O-Phosphoamino acid analysis of phosphorylated proteins by gas chromatography with flame photometric detection

Kataoka, Hiroyuki; Ueno, Yukizo; Makita, Masami

doi:10.1016/0731-7085(92)80054-q

Cited by 5 publications

(1 citation statement)

References 26 publications

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“…Although these are polar substances, their suitable derivatives can be determined by GC . For example, phosphoserine was determined by GC before the extensive use of MS detectors , because this is an important analyte, not only for lipidomics, but also for proteomics . This metabolite is monitored in connection with some degenerative changes in the organism .…”

Section: Introductionmentioning

confidence: 99%

Gas chromatography with mass spectrometry analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate

et al. 2014

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A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two-step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less-reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C12-C19 for the DB-5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 μg/mL (trimethylsilylated phosphate) and 72 μg/mL (trimethylsilylated phosphoethanolamine).

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Section: Introductionmentioning

confidence: 99%

Gas chromatography with mass spectrometry analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate

et al. 2014

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Analysis of free and bound O‐phosphoamino acids in urine by gas chromatography with flame photometric detection

Kataoka

Nakai

Katagiri

et al. 1993

Biomedical Chromatography

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A selective and sensitive method has been developed for the analysis of free and bound forms of O-phosphoamino acids, such as O-phosphoserine, O-phosphothreonine and O-phosphotyrosine, in urine samples by gas chromatography (GC). For free O-phosphoamino acid analysis, the urine sample was extracted with trichloroacetic acid and run through an ion exchange column. For total (free plus bound) O-phosphoamino acid analysis, the urine sample was hydrolysed in acid and base in order to release the O-phosphoamino acid from peptides or proteins. O-Phosphoamino acids in these prepared samples were converted into their N-isobutoxycarbonyl trimethyl ester derivatives and then measured by GC with flame photometric detection (FPD-GC). The calibration curve was linear in the range of 10-500 ng for each O-phosphoamino acid, and the detection limits were 30-80 pg as injection amounts. O-Phosphoamino acids in the urine samples could be selectively determined by the FPD-GC method without any influence from coexisting substances. The recoveries of O-phosphoamino acids added to urines and urine hydrolysates were 90-98% and relative standard deviations were 1.5-8.0%. By using this method, we investigated an age dependence of O-phosphoamino acid excretion in normal subjects.

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Identification of O-phosphoamino acids in urine hydrolysate by gas chromatography—mass spectrometry

Kataoka

Nakai

Makita

1993

Journal of Chromatography B: Biomedical Sciences and Applicatio

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O-Phosphoamino acid analysis of phosphorylated proteins by gas chromatography with flame photometric detection

Cited by 5 publications

References 26 publications

Gas chromatography with mass spectrometry analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate

Gas chromatography with mass spectrometry analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate

Analysis of free and bound O‐phosphoamino acids in urine by gas chromatography with flame photometric detection

Identification of O-phosphoamino acids in urine hydrolysate by gas chromatography—mass spectrometry

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