Ultraviolet Photodissociation of ESI- and MALDI-Generated Protein Ions on a Q-Exactive Mass Spectrometer

Abstract

The identification of molecular ions produced by MALDI or ESI strongly relies on their fragmentation to structurally informative fragments. The widely diffused fragmentation techniques for ESI multiply charged ions are either incompatible (ECD and ETD) or show lower efficiency (CID, HCD), with the predominantly singly charged peptide and protein ions formed by MALDI. In-source decay has been successfully adopted to sequence MALDI-generated ions, but it further increases spectral complexity, and it is not compatible with mass-spectrometry imaging. Excellent UVPD performances, in terms of number of fragment ions and sequence coverage, has been demonstrated for electrospray ionization for multiple proteomics applications. UVPD showed a much lower charge-state dependence, and so protein ions produced by MALDI may exhibit equal propensity to fragment. Here we report UVPD implementation on an Orbitrap Q-Exactive Plus mass spectrometer equipped with an ESI/EP-MALDI. UVPD of MALDI-generated ions was benchmarked against MALDI-ISD, MALDI-HCD, and ESI-UVPD. MALDI-UVPD outperformed MALDI-HCD and ISD, efficiently sequencing small proteins ions. Moreover, the singly charged nature of MALDI-UVPD avoids the bioinformatics challenges associated with highly congested ESI-UVPD mass spectra. Our results demonstrate the ability of UVPD to further improve tandem mass spectrometry capabilities for MALDI-generated protein ions. Data are available via ProteomeXchange with identifier PXD011526.

Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules

“…This landmark study opened a new frontier of top-down analysis using 193 nm UVPD, including adoption of the method for detailed characterization of proteins containing post-translational modifications, ,, engineered mutations, unnatural amino acids, , solvent-accessibility tags, and cross-linked sites . The successful characterization of proteins using 193 nm photoactivation also motivated the implementation of UVPD on other high-performance mass spectrometers including a 15 T FTICR platform and other variations of Orbitrap systems, ,, which offer sufficiently high mass accuracy and mass resolution to allow assignment of fragment ions in the dense spectra created by UVPD of intact proteins. UVPD (193 nm) has also been used to characterize native-like folded proteins in the context of structural biology applications, as described in the next section.…”

MS Amanda 2.0: Advancements in the standalone implementation

“…Several groups have reported the common occurrence of a+1, x+1, and y-1 ions in UVPD spectra [39], [40]. We wanted to investigate the applicability of these ion types to be used for scoring and tested various ion settings on HeLa samples measured on a Thermo Fisher QExactive using UVPD peptide fragmentation (PXD003109 [39]).…”

“…In addition to the common ions such as a, b, x, y, or z fragments, UVPD also often generates additional fragment ions such as a+1, c, x+1, or y-1 ions [39], [40]. Consideration of these ion types for scoring is now also supported by MS Amanda.…”

Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Adds Enhanced Functionalities to MALDI MS Profiling for Disease Diagnostics

“…In addition, multiply charged ions typically lead to superior fragmentation in collision-induced dissociation (CID)-based MS/MS (cf. peptide sequencing) and are a requirement for electron-mediated MS/MS analysis. , Ultraviolet photodissociation improves the fragmentation of singly charged protein ions, but the technology has limited availability on commercial instrumentation, and the requirement of high m / z mass analyzers remains for singly charged protein ions . Advantageously, liquid MALDI also benefits from virtually all of the advantages of solid MALDI, such as high tolerance toward sample additives/contaminants, post-analysis sample recovery and storage, and all of the advantages of a laser-based method, such as speed and a tightly controllable desorption event, while adding additional key features such as greater sample homogeneity, greater ion beam stability, and lower sample consumption …”