Top-"Double-Down" Mass Spectrometry of Histone H4 Proteoforms: Tandem Ultraviolet-Photon and Mobility/Mass-Selected Electron Capture Dissociations.

Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

Spatially Resolved Neuropeptide Characterization from Neuropathological Formalin-Fixed, Paraffin-Embedded Tissue Sections by a Combination of Imaging MALDI FT-ICR Mass Spectrometry Histochemistry and Liquid Extraction Surface Analysis-Trapped Ion Mobility Spectrometry-Tandem Mass Spectrometry.

To make the vast collections of well-documented human clinical samples archived in biobanks accessible for mass spectrometry imaging (MSI), recent developments have focused on the label-free top-down MS analysis of neuropeptides in sections of formalin-fixed, paraffin-embedded (FFPE) tissues. In analogy to immunohistochemistry (IHC), this variant of MSI has been designated MSHC (mass spectrometry histochemistry). Besides the detection and localization of neuropeptide and other biomolecular MS signals in these FFPE samples, there is great interest in their molecular identification and full characterization. We here used matrix assisted laser desorption ionization (MALDI) MSI employing ultrahigh-resolution FT-ICR MS on 2,5-dihydroxybenzoic acid (DHB) coated five-micron sections of human FFPE pituitary to demonstrate clear isotope patterns and elemental composition assignment of neuropeptides (with ∼1 ppm mass accuracy). Besides tandem MS fragmentation pattern analysis to deduce or confirm amino acid sequence information (Arg-vasopressin for the case presented here), there is a need for orthogonal primary structure characterization of the peptide-like MS signals of biomolecules desorbed directly off FFPE tissue sections. In the present work, we performed liquid extraction surface analysis (LESA) extractions on consecutive (uncoated) tissue slices. This enables the successful characterization by ion mobility MS of vasopressin present in FFPE material. Differences in sequence coverage are discussed on the basis of the mobility selected collision induced dissociation (CID), electron capture dissociation (ECD), and UV photodissociation (UVPD) MS/MS. Using Arg-vasopressin as model case (a peptide with a disulfide bridged ring structure), we illustrate the use of LESA in combination with a reduction agent for effective sequencing using mobility selected CID, ECD, and UVPD MS/MS.

Mapping chemotherapeutic drug distribution in cancer cell spheroids using 2D-TOF-SIMS and LESA-TIMS-MS.

Three-dimensional (3D) cancer cell cultures grown in the form of spheroids are effective models for the study of in vivo-like processes simulating cancer tumor pharmacological dynamics and morphology. In this study, we show the advantages of Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) combined with in situ Liquid Extraction Surface Analysis coupled to trapped Ion Mobility Spectrometry Mass Spectrometry (LESA-TIMS-TOF MS) for high spatial resolution mapping and quantitation of ABT-737, a chemotherapeutic drug, at the level of single human colon carcinoma cell spheroids (HCT 116 MCS). 2D-TOF-SIMS studies of consecutive sections (∼16 µm thick slices) showed that ABT-737 is homogenously distributed in the outer layers of the HCT 116 MCS. Complementary in situ LESA-TIMS-TOF MS/MS measurements confirmed the presence of the ABT-737 drug in the MCS slides by the observation of the molecular ion [M + H]+m/z and mobility, and the charateristic fragmentation pattern. LESA-TIMS-TOF MS allowed a quantitative assessment of the ABT-737 drug of the control MCS slice spiked with ABT-737 standard over the 0.4-4.1 ng range and MCS treated starting at 10 µM for 24 h. These experiments showcase an effective protocol for unambigous characterization and 3D mapping of chemotherapeutic drug distribution at the single MCS level.