The preparation and GC/MS analysis of biomass amino acids was performed as previously described by Antoniewicz et. al.18 Briefly, biomass pellets were hydrolyzed with 500 µL of 6N HCl at 110°C for 24 h, then dried under air at 65°C. Tert-butyldimethylsilyl (TBDMS) derivatives of amino acids were prepared by adding 35 µL of pyridine and 50 µL of MTBSTFA + 1% TBDMCS (Sigma 375934) and incubating for 30 minutes at 60°C, and were then transferred to injection vials for GC/MS analysis. 17 of the 20 amino acids were detected and quantified by this method. The three not measured, arginine, cysteine, and tryptophan, are estimated to constitute 12% of total protein mass.3 Since intracellular amino acid pools are known to be very small relative to proteinogenic amino acids19 (link), the measured signal will be dominated by the latter.
Tbdmcs
Manufactured by Merck Group
TBDMCS is a laboratory reagent used as a silylating agent in organic chemistry. It is commonly employed in the protection and derivatization of hydroxyl groups during various synthetic procedures.
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Lab products found in correlation
2 protocols using tbdmcs
1
GC/MS Analysis of Biomass Amino Acids
2
Intracellular Metabolite Profiling via GC-MS
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Cells were scraped using methanol-water. An equivalent volume of chloroform was then added, and the aqueous phase was collected and evaporated under airflow for polar intracellular metabolite analysis. After dissolution in 50 μL of 2% methoxyamine hydrochloride in pyridine, the tert-butylmethylsilyl derivative was prepared by adding 30 μL of N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MBTSTFA) + 1% tert-butyldimethylchlorosilane (TBDMCS; Sigma) and incubating at 55 °C for 1 h [22 ]. We monitored the ion clusters around m/z 459 (carbons 1-6 of citrate, electron impact ionization), m/z 174 (carbons 1–3 of pyruvate, electron impact ionization) and m/z 418 (carbons 1–4 of aspartate, electron impact ionization).
Mass spectral data were obtained on a 7890A mass spectrometer coupled with a 5675C gas chromatograph (Agilent Technologies). The settings are as follows: GC inlet 230 °C, transfer line 280 °C, MS source 230 °C, MS quad 150 °C. An HP-5 capillary column (30 m length, 250 μm diameter, 0.25 μm film thickness) was used for analysis of all metabolites.
Mass spectral data were obtained on a 7890A mass spectrometer coupled with a 5675C gas chromatograph (Agilent Technologies). The settings are as follows: GC inlet 230 °C, transfer line 280 °C, MS source 230 °C, MS quad 150 °C. An HP-5 capillary column (30 m length, 250 μm diameter, 0.25 μm film thickness) was used for analysis of all metabolites.
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