Data-Independent Acquisition Coupled to Visible Laser-Induced Dissociation at 473 nm (DIA-LID) for Peptide-Centric Specific Analysis of Cysteine-Containing Peptide Subset.

Thanks to comprehensive and unbiased sampling of all precursor ions, the interest to move toward bottom-up proteomic with data-independent acquisition (DIA) is continuously growing. DIA offers precision and reproducibility performances comparable to true targeted methods but has the advantage of enabling retrospective data testing with the hypothetical presence of new proteins of interest. Nonetheless, the chimeric nature of DIA MS/MS spectra inherent to concomitant transmission of a multiplicity of precursor ions makes the confident identification of peptides often challenging, even with spectral library-based extraction strategy. The introduction of specificity at the fragmentation step upon ultraviolet or visible laser-induced dissociation (LID) range targeting only the subset of cysteine-containing peptides (Cys-peptide) has been proposed as an option to streamline and reduce the search space. Here, we describe the first coupling between DIA and visible LID at 473 nm to test for the presence of Cys-peptides with a peptide-centric approach. As a test run, a spectral library was built for a pool of Cys-synthetic peptides used as surrogates of human kinases (1 peptide per protein). By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in an Escherichia coli proteome lysate, DIA-LID demonstrates a dynamic range of detection of at least 3 decades and coefficients of precision better than 20%. Finally, the spectral library was used to search for endogenous kinases in human cellular extract.

Fragmentation patterns of chromophore-tagged peptides in visible laser induced dissociation.

RATIONALE: Tandem mass spectrometry (MS/MS) is the pivotal tool for protein structural characterization and quantification. Identification relies on the fragmentation step of tryptic peptides in bottom-up strategy. Specificity of fragmentation can be obtained using laser-induced dissociation (LID) in the visible range, after tagging of the targeted peptides with an adequate chromophore. Backbone fragmentation is required to obtain specific fragments and confident identification. We present herein a study of fragmentation patterns of chromophore-tagged peptides in LID, showing the potential of LID methodology to provide the maximum number of fragments for further identification and quantification. METHODS: A total of 401 cysteine-containing tryptic peptides originating from the human proteome were derivatizated on the thiol group of cysteine with a Dabcyl maleimide chromophore, which has a high photo-absorption cross section at 473 nm. The derivatized peptides were then analyzed by LID at 473 nm on a Q Exactive instrument. RESULTS: LID spectra present a characteristic fragment at m/z 252.112 for all precursors. This product ion arises from the internal dissociation of the Dabcyl chromophore. Several peptide-backbone fragment ions are also detected. Results show the quasi absence of fragmentation at the cysteine site. This indicates that part of the energy must be redistributed across the entire system despite excitation initially localized at the chromophore. Indeed, the fragmentation mainly occurs at 3 to 5 amino acids from the derivatized cysteine residue. CONCLUSIONS: LID of derivatized cysteine-containing peptides displays the initial fragmentation of the chromophore. As energy is redistributed all along the peptide sequence, fragmentation of the peptide backbone is also observed. Thus, LID of chromophore-tagged peptides produces adequate fragment ions, allowing both good sequence coverage for a greater confidence of identification, and a large choice of transitions for specific quantification.