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MB Sample ID: SA217788

Local Sample ID:Tips_B
Subject ID:SU002357
Subject Type:Other organism
Subject Species:Xenopus tropicalis
Age Or Age Range:Stage 41-43

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Subject:

Subject ID:SU002357
Subject Type:Other organism
Subject Species:Xenopus tropicalis
Age Or Age Range:Stage 41-43

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Tips_BSA217788FL026436TipsTimepoint

Collection:

Collection ID:CO002350
Collection Summary:Tadpoles were anesthetized with 0.05% MS-222 in 1/9x MR and tested for response to touch prior to tissue collection. For uninjured tips (Tips), a sterilized scalpel was used to amputate 500µm of the posterior tip. For 0 hours post amputation (hpa) samples, the posterior third of the tail was amputated followed by collection of tail tissue 250µm anterior to the initial wound. For 3 and 24hpa, 250µm of tissue, including the regenerating tissue, was collected. 10 replicates of 25 tails (8 replicates for the 24hpa timepoint) were collected. For each sample, media was removed, and samples were frozen on dry ice within 5-8 minutes from the first amputation.
Sample Type:Tails

Treatment:

Treatment ID:TR002369
Treatment Summary:No applicable

Sample Preparation:

Sampleprep ID:SP002363
Sampleprep Summary:Aqueous metabolites for targeted LC-MS analysis were extracted using a protein precipitation method similar to the one described elsewhere (Mathon et al., 2019; Meador et al., 2020). Samples were first homogenized in 200 µL purified deionized water at 4 ˚C, and then 800 µL of methanol containing 6C13-glucose and 2C13-glutamate (reference internal standards) was added. Afterwards samples were vortexed, stored for 30 minutes at -20 ˚C, sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 14,000 rpm and 4 ˚C, and then 600 µL of supernatant was collected from each sample. Lastly, recovered supernatants were dried on a SpeedVac and reconstituted in 1.0 mL of LC-matching solvent containing 2C13-tyrosine and 3C13-lactate (reference internal standards).
Sampleprep Protocol Filename:JPatel_LC-MS_Methods.pdf

Combined analysis:

Analysis ID AN003712 AN003713
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Shimadzu Nexera X2 Shimadzu Nexera X2
Column Waters XBridge BEH Amide XP Waters XBridge BEH Amide XP
MS Type ESI ESI
MS instrument type Triple quadrupole Triple quadrupole
MS instrument name ABI Sciex 6500+ ABI Sciex 6500+
Ion Mode POSITIVE NEGATIVE
Units Peak Intensity Peak Intensity

Chromatography:

Chromatography ID:CH002750
Instrument Name:Shimadzu Nexera X2
Column Name:Waters XBridge BEH Amide XP
Chromatography Type:HILIC

MS:

MS ID:MS003461
Analysis ID:AN003712
Instrument Name:ABI Sciex 6500+
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. The LC-MS assay was targeting 361 metabolites (plus 4 spiked reference internal standards).
Ion Mode:POSITIVE
Analysis Protocol File:JPatel_LC-MS_Methods.pdf
  
MS ID:MS003462
Analysis ID:AN003713
Instrument Name:ABI Sciex 6500+
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. The LC-MS assay was targeting 361 metabolites (plus 4 spiked reference internal standards).
Ion Mode:NEGATIVE
Analysis Protocol File:JPatel_LC-MS_Methods.pdf
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