#METABOLOMICS WORKBENCH allisonjlevy_20180725_085830 DATATRACK_ID:1464 STUDY_ID:ST001027 ANALYSIS_ID:AN001685 PROJECT_ID:PR000685 VERSION 1 CREATED_ON July 30, 2018, 2:58 pm #PROJECT PR:PROJECT_TITLE Influence of Data-Processing Strategies on Normalized Lipid Levels using an PR:PROJECT_TITLE Open-Source LC-HRMS/MS Lipidomics Workflow PR:PROJECT_TYPE MS Data Processing PR:PROJECT_SUMMARY Lipidomics is an emerging field with significant potential for improving PR:PROJECT_SUMMARY clinical diagnosis and our understanding of health and disease. While the PR:PROJECT_SUMMARY diverse biological roles of lipids contribute to their clinical utility, the PR:PROJECT_SUMMARY unavailability of lipid internal standards representing each species, make lipid PR:PROJECT_SUMMARY quantitation analytically challenging. The common approach is to employ one or PR:PROJECT_SUMMARY more internal standards for each lipid class examined and use a single point PR:PROJECT_SUMMARY calibration for normalization (relative quantitation). To aid in standardizing PR:PROJECT_SUMMARY and automating this relative quantitation process, we developed LipidMatch PR:PROJECT_SUMMARY Normalizer (LMN) http://secim.ufl.edu/secim-tools/ which can be used in most PR:PROJECT_SUMMARY open source lipidomics workflows. While the effect of lipid structure on PR:PROJECT_SUMMARY relative quantitation has been investigated, applying LMN we show that PR:PROJECT_SUMMARY data-processing can significantly affect lipid semi-quantitative amounts. PR:PROJECT_SUMMARY Polarity and adduct choice had the greatest effect on normalized levels; when PR:PROJECT_SUMMARY calculated using positive versus negative ion mode data, one fourth of lipids PR:PROJECT_SUMMARY had greater than 50 % difference in normalized levels. Based on our study, PR:PROJECT_SUMMARY sodium adducts should not be used for statistics when sodium is not added PR:PROJECT_SUMMARY intentionally to the system, as lipid levels calculated using sodium adducts did PR:PROJECT_SUMMARY not correlate with lipid levels calculated using any other adduct. Relative PR:PROJECT_SUMMARY quantification using smoothing versus not smoothing, and peak area versus peak PR:PROJECT_SUMMARY height, showed minimal differences, except when using peak area for overlapping PR:PROJECT_SUMMARY isomers which were difficult to deconvolute. By characterizing sources or PR:PROJECT_SUMMARY variation introduced during data-processing and introducing automated tools, PR:PROJECT_SUMMARY this work helps increase through-put and improve data-quality for determining PR:PROJECT_SUMMARY relative changes across groups. PR:INSTITUTE University of Florida PR:DEPARTMENT Chemistry PR:LABORATORY Richard Yost Laboratory PR:LAST_NAME Levy PR:FIRST_NAME Allison PR:ADDRESS 214 Leigh Hall, PO Box 117200, Gainesville, Florida, 32611, USA PR:EMAIL allisonjlevy@ufl.edu PR:PHONE 352-392-0515 #STUDY ST:STUDY_TITLE Influence of Data-Processing Strategies on Normalized Lipid Levels using an ST:STUDY_TITLE Open-Source LC-HRMS/MS Lipidomics Workflow ST:STUDY_SUMMARY Lipidomics is an emerging field with significant potential for improving ST:STUDY_SUMMARY clinical diagnosis and our understanding of health and disease. While the ST:STUDY_SUMMARY diverse biological roles of lipids contribute to their clinical utility, the ST:STUDY_SUMMARY unavailability of lipid internal standards representing each species, make lipid ST:STUDY_SUMMARY quantitation analytically challenging. The common approach is to employ one or ST:STUDY_SUMMARY more internal standards for each lipid class examined and use a single point ST:STUDY_SUMMARY calibration for normalization (relative quantitation). To aid in standardizing ST:STUDY_SUMMARY and automating this relative quantitation process, we developed LipidMatch ST:STUDY_SUMMARY Normalizer (LMN) http://secim.ufl.edu/secim-tools/ which can be used in most ST:STUDY_SUMMARY open source lipidomics workflows. While the effect of lipid structure on ST:STUDY_SUMMARY relative quantitation has been investigated, applying LMN we show that ST:STUDY_SUMMARY data-processing can significantly affect lipid semi-quantitative amounts. ST:STUDY_SUMMARY Polarity and adduct choice had the greatest effect on normalized levels; when ST:STUDY_SUMMARY calculated using positive versus negative ion mode data, one fourth of lipids ST:STUDY_SUMMARY had greater than 50 % difference in normalized levels. Based on our study, ST:STUDY_SUMMARY sodium adducts should not be used for statistics when sodium is not added ST:STUDY_SUMMARY intentionally to the system, as lipid levels calculated using sodium adducts did ST:STUDY_SUMMARY not correlate with lipid levels calculated using any other adduct. Relative ST:STUDY_SUMMARY quantification using smoothing versus not smoothing, and peak area versus peak ST:STUDY_SUMMARY height, showed minimal differences, except when using peak area for overlapping ST:STUDY_SUMMARY isomers which were difficult to deconvolute. By characterizing sources or ST:STUDY_SUMMARY variation introduced during data-processing and introducing automated tools, ST:STUDY_SUMMARY this work helps increase through-put and improve data-quality for determining ST:STUDY_SUMMARY relative changes across groups. ST:INSTITUTE University of Florida ST:DEPARTMENT Chemistry ST:LABORATORY Richard Yost Laboratory ST:LAST_NAME Levy ST:FIRST_NAME Allison ST:ADDRESS 214 Leigh Hall, PO Box 117200, Gainesville, Florida, 32611, USA ST:EMAIL allisonjlevy@ufl.edu ST:PHONE 3523920515 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - QC2_33_ddtargetedneg type:QC SUBJECT_SAMPLE_FACTORS - QC2_47_ddtargetedneg type:QC SUBJECT_SAMPLE_FACTORS - QC2_48_ddtargetedneg type:QC SUBJECT_SAMPLE_FACTORS - QC3_34_ddtargetedneg type:QC SUBJECT_SAMPLE_FACTORS - QC3_49_ddtargetedneg type:QC SUBJECT_SAMPLE_FACTORS - QC1_26_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC1_28_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC1_51_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC1_53_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC2_27_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC2_29_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC2_54_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC3_30_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC3_52_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC3_55_ddtargetedpos type:QC SUBJECT_SAMPLE_FACTORS - QC1_32_ddtargetedneg type:QC SUBJECT_SAMPLE_FACTORS - QC1_14_fullAIFneg type:QC SUBJECT_SAMPLE_FACTORS - QC1_37_fullAIFneg type:QC SUBJECT_SAMPLE_FACTORS - QC1_02_fullAIFpos type:QC SUBJECT_SAMPLE_FACTORS - QC2_12_fullAIFpos type:QC SUBJECT_SAMPLE_FACTORS - QC3_01_fullAIFpos type:QC SUBJECT_SAMPLE_FACTORS - QC3_01b_fullAIFpos type:QC SUBJECT_SAMPLE_FACTORS - blank_13_neg type:Blank SUBJECT_SAMPLE_FACTORS - blank_31_neg type:Blank SUBJECT_SAMPLE_FACTORS - blank_01_pos type:Blank SUBJECT_SAMPLE_FACTORS - blank_01c_pos type:Blank SUBJECT_SAMPLE_FACTORS - blank_25_pos type:Blank SUBJECT_SAMPLE_FACTORS - blank_50_pos type:Blank #COLLECTION CO:COLLECTION_SUMMARY National Institute for Standards and Technology (NIST) standard reference CO:COLLECTION_SUMMARY material (SRM 1950) Metabolites in Frozen Human Plasma was purchased for use in CO:COLLECTION_SUMMARY this study. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY No treatments were applied to the NIST SRM 1950 materials. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were isolated from 20 µL of National Institute for Standards and SP:SAMPLEPREP_SUMMARY Technology (NIST) standard reference material (SRM 1950) Metabolites in Frozen SP:SAMPLEPREP_SUMMARY Human Plasma. Lipid internal standards purchased from Avanti Lipids (Alabaster, SP:SAMPLEPREP_SUMMARY AL), which included lysophosphatidylcholine (LPC(17:0)), phosphatidylcholine SP:SAMPLEPREP_SUMMARY (PC(17:0/17:0)), phosphatidylglycerol (PG(17:0/17:0)), phosphatidylethanolamine SP:SAMPLEPREP_SUMMARY (PE(17:0/17:0)), phosphatidylserine (PS(17:0/17:0)), triglyceride SP:SAMPLEPREP_SUMMARY (TG(15:0/15:0/15:0)), ceramide (Cer(d18:1/17:0)), and sphingomyelin SP:SAMPLEPREP_SUMMARY (SM(d18:1/17:0)), were spiked into the plasma at 1.4 nmol, 0.92 nmol, 0.93 nmol, SP:SAMPLEPREP_SUMMARY 0.97 nmol, 0.92 nmol, 0.26 nmol, 1.3 nmol, and 0.98 nmol, respectively. SP:SAMPLEPREP_SUMMARY 13C2-cholesterol was purchased from Cambridge Isotope Laboratories (Tewksbury, SP:SAMPLEPREP_SUMMARY MA), and spiked in at 1.8 nmol. The extraction was performed using the Matyash SP:SAMPLEPREP_SUMMARY method [1] and samples were reconstituted in 200 µL of isopropanol. [1] SP:SAMPLEPREP_SUMMARY Matyash, V., Liebisch, G., Kurzchalia, T.V., Shevchenko, A., Schwudke, D.: Lipid SP:SAMPLEPREP_SUMMARY extraction by methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid SP:SAMPLEPREP_SUMMARY Res. 49, 1137–1146 (2008). doi:10.1194/jlr.D700041-JLR200 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Liquid Chromatography Protocol Samples were injected onto a Waters (Milford, MA) CH:CHROMATOGRAPHY_SUMMARY BEH C18 UHPLC column (50 x 2.1 mm, 1.7 µm) held at 50 °C with mobile phase A CH:CHROMATOGRAPHY_SUMMARY consisting of acetonitrile:water (60:40, v/v) with 10 mM ammonium formate and CH:CHROMATOGRAPHY_SUMMARY 0.1% formic acid and mobile phase B consisting of isopropanol:acetonitrile:water CH:CHROMATOGRAPHY_SUMMARY (90:8:2) with 10 mM ammonium formate and 0.1% formic acid at a flow rate of 0.5 CH:CHROMATOGRAPHY_SUMMARY mL/min. A Dionex Ultimate 3000 RS UHLPC system (Thermo Scientific, San Jose, CA) CH:CHROMATOGRAPHY_SUMMARY coupled to a Thermo Q-Exactive mass spectrometer (San Jose, CA) was employed for CH:CHROMATOGRAPHY_SUMMARY data acquisition. The UHPLC gradient use in this experiment is shown in Table 1. CH:CHROMATOGRAPHY_SUMMARY Time (min) C (%) D (%) 0,,80,,20 1,,80,,20 3,,70,,30 4,,55,,45 6,,40, 60 CH:CHROMATOGRAPHY_SUMMARY 8,,35,,65 10 ,35,,65 15 ,,10,,90 17,,2,,98 18,,2,,98 19,,80,,20 23,,80, 20 CH:CHROMATOGRAPHY_SUMMARY Gradient for reverse phase liquid chromatography of lipids. Mobile phase C CH:CHROMATOGRAPHY_SUMMARY consisted of 60:40 acetonitrile:water and mobile phase D consisted of 90:8:2 CH:CHROMATOGRAPHY_SUMMARY isopropanol:acetonitrile:water, with both containing 0.1% formic acid 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium formate. The flow rate was 500 µL/min. CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST001027_AN001685_Results.txt UNITS:peak area #END