Quantification of intracellular phosphorylated carbohydrates in HT29 human colon adenocarcinoma cell line using liquid chromatography-electrospray ionization tandem mass spectrometry.
The quantitative understanding of the role of sugar phosphates in regulating tumor energetic metabolism at the proteomic and genomic level is a prerequisite for an efficient rational design in combined drug chemotherapy. Therefore, it is necessary to determine accurately the concentration of the main sugar phosphate pools at the lower concentrations present in the often-limited volume of tumor cell samples. Taking as an example the human adenocarcinoma cell line HT29, we here report a fast and reliable quantitative method based on the use of liquid nitrogen, a weak acid extraction, and liquid chromatography-electrospray ionization tandem mass spectrometry to quantify simultaneously the intracellular concentration of sugar phosphate pools. The method was set up using standard addition curves. Thus, it is possible to identify and quantify hexose phosphate, pentose phosphate, and triose phosphate pools up to 0.02-0.10 ng x microL(-1), depending on the analyte. The method developed was here used for the quantitative study of changes in phosphorylated carbohydrates of central carbon metabolism when high or low glucose concentration conditions are induced in vitro in the HT29 human colon adenocarcinoma cell line.
Vizán
Pedro
P
Department of Biochemistry and Molecular Biology, University of Barcelona, Av Diagonal 645, 08028 Barcelona, Spain.
Alcarraz-Vizán
Gema
G
Díaz-Moralli
Santiago
S
Rodríguez-Prados
Juan Carlos
JC
Zanuy
Míriam
M
Centelles
Josep J
JJ
Jáuregui
Olga
O
Cascante
Marta
M
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D005947:Glucose / Q000032:analysis / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000032:analysis / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D010427:Pentose Phosphate Pathway; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013403:Sugar Phosphates / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors
D013403:Sugar Phosphates; D019298:Glucose-6-Phosphate; D005947:Glucose