry Jianzhong Chen, PhD Assistant Professor School of Optometry UAB 1
2 Lipidome: A subset of Metabolome 2
3 Eight Categories of Lipids Fatty Acyls Glycerolipids Glycerophospholipids Sphingolipids Sterol lipids Prenol lipids Polyketides Saccharolipids Fahy E. et. al., J. Lipid Res. 2005,
4 Mass Spectrometric Analysis of Lipids Targeted Non targeted Lipids of Interest one or several specific lipid species one specific lipid class/subclass all lipid classes Mass Spectrometric Analysis multiple reaction monitoring (MRM) precursor ion scan Neutral loss scan SWATH (Sequential Window Acquisition of all Theoretical fragment ion spectra) MS/MS (Identification) combined with high resolution MS (Quantification) 4
5 Non targeted LipidomicAnalysis 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 5
6 Interference Peaks in Mass Spectrometric Analysis Contaminants from plastics (additive, polymer) Multiple adducts formation: H +, NH 4+, Na +, K + Non covalent adduct formation homo/hetero lipid dimers; between lipid and impurity In source dissociation Solvent degradation: CHCl 3 HCl Carryover: previous runs, glassware, calibrants 6
7 Plasticizers: phthalates Common Contaminants in Samples Polymers: esters of aliphatic dicarboxylic acids Detergents: Da PEG Da PPG Da silicone rubber Triton X Da Ingredients in cosmetics and hair conditioners Distearyldimethylammonium chloride m/z
8 Contamination from Detergent Da Detergent Triton 44 Da 44 Da 44 Da 44 Da 44 Da 44 Da 44 Da 8
9 Contamination from Silicone Rubber in LC/MS Da
10 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 10
11 Multiple adduct formation Chen JZ, et. al, IOVS, 2013, 54:
12 Non targeted Mass Spectrometry Analysis of Lipids Before optimization Dibutyl phthalate Bis(2 ethylhexyl) adipate 101 detergent Analyte peak 101 detergent 101 detergent After optimization 12
13 MS/MS all Analysis of All Lipid Peaks in a Sample Q1 Q2 Direct infusion, flow injection, and lipidclass targeted LC techniques Fast Q1 precursor selection step-wise through mass range CID Fragmentation Collection of High resolution MS/MS 13
14 SWATH: Full MS/MS Archive of Every Compound in a Sample MS 250ms MS/MS TIC MS/MS all 14
15 Pseudo Precursor Ion Scanning Extracted from MS/MS all Analysis 15
16 Pseudo Precursor Ion Scanning Extracted from MS/MS all Analysis
17 Summary Minimizing contaminants/interference peaks is particularly important for nontargeted 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. 17
18 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: Ende M, Spiteller G, Contaminants in mass spectrometry, Mass Spectrometry Review, 1982, 1: Useful websites for lipid analysis
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