#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