Lipididentifizierung in der LC-MS-basierten Lipidomik mittels einer Kombination aus SWATH und DMS Michael Witting, et al. Helmholtz Zentrum München Research Unit Analytical BioGeoChemistry Berlin, 14/3/17
Lipid identification in LC-MS based lipidomics using a combination of SWATH and DMS Michael Witting, et al. Helmholtz Zentrum München Research Unit Analytical BioGeoChemistry Berlin, 14/3/17
Motivation Complexity of the C. elegans lipidome Witting, M, et al. The C. elegans lipidome. Arch Biochem Biophys. 215 Jun 1. pii: S3-9861(15)267-2
Motivation Complexity of the C. elegans lipidome
Motivation Complexity of the C. elegans lipidome and ways to tackle it Witting, M, et al. The C. elegans lipidome. Arch Biochem Biophys. 215 Jun 1. pii: S3-9861(15)267-2
Motivation Complexity of the C. elegans lipidome and ways to tackle it Witting, M, et al. The C. elegans lipidome. Arch Biochem Biophys. 215 Jun 1. pii: S3-9861(15)267-2
Lipid identification strategies DDA, DIA and DIA-SWATH DDA DIA DIA-SWATH + - Precursor known - No reconstruction of spectra required - - Coverage problem - Precursor unknown - Reconstruction of spectra required - Large overlap of signals - No coverage problem - Precursor range known - No coverage problem - Exact Precursor mass unknown - Reconstruction of spectra required - Partial overlap of signals - Overlap in DIA causes merged spectra - Merged spectra reduce MS/MS scoring Analysis of single lipid classes in one run Use DMS to clean spectra on the fly Witting, M, et al., LipidFrag: Improving reliability of in silico fragmentation of lipids and application to the Caenorhabditis elegans lipidome, http://dx.doi.org/1.1371/journal.pone.172311,
Experimental setup LC conditions UPLC Instrument: Agilent 129 Column: Waters Acquity UPLC HSS T3 Column, 1Å, 1.8 μm, 2.1 mm X 1 mm Mobile phase A: 6:4 Water:ACN with 1 mm NH 4 HCO 2 and.1% formic acid Mobile phase B: 9:1 IPA:ACN with 1 mm NH 4 HCO 2 and.1% formic acid Column Temperature: 5 C Flow rate: 4 ul/min
Experimental setup LC conditions TOF Selexion Parameter Value Parameter Value Accumulation Time 1. ms Separation Voltage 4 Curtain Gas 25 Compensation Voltage Varies Gas 1 3 DMS offset -3 Gas 2 5 Ionization Voltage 55 Declustering Potential 8 Source Temperature 6 Start Mass 3 End Mass 1 Resolution enhancement DMS Temperature Modifier Modifier Composition Low Low 1-Propanol Low
Development of XCMS workflow Building a SWATH support for XCMS Acquisition with SWATH (one run per COV) Conversion to.ml XCMS for processing (workflow for SWATH required)
Development of XCMS workflow Building a SWATH support for XCMS first tests Split data into MS1 and MS2 data Sort MS2 data into different SWATH pockets Perform peak picking on each in individual Select MS1 precursor and search for respective SWATH pocket Find fitting peaks based on RT Get EICs for Precursor and each fragment ion in RT region of precursor Perform correlation analysis Data file MS1 MS2 MS2 MS2 MS1 MS2 MS2 MS2 intensity 2 15 1 5 1..75 497.5 5. 52.5 55. rt massdiff -2-4 -6 15 1 2 4 6 8 cor.5.25 5. 2 4 6 8 2 4 6 8
Development of XCMS workflow Spectra reconstruction: TG(12:/12:/12:) (m/z 656.59) 3e+5 Fatty Acyl Substituent Neutral Loss (NL) RCOOH + NH 3 2e+5-2 12: 217 intensity massdiff 14: 245 15: 259 1e+5-4 16:1 271 16: 273 17:1 285 e+ -6 17: 287 47 48 49 rt 2 4 6 18:3 295 6e+6 18:2 297.95 18:1 299 18: 31.9 4e+6 19: 315 2:5 319 cor.85 2e+6 2:4 321 2:2 325 2:1 327.8 2: 329 22:6 345.75 e+ 2 4 6 2 4 6
Development of XCMS workflow Spectra reconstruction: TG(18:1/18:1/16:) (m/z 876.8) 6 Fatty Acyl Substituent Neutral Loss (NL) RCOOH + NH 3 4-1 12: 217 intensity massdiff 14: 245 15: 259 2-2 16:1 271 16: 273-3 17:1 285 17: 287 56 58 6 rt 6 7 8 9 18:3 295 1. 4e+5 18:2 297 18:1 299.9 18: 31 3e+5 19: 315.8 2:5 319 cor 2e+5 2:4 321.7 2:2 325 1e+5 2:1 327.6 2: 329 22:6 345 e+ 6 7 8 9 6 7 8 9
Development of Workflow with standards TIC of all standards
Development of Workflow with standards PC samples fragment m/z 184.7
Development of Workflow with standards PC samples fragment m/z 184.7.8 V 1.6 V 2.4 V 3.2 V 4. V 4.8 V
Development of Workflow with standards C. elegans samples fragment m/z 184.7
Development of Workflow with standards C. elegans samples fragment m/z 184.7.2 V 1. V 1.8 V 2.6 V 3.4 V 4.2 V
What can DMS now add? PC standards 4e+6 3e+6 2 sample PC_SWATH_pos_DMS15-PC variable PC_SWATH_pos_DMS16-PC value 2e+6 PC.36.5. PC.36.4. PC.36.3. intensity PC_SWATH_pos_DMS17-PC PC_SWATH_pos_DMS18-PC PC_SWATH_pos_DMS19-PC PC.36.2. 1 PC_SWATH_pos_DMS2-PC PC_SWATH_pos_DMS21-PC 1e+6 PC_SWATH_pos_DMS22-PC e+ -1 1 2 3 4 5 Cov 3 325 35 375 4 rt
What can DMS no add? C. elegans samples PC(36:6) 1 3 Area 75 5 25 intensity 2 1 sample Pos_LC-DMS-SWATH16 Pos_LC-DMS-SWATH17 Pos_LC-DMS-SWATH18 Pos_LC-DMS-SWATH2 Pos_LC-DMS-SWATH21 Pos_LC-DMS-SWATH22 Pos_LC-DMS-SWATH23 Pos_LC-DMS-SWATH24 Pos_LC-DMS-SWATH26 Pos_LC-DMS-SWATH27-2 2 4 Cov 2 225 25 275 3 rt
What can DMS no add? C. elegans samples PC(36:6) 3-1 intensity 2 massdiff -2-3 1-4 -5 1. 25 21 215 22 rt 8e+5 73 74 75 76 77 78 6e+5.8 cor 4e+5.6 2e+5.4 e+ 73 74 75 76 77 78 73 74 75 76 77 78
What can DMS no add? C. elegans samples TG(55:6) Fatty Acyl Substituent Neutral Loss (NL) RCOOH + NH 3 4-1 12: 217 14: 245 intensity 2 massdiff -2 15: 259 16:1 271 16: 273-3 17:1 285 17: 287 49. 492.5 495. 497.5 rt 6 7 8 9 18:3 295 1. 18:2 297 15 18:1 299.8 18: 31 1 19: 315 cor.6 5 2:5 319 2:4 321 2:2 325.4 2:1 327 6 7 8 9 6 7 8 9 2: 329 22:6 345
Acknowledgment AB Sciex Cyrus Papan Bernd Müller TU München Andreas Dunkel IPB Halle Steffen Neumann BGC, especially Philippe Schmitt-Kopplin
Acknowledgment Funding sources