#METABOLOMICS WORKBENCH fernandezlab_20200729_082424 DATATRACK_ID:2106 STUDY_ID:ST001437 ANALYSIS_ID:AN002401 PROJECT_ID:PR000988 VERSION 1 CREATED_ON July 31, 2020, 3:44 pm #PROJECT PR:PROJECT_TITLE Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited PR:PROJECT_TITLE samples PR:PROJECT_SUMMARY The human metabolome provides a window into the mechanisms and biomarkers of PR:PROJECT_SUMMARY various diseases. However, because of limited availability, many sample types PR:PROJECT_SUMMARY are still difficult to study by metabolomic analyses. Here, we present a new PR:PROJECT_SUMMARY mass spectrometry (MS)-based metabolomics strategy that only consumes PR:PROJECT_SUMMARY sub-nanoliter sample volumes. The approach consists of combining a customized PR:PROJECT_SUMMARY metabolomics workflow with a pulsed MS ion generation method, known as PR:PROJECT_SUMMARY triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi PR:PROJECT_SUMMARY nanoESI) MS. Samples tested for this approach included exhaled breath PR:PROJECT_SUMMARY condensates (EBC) collected from cystic fibrosis (CF) patients as well as in PR:PROJECT_SUMMARY vitro-cultured human mesenchymal stromal cells (MSCs). Both test samples were PR:PROJECT_SUMMARY only available in minimum amounts. Experiments showed that picoliter-volume PR:PROJECT_SUMMARY spray pulses sufficed to generate high-quality spectral fingerprints, which PR:PROJECT_SUMMARY increased the information density produced per unit sample volume. This TENGi PR:PROJECT_SUMMARY nanoESI strategy has the potential to fill in the gap in metabolomics where PR:PROJECT_SUMMARY liquid chromatography-MS-based analyses cannot be applied. Our method could open PR:PROJECT_SUMMARY up new avenues for future investigations into understanding metabolic changes PR:PROJECT_SUMMARY caused by diseases or external stimuli. PR:INSTITUTE Georgia Institute of Technology PR:LAST_NAME Fernandez PR:FIRST_NAME Facundo PR:ADDRESS 901 Atlantic Dr NE, Atlanta, GA, 30332, USA PR:EMAIL fernandez@gatech.edu PR:PHONE 404-385-4432 #STUDY ST:STUDY_TITLE Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited ST:STUDY_TITLE samples ST:STUDY_SUMMARY The human metabolome provides a window into the mechanisms and biomarkers of ST:STUDY_SUMMARY various diseases. However, because of limited availability, many sample types ST:STUDY_SUMMARY are still difficult to study by metabolomic analyses. Here, we present a new ST:STUDY_SUMMARY mass spectrometry (MS)-based metabolomics strategy that only consumes ST:STUDY_SUMMARY sub-nanoliter sample volumes. The approach consists of combining a customized ST:STUDY_SUMMARY metabolomics workflow with a pulsed MS ion generation method, known as ST:STUDY_SUMMARY triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi ST:STUDY_SUMMARY nanoESI) MS. The first set of samples tested for this approach included exhaled ST:STUDY_SUMMARY breath condensates (EBC) collected from cystic fibrosis (CF) patients with ST:STUDY_SUMMARY impaired glucose tolerance to study the metabolome changes before and after the ST:STUDY_SUMMARY oral glucose tolerance test. ST:INSTITUTE Georgia Institute of Technology ST:LAST_NAME Facundo ST:FIRST_NAME Fernandez ST:ADDRESS 901 Atlantic Dr NW ST:EMAIL fernandez@gatech.edu ST:PHONE (404) 385-4432 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 11B01 Group:Post RAW_FILE_NAME=TENGI-11B-NEG-01 SUBJECT_SAMPLE_FACTORS - 11B02 Group:Post RAW_FILE_NAME=TENGI-11B-NEG-02 SUBJECT_SAMPLE_FACTORS - 12B01 Group:Post RAW_FILE_NAME=TENGI-12B-NEG-01 SUBJECT_SAMPLE_FACTORS - 12B02 Group:Post RAW_FILE_NAME=TENGI-12B-NEG-02 SUBJECT_SAMPLE_FACTORS - 13B01 Group:Post RAW_FILE_NAME=TENGI-13B-NEG-01 SUBJECT_SAMPLE_FACTORS - 13B03 Group:Post RAW_FILE_NAME=TENGI-13B-NEG-03 SUBJECT_SAMPLE_FACTORS - 14B01 Group:Post RAW_FILE_NAME=TENGI-14B-NEG-01 SUBJECT_SAMPLE_FACTORS - 14B02 Group:Post RAW_FILE_NAME=TENGI-14B-NEG-02 SUBJECT_SAMPLE_FACTORS - 15B01 Group:Post RAW_FILE_NAME=TENGI-15B-NEG-01 SUBJECT_SAMPLE_FACTORS - 15B02 Group:Post RAW_FILE_NAME=TENGI-15B-NEG-02 SUBJECT_SAMPLE_FACTORS - 16B01 Group:Post RAW_FILE_NAME=TENGI-16B-NEG-01 SUBJECT_SAMPLE_FACTORS - 16B02 Group:Post RAW_FILE_NAME=TENGI-16B-NEG-02 SUBJECT_SAMPLE_FACTORS - 17B02 Group:Post RAW_FILE_NAME=TENGI-17B-NEG-02 SUBJECT_SAMPLE_FACTORS - 17B03 Group:Post RAW_FILE_NAME=TENGI-17B-NEG-03 SUBJECT_SAMPLE_FACTORS - 8B02 Group:Post RAW_FILE_NAME=TENGI-8B-NEG-02 SUBJECT_SAMPLE_FACTORS - 8B03 Group:Post RAW_FILE_NAME=TENGI-8B-NEG-03 SUBJECT_SAMPLE_FACTORS - 9B01 Group:Post RAW_FILE_NAME=TENGI-9B-NEG-01 SUBJECT_SAMPLE_FACTORS - 9B03 Group:Post RAW_FILE_NAME=TENGI-9B-NEG-03 SUBJECT_SAMPLE_FACTORS - 10A01 Group:Pre RAW_FILE_NAME=TENGI-10A-NEG-01 SUBJECT_SAMPLE_FACTORS - 10A02 Group:Pre RAW_FILE_NAME=TENGI-10A-NEG-02 SUBJECT_SAMPLE_FACTORS - 11A01 Group:Pre RAW_FILE_NAME=TENGI-11A-NEG-01 SUBJECT_SAMPLE_FACTORS - 11A02 Group:Pre RAW_FILE_NAME=TENGI-11A-NEG-02 SUBJECT_SAMPLE_FACTORS - 12A01 Group:Pre RAW_FILE_NAME=TENGI-12A-NEG-01 SUBJECT_SAMPLE_FACTORS - 12A02 Group:Pre RAW_FILE_NAME=TENGI-12A-NEG-02 SUBJECT_SAMPLE_FACTORS - 13A01 Group:Pre RAW_FILE_NAME=TENGI-13A-NEG-01 SUBJECT_SAMPLE_FACTORS - 13A03 Group:Pre RAW_FILE_NAME=TENGI-13A-NEG-03 SUBJECT_SAMPLE_FACTORS - 14A01 Group:Pre RAW_FILE_NAME=TENGI-14A-NEG-01 SUBJECT_SAMPLE_FACTORS - 14A02 Group:Pre RAW_FILE_NAME=TENGI-14A-NEG-02 SUBJECT_SAMPLE_FACTORS - 16A01 Group:Pre RAW_FILE_NAME=TENGI-16A-NEG-01 SUBJECT_SAMPLE_FACTORS - 16A02 Group:Pre RAW_FILE_NAME=TENGI-16A-NEG-02 SUBJECT_SAMPLE_FACTORS - 17A01 Group:Pre RAW_FILE_NAME=TENGI-17A-NEG-01 SUBJECT_SAMPLE_FACTORS - 17A02 Group:Pre RAW_FILE_NAME=TENGI-17A-NEG-02 SUBJECT_SAMPLE_FACTORS - 2A02 Group:Pre RAW_FILE_NAME=TENGI-2A-NEG-02 SUBJECT_SAMPLE_FACTORS - 2A03 Group:Pre RAW_FILE_NAME=TENGI-2A-NEG-03 SUBJECT_SAMPLE_FACTORS - 8A02 Group:Pre RAW_FILE_NAME=TENGI-8A-NEG-02 SUBJECT_SAMPLE_FACTORS - 8A03 Group:Pre RAW_FILE_NAME=TENGI-8A-NEG-03 SUBJECT_SAMPLE_FACTORS - 9A01 Group:Pre RAW_FILE_NAME=TENGI-9A-NEG-01 SUBJECT_SAMPLE_FACTORS - 9A02 Group:Pre RAW_FILE_NAME=TENGI-9A-NEG-02 SUBJECT_SAMPLE_FACTORS - Blank02 Group:Blank RAW_FILE_NAME=TENGI-BLANK-NEG-02 SUBJECT_SAMPLE_FACTORS - Blank03 Group:Blank RAW_FILE_NAME=TENGI-BLANK-NEG-03 SUBJECT_SAMPLE_FACTORS - QC_A01 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-A01 SUBJECT_SAMPLE_FACTORS - QC_A03 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-A03 SUBJECT_SAMPLE_FACTORS - QC_B02 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-B02 SUBJECT_SAMPLE_FACTORS - QC_B03 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-B03 SUBJECT_SAMPLE_FACTORS - QC_C01 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-C01 SUBJECT_SAMPLE_FACTORS - QC_C02 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-C02 SUBJECT_SAMPLE_FACTORS - QC_D01 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-D01 SUBJECT_SAMPLE_FACTORS - QC_D02 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-D02 SUBJECT_SAMPLE_FACTORS - QC_E02 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-E02 SUBJECT_SAMPLE_FACTORS - QC_E03 Group:QC RAW_FILE_NAME=TENGI-QC-NEG-E03 #COLLECTION CO:COLLECTION_SUMMARY EBC from 11 CF patients with abnormalities in glucose homeostasis (as defined by CO:COLLECTION_SUMMARY having prediabetes/impaired glucose tolerance [CF IGT] on an oral glucose CO:COLLECTION_SUMMARY tolerance test) was collected both fasting and 2 hours following ingestion of a CO:COLLECTION_SUMMARY glucose drink. They were termed as “Pre” and “Post”, respectively. Of CO:COLLECTION_SUMMARY these 11 subjects, one did not have an adequate sample for the “Pre” and CO:COLLECTION_SUMMARY another did not have a sample for the “Post” study giving a total of 20 CO:COLLECTION_SUMMARY samples analyzed (one of then failed to purduce enough features as others and CO:COLLECTION_SUMMARY was regarded as an outlier and removed). EBC sample collection followed the CO:COLLECTION_SUMMARY guidelines approved by the Georgia Institute of Technology and the Emory CO:COLLECTION_SUMMARY University Institutional Review Boards (approval number IRB00000372). Samples CO:COLLECTION_SUMMARY were collected with an R-Tube collector (Respiratory Research, Inc., Austin, TX, CO:COLLECTION_SUMMARY USA). After collection, samples were immediately frozen at -80℃ until CO:COLLECTION_SUMMARY processed. CO:SAMPLE_TYPE Exhaled Breath condensate #TREATMENT TR:TREATMENT_SUMMARY A total of 20 EBC samples (10 for each group), each 50 µL in volume before TR:TREATMENT_SUMMARY concentration, were phenotyped. A pooled sample, which was used as a QC, was TR:TREATMENT_SUMMARY prepared by taking 5 µL from each EBC sample and mixing the aliquots together. TR:TREATMENT_SUMMARY Then, the 20 samples, together with the pooled QC sample and the blank sample TR:TREATMENT_SUMMARY (containing only ultrapure water) were lyophilized at -40 C and 100 mTorr for TR:TREATMENT_SUMMARY 24h in a VirTis Benchtop freeze-drier (LP Industries, Stone Ridge, NY, USA). TR:TREATMENT_SUMMARY Residues were then reconstituted in 9 μL of methanol/water 1:9 (v:v) with TR:TREATMENT_SUMMARY 1×10-6 M 13C-tyrosine spiked in. This resulted in a 5-fold concentration TR:TREATMENT_SUMMARY factor. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A total of 20 EBC samples (10 for each group), each 50 µL in volume before SP:SAMPLEPREP_SUMMARY concentration, were phenotyped. A pooled sample, which was used as a QC, was SP:SAMPLEPREP_SUMMARY prepared by taking 5 µL from each EBC sample and mixing the aliquots together. SP:SAMPLEPREP_SUMMARY Then, the 20 samples, together with the pooled QC sample and the blank sample SP:SAMPLEPREP_SUMMARY (containing only ultrapure water) were lyophilized at -40 C and 100 mTorr for SP:SAMPLEPREP_SUMMARY 24h in a VirTis Benchtop freeze-drier (LP Industries, Stone Ridge, NY, USA). SP:SAMPLEPREP_SUMMARY Residues were then reconstituted in 9 μL of methanol/water 1:9 (v:v) with SP:SAMPLEPREP_SUMMARY 1×10-6 M 13C-tyrosine spiked in. This resulted in a 5-fold concentration SP:SAMPLEPREP_SUMMARY factor. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 S QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Negative ion mode in the 50-750 m/z range was used for experiments Detailed data MS:MS_COMMENTS process see attached Method file MS:MS_RESULTS_FILE ST001437_AN002401_Results.txt UNITS:Normalized Intensity (Intensity ratio against internal standard signal)) Has m/z:Yes Has RT:No RT units:No RT data #END