Premium
Tandem mass spectrometry of multiply phosphorylated forms of a ‘histidine‐tag’ derived from a recombinant protein kinase expressed in bacteria
Author(s) -
Du Ping,
Loulakis Pat,
Xie Zhi,
Simons Samuel P.,
Geoghegan Kieran F.
Publication year - 2005
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1821
Subject(s) - chemistry , recombinant dna , phosphorylation , tandem , tandem mass spectrometry , bacteria , mass spectrometry , histidine , biochemistry , chromatography , enzyme , gene , materials science , genetics , biology , composite material
When a histidine‐tagged form of the protein kinase Aurora‐2 was expressed in Escherichia coli , the purified product carried four to nine phosphate groups, although many fewer were expected. The amino‐terminal tag had the sequence GSSSSGLVPRGSHMK‐. Tryptic digestion of the product followed by analysis by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) showed that phosphorylation could occur on the five serine residues of the tag. Mono‐, bis‐, tris‐, tetra‐ and pentaphosphorylated forms of the tag were detected, and their behavior in MS/MS was studied using a quadrupole/time‐of‐flight mass spectrometer. The MS/MS spectra were dominated by the products of neutral loss events (in 98 Da increments, each equivalent to loss of H 3 PO 4 ), but sufficient b‐ and y‐type sequence ions were detected to allow the locations of the phosphates to be specified in some cases. The assignment of phosphorylation sites for incompletely phosphorylated forms of the tag peptide was challenging, but it appeared that Ser‐10 and Ser‐11 of the tag were more likely to be phosphorylated than Ser‐2 and Ser‐3. Copyright © 2005 John Wiley & Sons, Ltd.