Femtosecond laser‐induced ionization/dissociation tandem mass spectrometry (fsLID‐MS/MS) of deprotonated phosphopeptide anions

Abstract

Multiply deprotonated phosphopeptide anions were sequenced via negative-mode fsLID-MS/MS, with phosphosite localization facilitated by a/x ion series in addition to diagnostic x(n)-98 ions. fsLID-MS/MS is qualitatively competitive with other techniques. Further efficiency enhancements (e.g., implementation on a linear trap or/and higher pulse frequencies) may permit sequence analyses on chromatographic timescales.

Photodissociation mass spectrometry: new tools for characterization of biological molecules

“…[18][19][20] Photodissociation has also been demonstrated using a femtosecond titanium sapphire laser which produces near IR photons at 800 nm (1.5 eV per photon). [50][51][52] In the UV/vis range, Nd:YAG, excimer, and optical parametric oscillator (OPO):YAG lasers have been utilized for photodissociation. 17 Owing to the fact that the energies of UV photons are 20-100Â greater than IR photons, photodissociation can occur upon absorption of a single photon.…”

“…For example, recently a near-IR Ti-sapphire femtosecond laser (800 nm wavelength, 1.55 eV per photon) was used for multiphoton photodissociation. [50][51][52] The method was termed femtosecond laser-induced ionization/dissociation (fs-LID) because the process entailed escape of an electron from a protonated peptide prior to dissociation of the incipient oxidized radical intermediate. The resulting spectra for peptides contained a rich array of sequence ions, including a, b, c, x, y, and z ions.…”

193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes

“…FsLID has also been applied in negative ion mode for the analysis of multiply deprotonated phosphopeptide anions, where phosphate retaining a / x ions facilitate sequencing and modification localization. 24 Long activation times and narrow charge state restrictions ultimately have limited the adoption of fsLID for large-scale analysis.…”

“…No phosphate neutral loss was detected from c / z or a / x ions with the exception of potentially diagnostic a + 1–98 ions observed C-terminal to the site of phosphorylation. FsLID has also been applied in negative ion mode for the analysis of multiply deprotonated phosphopeptide anions, where phosphate retaining a / x ions facilitate sequencing and modification localization . Long activation times and narrow charge state restrictions ultimately have limited the adoption of fsLID for large-scale analysis.…”

Phosphoproteomics in the Age of Rapid and Deep Proteome Profiling

“…The vast majority of proteomics workflows use positive electrospray ionization with LC–MS/MS to fragment peptide cations with collisional activation, which limits detection of species that prefer deprotonation over protonation. While collision-based dissociation approaches do not generate reproducible sequence-informative product ion spectra of negatively charged peptides, several photodissociation and electron-driven fragmentation methods have emerged to facilitate high-throughput proteomics in the negative mode. − Because the negative charge of phosphoryl groups can lead to preferential ionization of phosphopeptides as anions, , these negative mode approaches have the potential to provide a new dimension to phosphoproteomic experiments. Holistically, there are certainly many interesting avenues to explore with phosphopeptide fragmentation.…”