Detection of four oxidation sites in viral prolyl‐4‐hydroxylase by top‐down mass spectrometry

• Published in: Protein science 2003-10, Vol.12 (10), p.2320-2326
• Main Authors: Ge, Ying, Lawhorn, Brian G., ElNaggar, Mariam, Sze, Siu Kwan, Begley, Tadhg P., McLafferty, Fred W.
• Format: Article
• Language: English
• Subjects: 5‐oxaproline; Amino Acid Sequence; Ascorbic Acid - metabolism; Catalytic Domain; Hydroxylation; Kinetics; Oxidation-Reduction; Oxygen - chemistry; Oxygen - metabolism; Peptides - metabolism; Phycodnaviridae - enzymology; Procollagen-Proline Dioxygenase - chemistry; Procollagen-Proline Dioxygenase - genetics; Procollagen-Proline Dioxygenase - metabolism; Proline - analogs & derivatives; Proline - metabolism; prolyl 4‐hydroxylase; Protein oxidation; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization - methods; Time Factors; top‐down mass spectrometry
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.03244403

Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl‐4‐hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 ± 3, and