Identification of a gluconic acid derivative attached to the N-terminus of histidine-tagged proteins expressed in bacteria.
Our previous studies have shown that the His tag cleaved from fusion proteins contained two distinct components P1 and P2. P1 has been identified to be a His-tagged peptide of G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R resulted from initiator methionine deletion, and P2 contains an unknown moiety at the second residue glycine of the tag (x-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R, x = 178.0 Da). This study aimed to determine the structure of the modification by using a combination of protein isotope labeling and mass spectrometry. His-tagged FKBP was expressed in (15)N and (13)C labeling growth media respectively. Isotopic labeled His-tagged proteins ((15)N-His-FKBP and (13)C-His-FKBP) were isolated by affinity chromatography and subjected to Xa digestions to release the labeled His tag. Subsequent analyses of the released His tag by MALDI-TOF-MS indicated a mass difference of 178.0 +/- 0.2 Da, between the two (15)N-labeled peptides P1 and P2, suggesting that the modification moiety contained no nitrogen. A mass difference of 184.0 +/- 0.2 Da was observed on MALDI between (13)C-labeled peptide P1 and P2, indicating six carbons in the modification group. Also, comparing the mass shift on MALDI spectra of P1 and P2 after hydrogen/deuterium exchange revealed that the modification moiety had five hydroxyl groups. It was concluded that the modification was a gluconic acid derivative attached to the N-terminus of His-tagged proteins expressed in bacteria. The proposed structure was further confirmed by MALDI analysis of periodate oxidation products of His-tagged peptides.
Yan
Z
Z
R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, 19477-0776, USA.
Caldwell
G W
GW
McDonell
P A
PA
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005942:Gluconates; D006639:Histidine; D020104:Immunophilins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D022021:Tacrolimus Binding Proteins
D002247:Carbon Isotopes; D005942:Gluconates; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D006639:Histidine; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; C030691:gluconic acid