Separation and identification of enzymatically prepared dephosphorylated products of myo-inositolhexakisphosphate using LC-MS.
LC-MS technique described here is a new way for the separation and direct determination of UV-Vis insensitive inositol phosphates (InsP(2)-InsP(6)). This circumvents the need of radioisotopic labeling and post-column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP(6) on C(18)-column using MeOH/H(2)O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI-MS). The LC-MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP(6 )in a sequential manner leading to InsP(2 )(InsP(2) x 2Na, t(R) 4.4-4.54 min, base peak m/z 382.9) as the end product.
Vats
Purva
P
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA.
Bhushan
Bharat
B
Chakarborti
Asit K
AK
Banerjee
Uttam C
UC
D010832:6-Phytase / Q000737:chemistry*; D001234:Aspergillus niger / Q000201:enzymology; D002853:Chromatography, Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D010766:Phosphorylation; D010833:Phytic Acid / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010833:Phytic Acid; D010832:6-Phytase