Trace detection of styphnate from pre- and post-blast samples by hydrophilic interaction chromatography with tandem mass spectrometry.

RATIONALE: Although ubiquitous in explosives and ammunition, few trace methods for detection of heavy metal-containing primary explosives from forensic samples are currently in practice. METHODS: Extracts of cotton swabs or direct sampling of items were cleaned up using solid-phase extraction to remove heavy metal contaminants (i.e., lead) while retaining the organic styphnate component. The styphnate was chromatographically separated using hydrophilic interaction chromatography (HILIC) and detected via high-resolution tandem mass spectrometry (MS/MS) using a sensitive, targeted approach in five minutes or less. RESULTS: A mass spectrometric method for the detection of styphnate, including limit of detection (LOD), sample stability, and interferences was developed. We present a validated method for the extraction, separation, and detection of styphnate from lead(II) styphnate with an estimated LOD of 257 ppt (pg/mL). CONCLUSIONS: We detail an improved LOD relative to previous reports for trace detection of styphnate and, for the first time to our knowledge, the post-blast analysis of styphnate.

Separation and Collision Cross Section Measurements of Protein Complexes Afforded by a Modular Drift Tube Coupled to an Orbitrap Mass Spectrometer.

New developments in analytical technologies and biophysical methods have advanced the characterization of increasingly complex biomolecular assemblies using native mass spectrometry (MS). Ion mobility methods, in particular, have enabled a new dimension of structural information and analysis of proteins, allowing separation of conformations and providing size and shape insights based on collision cross sections (CCSs). Based on the concepts of absorption-mode Fourier transform (aFT) multiplexing ion mobility spectrometry (IMS), here, a modular drift tube design proves capable of separating native-like proteins up to 148 kDa with resolution up to 45. Coupled with high-resolution Orbitrap MS, binding of small ligands and cofactors can be resolved in the mass domain and correlated to changes in structural heterogeneity observed in the ion-neutral CCS distributions. We also demonstrate the ability to rapidly determine accurate CCSs for proteins with 1-min aFT-IMS-MS sweeps without the need for calibrants or correction factors.

Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase.

The recent discovery of asymmetric arrangements of trimers in the tautomerase superfamily (TSF) adds structural diversity to this already mechanistically diverse superfamily. Classification of asymmetric trimers has previously been determined using X-ray crystallography. Here, native mass spectrometry (MS) and ultraviolet photodissociation (UVPD) are employed as an integrated strategy for more rapid and sensitive differentiation of symmetric and asymmetric trimers. Specifically, the unfolding of symmetric and asymmetric trimers initiated by collisional heating was probed using UVPD, which revealed unique gas-phase unfolding pathways. Variations in UVPD patterns from native-like, compact trimeric structures to unfolded, extended conformations indicate a rearrangement of higher-order structure in the asymmetric trimers that are believed to be stabilized by salt-bridge triads, which are absent from the symmetric trimers. Consequently, the symmetric trimers were found to be less stable in the gas phase, resulting in enhanced UVPD fragmentation overall and a notable difference in higher-order re-structuring based on the extent of hydrogen migration of protein fragments. The increased stability of the asymmetric trimers may justify their evolution and concomitant diversification of the TSF. Facilitating the classification of TSF members as symmetric or asymmetric trimers assists in delineating the evolutionary history of the TSF.

Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.

Enhanced Ion Mobility Separation and Characterization of Isomeric Phosphatidylcholines Using Absorption Mode Fourier Transform Multiplexing and Ultraviolet Photodissociation Mass Spectrometry.

The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon-carbon double bonds (C═C) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.

Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation.

Analysis of native-like protein structures in the gas phase via native mass spectrometry and auxiliary techniques has become a powerful tool for structural biology applications. In combination with ultraviolet photodissociation (UVPD), native top-down mass spectrometry informs backbone flexibility, topology, hydrogen bonding networks, and conformational changes in protein structure. Although it is known that the primary structure affects dissociation of peptides and proteins in the gas phase, its effect on the types and locations of backbone cleavages promoted by UVPD and concomitant influence on structural characterization of native-like proteins is not well understood. Here, trends in the fragmentation of native-like proteins were evaluated by tracking the propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top down UVPD data set encompassing >9600 fragment ions. Differing fragmentation trends are reported for the production of distinct fragment types, attributed to a combination of both direct dissociation pathways from excited electronic states and those surmised to involve intramolecular vibrational energy redistribution after internal conversion. The latter pathways were systematically evaluated to evince the role of proton mobility in the generation of "CID-like" fragments through UVPD, providing pertinent insight into the characterization of native-like proteins. Fragmentation trends presented here are envisioned to enhance analysis of the protein higher-order structure or augment scoring algorithms in the high-throughput analysis of intact proteins.

Enhanced Characterization of Membrane Protein Complexes by Ultraviolet Photodissociation Mass Spectrometry.

Development of chemical chaperones to solubilize membrane protein complexes in aqueous solutions has allowed for gas-phase analysis of their native-like assemblies, including rapid evaluation of stability and interacting partners. Characterization of protein primary sequence, however, has thus far been limited. Ultraviolet photodissociation (UVPD) generates a multitude of sequence ions for the E. coli ammonia channel (AmtB), provides improved localization of a possible post-translational modification of aquaporin Z (AqpZ), and surpasses previous reports of sequence coverage for mechanosensitive channel of large conductance (MscL). Variations in UVPD sequence ion abundance have been shown to correspond to structural changes induced upon some perturbation. Preliminary results are reported here for elucidating increased rigidity or flexibility of MscL when bound to various phospholipids.

Mapping RNAPII CTD Phosphorylation Reveals That the Identity and Modification of Seventh Heptad Residues Direct Tyr1 Phosphorylation.

The C-terminal domain (CTD) of the largest subunit in eukaryotic RNA polymerase II has a repetitive heptad sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 which is responsible for recruiting transcriptional regulatory factors. The seventh heptad residues in mammals are less conserved and subject to various post-translational modifications, but the consequences of such variations are not well understood. In this study, we use ultraviolet photodissociation mass spectrometry, kinetic assays, and structural analyses to dissect how different residues or modifications at the seventh heptad position alter Tyr1 phosphorylation. We found that negatively charged residues in this position promote phosphorylation of adjacent Tyr1 sites, whereas positively charged residues discriminate against it. Modifications that alter the charges on seventh heptad residues such as arginine citrullination negate such distinctions. Such specificity can be explained by conserved, positively charged pockets near the active sites of ABL1 and its homologues. Our results reveal a novel mechanism for variations or modifications in the seventh heptad position directing subsequent phosphorylation of other CTD sites, which can contribute to the formation of various modification combinations that likely impact transcriptional regulation.

Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb.

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD's coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription.

The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, whereas Ser2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. We found that Ssu72 dephosphorylates pSer5 effectively but only has low activities toward pSer7 and pSer2 The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ß-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. Our results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II that occur at different stages of transcription.

Impact of charge state on 193 nm ultraviolet photodissociation of protein complexes.

As applications in mass spectrometry continue to expand into the field of structural biology, there have been an increasing number of studies on noncovalent biological assemblies. Ensuring that protein complexes maintain native-like conformations and architectures during the transition from solution to the gas phase is a key aim. Probing composition and arrangement of subunits of multi-charged complexes via tandem mass spectrometry (MS/MS) may lead to protein unfolding and the redistribution of charges on the constituent subunits, leading to asymmetric charge partitioning and ejection of a high-charged monomer. Additionally, the overall dissociation efficiency of many ion activation methods is often suppressed for low charge states, hindering the effectiveness of MS/MS for complexes that have low charge density. Ultraviolet photodissociation (UVPD) of proteins using 193 nm photons is a high-energy alternative to collisional activation and demonstrates little to no charge state dependence. Here the symmetry of charge partitioning upon UVPD is evaluated for an array of multimeric protein complexes as a function of initial charge state. The results demonstrate that high laser energies (3 mJ) for UVPD induces more symmetric charge partitioning and ejection of low-charged, presumably compact monomers than higher-energy collisional dissociation (HCD).