Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA083384

Local Sample ID:	39-Synechocystis_6803-cell-630p-3
Subject ID:	SU001265
Subject Type:	Bacteria
Subject Species:	Synechocystis sp. PCC 6803
Taxonomy ID:	1148
Genotype Strain:	NCBI:txid1148
Cell Biosource Or Supplier:	ATCC

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Sample Preparation:

Sampleprep ID:	SP001273
Sampleprep Summary:	Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and the bottom, clear MeOH:H2O layer were separated into separate tubes and dried under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and dried under N2,gas. The protocol outlined above is suitable for filter-quenched cyanobacteria samples and centrifuged cell pellets. The polar methanol/water fraction resulting from the biphasic extraction was processed for analysis by hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was mixed and aliquoted into twelve vials. A QC injection was run every tenth injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC until derivatization, immediately prior to MS analysis. Samples were derivatized in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following section. Ten microliters were removed from each sample to create a pooled QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection. The non-polar MTBE phase was processed for non-targeted LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and aliquoted into thirteen pooled QC samples. QC injections were placed after every sixth injection. An acid hydrolysis protocol was developed for identification of amino acids, nucleosides, and carbohydrates bound in insoluble pellet of protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the biphasic extraction were removed from storage at -80 degrees C and residual solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. The resulting suspension was a bright teal. The suspension was transferred equally two three separate glass vials for hydrolysis of the separate polymer constituents. Hydrolysis of proteins to amino acids was completed with a hydrochloric acid (HCl) hydrolysis, based on previously published protocols (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for with a loose cap seal. After 4 hours, the acid in each vial was entirely evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, and returned to 110 degrees C. After a total of 24 hours, vials were removed, and remaining acid was evaporated under nitrogen gas. Samples were resuspended in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the pooled QC was aliquoted into fourteen vials, and the solvent was evaporated under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid in each sample was manually checked and curated in the software Chromeleon™. Aspartic acid with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Threonine with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and allowed to cool, and 100 uL was transferred to a glass teardrop vial. The remaining pellet was incubated at 160 degrees C for 40 minutes, removed and allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was transferred to the glass teardrop vial which contained purines from the 130 degree C incubation. The acid was evaporated under nitrogen gas. Samples were removed after the 10 minute 130ºC incubation to preserve purines (guanine and adenine) from degradation during the 40 minute 160ºC incubation.

Sampleprep ID:

SP001273

Sampleprep Summary:

Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and the bottom, clear MeOH:H2O layer were separated into separate tubes and dried under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and dried under N2,gas. The protocol outlined above is suitable for filter-quenched cyanobacteria samples and centrifuged cell pellets. The polar methanol/water fraction resulting from the biphasic extraction was processed for analysis by hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was mixed and aliquoted into twelve vials. A QC injection was run every tenth injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC until derivatization, immediately prior to MS analysis. Samples were derivatized in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following section. Ten microliters were removed from each sample to create a pooled QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection. The non-polar MTBE phase was processed for non-targeted LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and aliquoted into thirteen pooled QC samples. QC injections were placed after every sixth injection. An acid hydrolysis protocol was developed for identification of amino acids, nucleosides, and carbohydrates bound in insoluble pellet of protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the biphasic extraction were removed from storage at -80 degrees C and residual solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. The resulting suspension was a bright teal. The suspension was transferred equally two three separate glass vials for hydrolysis of the separate polymer constituents. Hydrolysis of proteins to amino acids was completed with a hydrochloric acid (HCl) hydrolysis, based on previously published protocols (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for with a loose cap seal. After 4 hours, the acid in each vial was entirely evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, and returned to 110 degrees C. After a total of 24 hours, vials were removed, and remaining acid was evaporated under nitrogen gas. Samples were resuspended in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the pooled QC was aliquoted into fourteen vials, and the solvent was evaporated under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid in each sample was manually checked and curated in the software Chromeleon™. Aspartic acid with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Threonine with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and allowed to cool, and 100 uL was transferred to a glass teardrop vial. The remaining pellet was incubated at 160 degrees C for 40 minutes, removed and allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was transferred to the glass teardrop vial which contained purines from the 130 degree C incubation. The acid was evaporated under nitrogen gas. Samples were removed after the 10 minute 130ºC incubation to preserve purines (guanine and adenine) from degradation during the 40 minute 160ºC incubation.