Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation

Abstract

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer’s disease and in cancer pathogenesis. The 5 nanosecond, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision-based and electron-based dissociation methods.

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis

“…Unit mass resolving power is achieved at 2 Hz spectral acquisition rate for proteins as large as bovine serum albumin (66 kDa), and 2,000,000 resolving power is demonstrated for a 12 s detection period. Collisional and electron transfer dissociation offer efficient protein structural characterization, and UV photodissociation [34] is planned. We expect the first applications of the instrument to focus on top-down proteomics [35], mapping contact surfaces in large protein complexes by hydrogen-deuterium exchange [36], and petroleomics [37].…”

“…Future work will focus on increasing the speed and efficiency of fragmentation and identification of additional fragments (e.g., internal fragments and/or neutral losses other than water). A barium fluoride window on the ICR cell flange enables future implementation of photodissociation induced by UV (193 [34] or 266 nm), vis (532 nm), or IR (10.6 micron) photons.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…As a result, multiply-charged species are formed during ESI and the total analyte signal is distributed across multiple channels corresponding to the individual charge states. For proteins, which may range in molecular weights from 4,000 Da, in the case of some ribosomal proteins [37], to approximately 150,000 Da, in the case of mAbs [38], ESI enables detection of intact species on most commercial instruments. Depending on the instrument, signal distribution spans across multiple charge states in the m/z range between 100 -4,000 Th.…”

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

“…All MS experiments were performed on a Thermo Scientific Orbitrap Elite mass spectrometer (San Jose, CA, USA) custom fit with an unfocused, non-collimated Coherent ExciStar 193 nm excimer laser (Santa Clara, CA, USA) to perform ultraviolet photodissociation, as previously described [46,47]. Peptides, both modified and unmodified, w ere analyzed by direct infusion electrospray ionization with a spray voltage of 4 kV and a capillary temperature of 275°C.…”