Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization.

Owing to its ability to generate extensive fragmentation of proteins, ultraviolet photodissociation (UVPD) mass spectrometry (MS) has emerged as a versatile ion activation technique for the structural characterization of native proteins and protein complexes. Interpreting these fragmentation patterns provides insight into the secondary and tertiary structures of protein ions. However, the inherent complexity and diversity of proteins often pose challenges in resolving their numerous conformations. To address this limitation, we combined UVPD-MS with drift tube ion mobility, offering potential to acquire conformationally selective MS/MS information. A low-pressure drift tube (LPDT) Orbitrap mass spectrometer equipped with 193 nm UVPD capabilities enables the analysis of protein conformers through the analysis of arrival time distributions (ATDs) of individual fragment ions. ATDs of fragment ions are compared for different backbone cleavage sites of the protein or different precursor charge states to give information about regions of potential folding or elongation. This integrated platform offers promise for advancing our understanding of protein structures in the gas phase.

UniChromCD for Demultiplexing Time-Resolved Charge Detection-Mass Spectrometry Data.

Charge detection mass spectrometry (CD-MS) enables characterization of large, heterogeneous analytes through the analysis of individual ion signals. Because hundreds to thousands of scans must be acquired to produce adequate ion statistics, CD-MS generally requires long analysis times. The slow acquisition speed of CD-MS complicates efforts to couple it with time-dispersive techniques, such as chromatography and ion mobility, because it is not always possible to acquire enough scans from a single sample injection to generate sufficient ion statistics. Multiplexing methods based on Hadamard and Fourier transforms offer an attractive solution to this problem by improving the duty cycle of the separation while preserving retention/drift time information. However, integrating multiplexing with CD-MS data processing is complex. Here, we present UniChromCD, a new module in the open-source UniDec package that incorporates CD-MS time-domain data processing with demultiplexing tools. Following a detailed description of the algorithm, we demonstrate its capabilities using two multiplexed CD-MS workflows: Hadamard-transform size-exclusion chromatography and Fourier-transform ion mobility. Overall, UniChromCD provides a user-friendly interface for analysis and visualization of time-resolved CD-MS data.

MS-TAFI: A Tool for the Analysis of Fragment Ions Generated from Intact Proteins.

Tandem mass spectrometry (MS/MS) spectra of intact proteins can be difficult to interpret owing to the variety of fragment ion types and abundances. This information is crucial for maximizing the information derived from top-down mass spectrometry of proteins and protein complexes. MS-TAFI (Mass Spectrometry Tool for the Analysis of Fragment Ions) is a free Python-based program which offers a streamlined approach to the data analysis and visualization of deconvoluted MS/MS data of intact proteins. The application also contains tools for native mass spectrometry experiments with the ability to search for fragment ions that retain ligands (holo ions) as well as visualize the location of charge sites obtained from 193 nm ultraviolet photodissociation data. The source code and complete application for MS-TAFI is available for download at https://github.com/kylejuetten.

Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

Top-Down Analysis of Supercharged Proteins Using Collision-, Electron-, and Photon-Based Activation Methods.

The impact of supercharging on the fragmentation patterns of six proteins, ubiquitin, cytochrome c, staph nuclease, myoglobin, dihydrofolate reductase, and carbonic anhydrase, was investigated for five activation methods, HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD under denaturing conditions. Changes in sequence coverage, alterations in the number and abundance of preferential cleavages (N-terminal to proline, C-terminal to aspartic or glutamic acid, adjacent to aromatic residues), and changes in individual fragment ion abundances were evaluated. Large decreases in sequence coverage were observed upon supercharging of proteins activated by HCD, whereas modest gains were observed for ETD. Minimal changes in sequence coverage were observed when using EThcD, 213 nm UVPD, and 193 nm UVPD, all of which tended to display the highest sequence coverages of the activation methods. Specific preferential backbone cleavage sites were increasingly enhanced for all proteins in supercharged states for all activation methods, particularly for HCD, 213 nm UVPD, and 193 nm UVPD. Even if large gains in sequence coverages were not apparent for the highest charge states, supercharging consistently led to at least a few new backbone cleavage sites for ETD, EThcD, 213 nm UVPD, and 193 nm UVPD for all proteins.

Distinctive interactomes of RNA polymerase II phosphorylation during different stages of transcription.

During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription.