#METABOLOMICS WORKBENCH fernandezlab_20200802_095818 DATATRACK_ID:2108 STUDY_ID:ST001438 ANALYSIS_ID:AN002402 PROJECT_ID:000000 VERSION 1 CREATED_ON August 2, 2020, 12:27 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 TENGi_MSC 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. A second example to illustrate TENGi MS capabilities involves rare ST:STUDY_SUMMARY cell metabolomics of cultured mesenchymal stromal cells (MSCs), a cell type that ST:STUDY_SUMMARY has shown potential for treating a variety of chronic diseases. Examination of ST:STUDY_SUMMARY metabolic changes of MSCs cultured under conditions that may impact in vitro ST:STUDY_SUMMARY therapeutic activity, such as aggregate culture, or preconditioning with ST:STUDY_SUMMARY interferon gamma (IFN- γ)13, is critical for identifying attributes of cell ST:STUDY_SUMMARY quality. Reducing cell numbers required to perform MSC metabolomic analysis is ST:STUDY_SUMMARY essential for improving the manufacturing of highly therapeutic MSCs without ST:STUDY_SUMMARY significantly impeding production. ST:INSTITUTE Georgia Institute of Technology ST:LAST_NAME Fernandez ST:FIRST_NAME Facundo ST:ADDRESS 901 Atlantic Dr NE, Atlanta, GA, 30332, USA ST:EMAIL fernandez@gatech.edu ST:PHONE 404-385-4432 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - S1_1 Group:Stimulated RAW_FILE_NAME=S1-Posi-1 SUBJECT_SAMPLE_FACTORS - S1_2 Group:Stimulated RAW_FILE_NAME=S1-Posi-2 SUBJECT_SAMPLE_FACTORS - S2_1 Group:Stimulated RAW_FILE_NAME=S2-Posi-1 SUBJECT_SAMPLE_FACTORS - S2_3 Group:Stimulated RAW_FILE_NAME=S2-Posi-3 SUBJECT_SAMPLE_FACTORS - S3_1 Group:Stimulated RAW_FILE_NAME=S3-Posi-1 SUBJECT_SAMPLE_FACTORS - S3_2 Group:Stimulated RAW_FILE_NAME=S3-Posi-2 SUBJECT_SAMPLE_FACTORS - S4_2 Group:Stimulated RAW_FILE_NAME=S4-Posi-2 SUBJECT_SAMPLE_FACTORS - S4_3 Group:Stimulated RAW_FILE_NAME=S4-Posi-3 SUBJECT_SAMPLE_FACTORS - S5_1 Group:Stimulated RAW_FILE_NAME=S5-Posi-1 SUBJECT_SAMPLE_FACTORS - S5_3 Group:Stimulated RAW_FILE_NAME=S5-Posi-3 SUBJECT_SAMPLE_FACTORS - S6_1 Group:Stimulated RAW_FILE_NAME=S6-Posi-1 SUBJECT_SAMPLE_FACTORS - S6_2 Group:Stimulated RAW_FILE_NAME=S6-Posi-2 SUBJECT_SAMPLE_FACTORS - U1_1 Group:Unstimulated RAW_FILE_NAME=U1-Posi-1 SUBJECT_SAMPLE_FACTORS - U1_2 Group:Unstimulated RAW_FILE_NAME=U1-Posi-2 SUBJECT_SAMPLE_FACTORS - U2_2 Group:Unstimulated RAW_FILE_NAME=U2-Posi-2 SUBJECT_SAMPLE_FACTORS - U2_3 Group:Unstimulated RAW_FILE_NAME=U2-Posi-3 SUBJECT_SAMPLE_FACTORS - U3_1 Group:Unstimulated RAW_FILE_NAME=U3-Posi-1 SUBJECT_SAMPLE_FACTORS - U3_3 Group:Unstimulated RAW_FILE_NAME=U3-Posi-3 SUBJECT_SAMPLE_FACTORS - U4_1 Group:Unstimulated RAW_FILE_NAME=U4-Posi-1 SUBJECT_SAMPLE_FACTORS - U4_2 Group:Unstimulated RAW_FILE_NAME=U4-Posi-2 SUBJECT_SAMPLE_FACTORS - U5_2 Group:Unstimulated RAW_FILE_NAME=U5-Posi-2 SUBJECT_SAMPLE_FACTORS - U5_3 Group:Unstimulated RAW_FILE_NAME=U5-Posi-3 SUBJECT_SAMPLE_FACTORS - U6_1 Group:Unstimulated RAW_FILE_NAME=U6-Posi-1 SUBJECT_SAMPLE_FACTORS - U6_3 Group:Unstimulated RAW_FILE_NAME=U6-Posi-3 SUBJECT_SAMPLE_FACTORS - Blank_2 Group:Blank RAW_FILE_NAME=Blank-Posi-2 SUBJECT_SAMPLE_FACTORS - Blank_3 Group:Blank RAW_FILE_NAME=Blank-Posi-3 SUBJECT_SAMPLE_FACTORS - QC_A1 Group:QC RAW_FILE_NAME=QC-Posi-A1 SUBJECT_SAMPLE_FACTORS - QC_A2 Group:QC RAW_FILE_NAME=QC-Posi-A2 SUBJECT_SAMPLE_FACTORS - QC_B2 Group:QC RAW_FILE_NAME=QC-Posi-B2 SUBJECT_SAMPLE_FACTORS - QC_B3 Group:QC RAW_FILE_NAME=QC-Posi-B3 SUBJECT_SAMPLE_FACTORS - QC_C1 Group:QC RAW_FILE_NAME=QC-Posi-C1 SUBJECT_SAMPLE_FACTORS - QC_C3 Group:QC RAW_FILE_NAME=QC-Posi-C2 #COLLECTION CO:COLLECTION_SUMMARY Bone marrow-derived MSCs (RoosterBio Inc., Lot #000139) were expanded for two CO:COLLECTION_SUMMARY passages in culture after being received. They were frozen in ~5x105 aliquots in CO:COLLECTION_SUMMARY Cryostor CS10 freeze media (BioLife). Frozen aliquots were revived and plated in CO:COLLECTION_SUMMARY tissue culture polystyrene flasks (Corning) for 3-4 days prior to seeding onto CO:COLLECTION_SUMMARY test surfaces. MSCs were cultured in low-glucose DMEM (Gibco) supplemented with CO:COLLECTION_SUMMARY 10% fetal bovine serum (FBS, Atlanta Biologicals, lot E16063), and 1% CO:COLLECTION_SUMMARY antibiotic/antimycotic solution (Gibco). Once confluent, MSCs were washed with CO:COLLECTION_SUMMARY sterile-filtered phosphate-buffered saline (PBS, Thermo Fisher) and detached CO:COLLECTION_SUMMARY from flasks using TrypLE express (Thermo Fisher). Dissociated cells were counted CO:COLLECTION_SUMMARY using a hemacytometer and replated at 13,000 cells/cm2 in T-75 tissue culture CO:COLLECTION_SUMMARY flasks. After overnight incubation, MSCs then were exposed to 48 hours of CO:COLLECTION_SUMMARY culture media (control conditions), or culture media supplemented with 50 ng/mL CO:COLLECTION_SUMMARY IFN- γ (Thermo Fisher). MSCs were then washed with PBS and trypsinized as CO:COLLECTION_SUMMARY described above, and the number of harvested cells counted using a CO:COLLECTION_SUMMARY hemocytometer. CO:SAMPLE_TYPE Mesenchymal stromal cells #TREATMENT TR:TREATMENT_SUMMARY MSCs were then washed with PBS and trypsinized as described above, and the TR:TREATMENT_SUMMARY number of harvested cells counted using a hemocytometer. MSCs were then TR:TREATMENT_SUMMARY resuspended in 155 mM ammonium acetate (Fluka) at a concentration of 1.6x106 TR:TREATMENT_SUMMARY cells/mL and aliquoted into 50-µL samples (8x104 cells per aliquot). Cells were TR:TREATMENT_SUMMARY then quenched by adding 200-µL MeOH into each sample vial and stored at -80 TR:TREATMENT_SUMMARY C until metabolite extraction. Frozen cells were subject to three freeze-thaw TR:TREATMENT_SUMMARY cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. TR:TREATMENT_SUMMARY Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate TR:TREATMENT_SUMMARY proteins. From the supernatant, 200 µL was transferred into a new vial for TR:TREATMENT_SUMMARY lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. TR:TREATMENT_SUMMARY All cell extracts and the QC sample were then lyophilized at -40 C and 100 TR:TREATMENT_SUMMARY mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, TR:TREATMENT_SUMMARY USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic TR:TREATMENT_SUMMARY solution to a final volume of 10 µL (for samples), and 18 µL (for QCs). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for SP:SAMPLEPREP_SUMMARY freezing and ice-water sonication for thawing. Cell samples were then SP:SAMPLEPREP_SUMMARY centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the SP:SAMPLEPREP_SUMMARY supernatant, 200 µL was transferred into a new vial for lyophilization. The SP:SAMPLEPREP_SUMMARY pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts SP:SAMPLEPREP_SUMMARY and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a SP:SAMPLEPREP_SUMMARY VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were SP:SAMPLEPREP_SUMMARY reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final SP:SAMPLEPREP_SUMMARY volume of 10 µL (for samples), and 18 µL (for QCs). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS See attached Method file MS:MS_RESULTS_FILE ST001438_AN002402_Results.txt UNITS:Normalized Intensity (Intensity ratio against internal standard signal)) Has m/z:Yes Has RT:No RT units:No RT data #END