Abstract. We describe the design and initial performance of the first 21 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The 21 tesla magnet is the highest field superconducting magnet ever used for FT-ICR and features high spatial homogeneity, high temporal stability, and negligible liquid helium consumption. The instrument includes a commercial dual linear quadrupole trap front end that features high sensitivity, precise control of trapped ion number, and collisional and electron transfer dissociation. A third linear quadrupole trap offers high ion capacity and ejection efficiency, and rf quadrupole ion injection optics deliver ions to a novel dynamically harmonized ICR cell. Mass resolving power of 150,000 (m/Δm 50% ) is achieved for bovine serum albumin (66 kDa) for a 0.38 s detection period, and greater than 2,000,000 resolving power is achieved for a 12 s detection period. Externally calibrated broadband mass measurement accuracy is typically less than 150 ppb rms, with resolving power greater than 300,000 at m/z 400 for a 0.76 s detection period. Combined analysis of electron transfer and collisional dissociation spectra results in 68% sequence coverage for carbonic anhydrase. The instrument is part of the NSF High-Field FT-ICR User Facility and is available free of charge to qualified users.

Protein interaction topologies are critical determinants of biological function. Large-scale or proteome-wide measurements of protein interaction topologies in cells currently pose an unmet challenge that could dramatically improve understanding of complex biological systems. A primary impediment includes direct protein topology and interaction measurements from living systems since interactions that lack biological significance may be introduced during cell lysis. Furthermore, many biologically relevant protein interactions will likely not survive the lysis/sample preparation and may only be measured with in vivo methods. As a step toward meeting this challenge, a new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed that enables assignment of cross-linked peptides “on-the-fly”. Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were identified from cross-linked E. coli cells. These data allow assembly of the first protein interaction network that also contains topological features of every interaction, as it existed in cells during cross-linker application. Of the identified interprotein cross-linked peptide pairs, 40% are derived from known interactions and provide new topological data that can help visualize how these interactions exist in cells. Other identified cross-linked peptide pairs are from proteins known to be involved within the same complex, but yield newly discovered direct physical interactors. ReACT enables the first view of these interactions inside cells, and the results acquired with this method suggest cross-linking can play a major role in future efforts to map the interactome in cells.

The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring possibility to study details of the protein structure to function relationships as they exist within living cells. Here we apply the latest technological advances in chemical cross-linking combined with mass spectrometry to study protein topologies and interactions from living human cells identifying a total of 368 cross-links. These include cross-links from all major cellular compartments including membrane, cytosolic and nuclear proteins. Intraprotein and interprotein cross-links were also observed for core histone proteins, including several cross-links containing post-translational modifications which are known histone marks conferring distinct epigenetic functions. Proteins are the principal operatives within cells, involved in carrying out essentially all biological functions. A complex network of intra-and intermolecular interactions, post-translational modifications and abundance levels is required to maintain the delicate balance of function essential for life. Subtle changes within this network can give rise to specific biological responses to environmental factors, onset of disease, normal aging, and other biological processes. Therefore, direct experimental observation of protein structures and interactions in relation to biological function is paramount to improved understanding of living systems.Chemical cross-linking has long been used as a method of fixation to preserve biological samples in the fields of histology and pathology (1). Protein interactions and topologies have also been studied with chemical cross-linking methods for many years (2-4). Chemical cross-linking with mass spectrometry (XL-MS) 1 is emerging as a powerful technology to study protein structures and interactions in complex biological systems (5). Technological advances in chemistry, analytical instrumentation, and informatics are beginning to allow the successful application of XL-MS to study protein topologies and interactions on a large scale in complex biological systems. These methods are able to provide low resolution spatial information on protein topologies through distance constraints imposed by the chemical linker arm distance. The resultant distance constraints are often used to refine crystal structure measurements and to assist de novo structure prediction with molecular modeling techniques (6, 7). Structural information derived through cross-linking experiments is largely complementary to structural information obtained through other techniques including hydrogen-deuterium exchange mass spectrometry, NMR, and x-ray cryst...

Protein interactions and topologies are key features that enable specificity, function and the evolution of highly integrated, regulated networks in biological systems. Primary challenges associated with the study of biological systems include identification of protein interactions and measurement of topological features of proteins and their interactions in vivo. Advancements such as the Yeast Two-Hybrid (1), coimmunoprecipitation (2), and Tandem Affinity Purification tags (3) have greatly increased the ability to identify hundreds or even thousands of interactions from complex biological samples (2, 4 -6). Despite the many thousands of protein interactions that are now known (7) however, for only a tiny fraction is there any knowledge of their in vivo topology. On the other hand, if topologies of interactions were more widely known, this information could improve understanding of underlying fundamental factors that drive interactions, improve development of highly specific modulators of protein interactions, improve interaction prediction capabilities, and improve comprehension on biological systems. Unfortunately, exceedingly few methods exist to allow unbiased measurement of proteinprotein interaction topological features in cells.Chemical cross-linking has great potential for in vivo interaction topological studies (8 -10). Cross-linked peptides contain information about interacting protein identities and can uniquely define regions of protein sequences that are near one another when proteins are present within the native cellular environment. Challenges associated with in vivo crosslinking analysis that have precluded this achievement include the difficulty in identification of cross-linked peptides and the severe dynamic range constraints resultant from the overwhelming majority of noncross-linked peptides. Our efforts to overcome these challenges resulted in development of Protein Interaction Reporter (PIR) 1 technology (11) that uses a novel type of cross-linker and mass spectrometry to identify peptides that are close to one another within protein complexes in cells. These efforts resulted in the first reported identification of cross-linked peptides from live cells (9) including the first in vivo identification of an interaction among two outer membrane cytochrome c proteins, an interaction that appears to be critical to electron transport properties of Shewanella oneidensis (12).Here we present the first application of PIR technology to the study of interactions in E. coli cells where 65 cross-linked peptide pairs were unambiguously identified. To date, this constitutes the largest in vivo cross-linked peptide data set ever produced. In this system, we are also able to compare many of our results with known protein and protein complex crystal structures that demonstrate excellent agreement with our in vivo data. Importantly, this comparative analysis was also used to define distance constraints that enable refinements of structural prediction of in vivo protein complexes never before possible. Furthe...

A-kinase anchoring protein 79 (AKAP79) is a human anchoring protein that organizes cAMP-dependent protein kinase (PKA), Ca 2þ ∕calmodulin (CaM)-dependent protein phosphatase (PP2B), and protein kinase C (PKC) for phosphoregulation of synaptic signaling. Quantitative biochemical analyses of selected AKAP79 complexes have determined the quaternary structure of these signaling complexes. We show that AKAP79 dimerizes, and we demonstrate that, upon addition of a lysine-reactive cross-linker, parallel homomeric dimers are stabilized through K328-K328 and K333-K333 cross-links. An assembly of greater complexity comprising AKAP79, PP2B, a type II regulatory subunit fragment (RII 1-45) of PKA, and CaM was reconstituted in vitro. Using native MS, we determined the molecular mass of this complex as 466 kDa. This indicates that dimeric AKAP79 coordinates two RII 1-45 homodimers, four PP2B heterodimers, and two CaM molecules. Binding of Ca 2þ ∕CaM to AKAP79 stabilizes the complex by generating a second interface for PP2B. This leads to activation of the anchored phosphatases. Our architectural model reveals how dimeric AKAP79 concentrates pockets of second messenger responsive enzyme activities at the plasma membrane. F ollowing its discovery, cyclic AMP (cAMP) was thought to be freely diffusible within the cell (1). Subsequently, it was demonstrated that utilization of this second messenger is more sophisticated. For example, treatment of cardiac myocytes with norepinephrine, but not prostaglandin E1, stimulates contraction and glycogen metabolism, despite the fact that both hormones elevate cAMP to activate cAMP-dependent protein kinase (PKA) (2). It is now recognized that intracellular cAMP levels fluctuate in cellular microdomains where cAMP effector proteins such as PKA, Epac guanine nucleotide exchange factors, and cyclic nucleotide-gated ion channels reside (3-5). Likewise, other second messengers such as Ca 2þ and certain phospholipids operate in cellular microdomains where they interface with their own effector proteins (6).A-kinase anchoring proteins (AKAPs) organize responses to these second messengers. AKAP79 is a prototypical AKAP, exemplifying the three properties that are characteristic of the family: (i) an amphipathic α-helix (residues 387-406) that binds to the D∕D domain (residues 1-45) of PKA RII subunits (7); (ii) a subcellular localization signal, in its case, three tandem membrane-binding basic regions (MBBRs) that bind to PIP 2 (8); and (iii) the ability to interact with multiple signaling molecules. AKAP79 targets PKA, the Ca 2þ -dependent Ser/Thr protein phosphatase PP2B (calcineurin) (9), and protein kinase C (PKC) (10) to substrates including transmembrane receptors and ion channels. A more comprehensive list of AKAP79 binding partners is shown in Fig. S1. Ancillary protein-lipid and proteinprotein interactions serve to localize AKAP79 with particular membrane substrates (8). For example, a leucine zipper-like motif in AKAP79 associates with the cytoplasmic tail of L-type Ca 2þ channels, enabling...

Protein interactions are critical determinants of insect-transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and that are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.

Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately three fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.