Combined Infrared Multiphoton Dissociation with Ultraviolet Photodissociation for Ubiquitin Characterization.
Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C-Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C-N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization. Graphical Abstract ᅟ.
Institut Lumière Matière, Université Lyon 1 - CNRS, Université de Lyon, 69622, Villeurbanne, France.
Mohammad A
MA
Halim
Université de Lyon, Institut des Sciences Analytiques, UMR 5280, CNRS, Université Lyon 1, ENS Lyon, 69100, Villeurbanne, France.
Marion
M
Girod
Institut Lumière Matière, Université Lyon 1 - CNRS, Université de Lyon, 69622, Villeurbanne, France.
Luke
L
MacAleese
Université de Lyon, Institut des Sciences Analytiques, UMR 5280, CNRS, Université Lyon 1, ENS Lyon, 69100, Villeurbanne, France.
Jérôme
J
Lemoine
Institut Lumière Matière, Université Lyon 1 - CNRS, Université de Lyon, 69622, Villeurbanne, France.
Rodolphe
R
Antoine
Institut Lumière Matière, Université Lyon 1 - CNRS, Université de Lyon, 69622, Villeurbanne, France. philippe.dugourd@univ-lyon1.fr.
Philippe
P
Dugourd
IRMPD; Photodissociation; Top-down proteomics; UVPD; Ubiquitin