title: Identification of a gluconic acid derivative attached to the N-terminus of histidine-tagged proteins expressed in bacteria.
abstract: 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.
authors.affiliation: R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, 19477-0776, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Yan
authors.forename: G W
authors.initials: GW
authors.lastname: Caldwell
authors.forename: P A
authors.initials: PA
authors.lastname: McDonell
mesh_terms: 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
chemical_list: 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