Top-down mass analysis of protein tyrosine nitration: comparison of electron capture dissociation with "slow-heating" tandem mass spectrometry methods.
Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the "slow-heating" threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the "slow-heating" methods.
Mikhailov
Victor A
VA
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Iniesta
Jesus
J
Cooper
Helen J
HJ
D045304:Cytochromes c / Q000737:chemistry; D005583:Fourier Analysis; D009113:Muramidase / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D009574:Nitro Compounds / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry*
D009211:Myoglobin; D009574:Nitro Compounds; D011506:Proteins; D014443:Tyrosine; D045304:Cytochromes c; D009113:Muramidase