Multidimensional Cross-Linking and Real-Time Informatics for Multiprotein Interaction Studies.

Chemical cross-linking combined with mass spectrometry is a technique used to study protein structures and identify protein complexes. Traditionally, chemical cross-linkers contain two reactive groups, allowing them to covalently bond a pair of proximal residues, either within a protein or between two proteins. The output of a cross-linking experiment is a list of interacting site pairs that provide structural constraints for modeling of new structures and complexes. Due to the binary reactive nature of cross-linking reagents, only pairs of interacting sites can be directly observed, and assembly of higher-order structures typically requires prior knowledge of complex composition or iterative docking to produce a putative model. Here, we describe a new tetrameric cross-linker bearing four amine-reactive groups, allowing it to covalently link up to four proteins simultaneously and a real-time instrument method to facilitate the identification of these tetrameric cross-links. We applied this new cross-linker to isolated mitochondria and identified a number of higher-order cross-links in various OXPHOS complexes and ATP synthase, demonstrating its utility in characterizing complex interfaces. We also show that higher-order cross-links can be used to effectively filter models of large protein assemblies generated by using Alphafold. Higher-dimensional cross-linking provides a new avenue for characterizing multiple protein interfaces, even in complex samples such as intact mitochondria.

Applications and advancements of FT-ICR-MS for interactome studies.

The set of all intra- and intermolecular interactions, collectively known as the interactome, is currently an unmet challenge for any analytical method, but if measured, could provide unparalleled insight on molecular function in living systems. Developments and applications of chemical cross-linking and high-performance mass spectrometry technologies are beginning to reveal details on how proteins interact in cells and how protein conformations and interactions inside cells change with phenotype or during drug treatment or other perturbations. A major contributor to these advances is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) technology and its implementation with accurate mass measurements on cross-linked peptide-pair precursor and fragment ions to enable improved identification methods. However, these applications place increased demands on mass spectrometer performance in terms of high-resolution spectral acquisition rates for on-line MSn experiments. Moreover, FT-ICR-MS also offers unique opportunities to develop and implement parallel ICR cells for multiplexed signal acquisition and the potential to greatly advance accurate mass acquisition rates for interactome studies. This review highlights our efforts to exploit accurate mass FT-ICR-MS technologies with chemical cross-linking and developments being pursued to realize parallel MS array capabilities that will further advance visualization of the interactome.

A planar quadrupole device for transmitting and trapping ions in high vacuum.

RATIONALE: Hybrid mass spectrometers combine multiple mass analyzers to achieve optimal performance in terms of tandem mass spectrometry, high mass resolving power, and mass measurement accuracy for studying highly complex samples. As a result, the need for transport, trapping, and control of ion kinetic energies is critical for the successful integration of multiple mass analyzers and hybrid instrument operation. In addition, transportation of ion populations between two physically distinct locations can result in time-of-flight (TOF) discrimination against ions with widely disparate m/z values, compromising full mass spectral performance. In this work, we demonstrated a new ion guide, referred to as a planar quadrupole (PQ) ion guide, composed of two parallel printed circuit boards (PCB) that allow radiofrequency (RF) and direct current (DC) voltages to be combined to enable both axial transport and trapping of ion populations in the ultrahigh vacuum region of the mass spectrometer. As compared with a conventional multipole ion guide, the PQ ion guide showed comparable performance in ion m/z values, signal-to-noise, and intensity and effectively reduced mass discrimination caused by TOF effects. METHODS: A PQ device was developed with two PCBs and simulated with SIMION 8.1. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry instrumentation were used for the testing of PQ performance. RESULTS: .In this work, we demonstrated a planar quadrupole (PQ) ion guide composed of two parallel PCB plates. The PQ enables both axial ion transport and trapping of ion populations throughout the ion transfer process from a LTQ to an ICR cell. As compared with a conventional multipole ion guide, the PQ showed comparable ion transmission efficiency and effectively reduced mass discrimination caused by TOF effects. CONCLUSIONS: The PQ is a simple design that can be implemented for ion transmission and trapping on virtually any mass spectrometer.

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically or into the cross-linker molecules. Isotope-labeled cross-linkers have primarily utilized deuterium and rely on MS1-based quantitation of precursor ion extracted ion chromatograms. Recently the development and application of isobaric quantitative protein interaction reporter (iqPIR) cross-linkers were reported, which utilize 13C and 15N isotope labels. Quantitation is accomplished using relative fragment ion isotope abundances in tandem mass spectra. Here we describe the synthesis and initial evaluation of a multiplexed set of iqPIR molecules, allowing for up to six cross-linked samples to be quantified simultaneously. To analyze data for such cross-linkers, the two-channel mode of iqPIR quantitative analysis was adapted to accommodate any number of channels with defined ion isotope peak mass offsets. The summed ion peak intensities in the overlapping channel isotope envelopes are apportioned among the channels to minimize the difference with respect to the predicted ion isotope envelopes. The result is accurate and reproducible relative quantitation enabling direct comparison among six differentially labeled cross-linked samples. The approach described here is generally extensible for the iqPIR strategy, accommodating future iqPIR reagent design, and enables large-scale in vivo quantitative XL-MS investigation of the interactome.

Application of frequency multiple FT-ICR-MS signal acquisition for improved proteome research.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with liquid chromatography (LC) is a powerful combination useful in many research areas due to the utility of high mass resolving power and mass measurement accuracy for studying highly complex samples. Ideally, every analyte in a complex sample can be subjected to accurate mass MS/MS analysis to aid in identification. FT-ICR MS can provide high mass resolving power and mass accuracy at the cost of long data acquisition periods, reducing the number of spectra that can be acquired per unit time. Frequency multiple signal acquisition has long been realized as an attractive method to obtain high mass resolving power and mass accuracy with shorter data acquisition periods. However, one of the limitations associated with frequency multiple signal acquisition is reduced signal intensity as compared to a traditional dipole detector. In this study, we demonstrated the use of a novel ICR cell to improve frequency multiple signal intensity and investigated the potential use of frequency multiple acquisition for proteome measurements. This novel ICR cell containing both dipole and frequency multiple detection electrodes was installed on a 7T FT-ICR MS coupled to an LC system. Tryptic digests of HeLa cell lysates were analyzed using dipole and frequency multiple detectors by holding either the mass resolving power or signal acquisition time constant. Compared to dipole detection, second frequency multiple detection yielded 36% or 45% more unique identified peptides from HeLa cell lysates at twice the scan rate or twice the mass resolving power, respectively. These results indicate that frequency multiple signal acquisition with either the same resolving power or the same signal acquisition duration as used with dipole signals can produce a significant increase in the number of peptides identified in complex proteome samples.

Isobaric Quantitative Protein Interaction Reporter Technology for Comparative Interactome Studies.

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful tool for the large-scale study of protein structures and interactions from complex biological samples including intact cells and tissues. Quantitative XL-MS (qXL-MS) provides unique information on protein conformational and interaction changes resulting from perturbations such as drug treatment and disease state. Previous qXL-MS studies relied on the incorporation of stable isotopes into the cross-linker (primarily deuterium) or metabolic labeling with SILAC. Here, we introduce isobaric quantitative protein interaction reporter (iqPIR) technology which utilizes stable isotopes selectively incorporated into the cross-linker design, allowing for isobaric cross-linked peptide pairs originating from different samples to display distinct quantitative isotope signatures in tandem mass spectra. This enables improved quantitation of cross-linked peptide levels from proteome-wide samples because of the reduced complexity of tandem mass spectra relative to MS1 spectra. In addition, because of the isotope incorporation in the reporter and the residual components of the cross-linker that remain on released peptides, each fragmentation spectrum can offer multiple independent opportunities and, therefore, improved confidence for quantitative assessment of the cross-linker pair level. Finally, in addition to providing information on solvent accessibility of lysine sites, dead end iqPIR cross-linked products can provide protein abundance and/or lysine site modification level information all from a single in vivo cross-linking experiment.

Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification.

Chemical cross-linking combined with mass spectrometry provides a method to study protein structures and interactions. The introduction of cleavable bonds in a cross-linker provides an avenue to decouple released peptide masses from their precursor species, greatly simplifying the downstream search, allowing for whole proteome investigations to be performed. Typically, these experiments have been challenging to carry out, often utilizing nonstandard methods to fully identify cross-linked peptides. Mango is an open source software tool that extracts precursor masses from chimeric spectra generated using cleavable cross-linkers, greatly simplifying the downstream search. As it is designed to work with chimeric spectra, Mango can be used on traditional high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers without the need for additional modifications. When paired with a traditional proteomics search engine, Mango can be used to identify several thousand cross-linked peptide pairs searching against the entire Escherichia coli proteome. Mango provides an avenue to perform whole proteome cross-linking experiments without specialized instrumentation or access to nonstandard methods.

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart.

Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondria respiration; and conformational enrichment of ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondria remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.

Systems structural biology measurements by in vivo cross-linking with mass spectrometry.

This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.

Elusive Conformational Dynamics of PPARγ Inactivation Tied Down by Chemical Cross-Linking.

In this issue of Structure, Zheng et al. (2018) have described the dynamics of PPARγ in complex with a non-agonist by exploring its solution-phase conformational landscape through chemical cross-linking in combination with a multitude of different treatment conditions, including their new synthetic anti-diabetic non-agonist, revealing the physical mechanism of PPARγ inactivation.

Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry.

Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.