Reversible Photochromism of 4,4'-Disubstituted 2,2'-Bipyridine in the Presence of SO3.

We report an unusual photochromic behavior of 4,4'-disubstituted-2,2'-bipyridine. It was found that in the presence of a SO3 source and HCl, 2,2'-bipyridine-4,4'-dibutyl ester undergoes a color change from yellow to magenta in solution with maximum absorbance at 545 nm upon irradiation with 395 nm light. The photochromism is thermally reversible in solution. Different from the known bipyridine-based photoswitching pathways, the photo response does not involve any metal which form colored complexes or the formation of colored free radical cations like the photo-reduction of viologens. A combination of experimental and computational analysis was used to probe the mechanism. The results suggest the colored species to be a complex formed between N-oxide of the 2,2'-bipyridine-4,4'-dibutyl ester and SO2; the N-oxide and SO2 are formed from photoactivated oxidation of the bipyridine with SO3 serving as the oxygen source. This complex represents a new addition to the library of photoswitches that is easy to synthesize, reversible in solution, and of high fatigue resistance, making it a promising candidate for applications in photo-switchable materials and SO3 detection. We also demonstrated experimentally similar photochromic behaviors with 2,2'-bipyridine-containing polymers.

Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF.

Mass spectrometry (MS)-based proteomics workflows of intact protein ions have increasingly been utilized to study biological systems. These workflows, however, frequently result in convoluted and difficult to analyze mass spectra. Ion mobility spectrometry (IMS) is a promising tool to overcome these limitations by separating ions by their mass- and size-to-charge ratios. In this work, we further characterize a newly developed method to collisionally dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device. Dissociation occurs prior to ion mobility separation and thus, all product ions are distributed throughout the mobility dimension, enabling facile assignment of near isobaric product ions. We demonstrate that collisional activation within a TIMS device is capable of dissociating protein ions up to 66 kDa. We also demonstrate that the ion population size within the TIMS device significantly influences the efficiency of fragmentation. Lastly, we compare CIDtims to the other modes of collisional activation available on the Bruker timsTOF and demonstrate that the mobility resolution in CIDtims enables the annotation of overlapping fragment ions and improves sequence coverage.

Fragmentation and Mobility Separation of Peptide and Protein Ions in a Trapped-Ion Mobility Device.

Ion mobility separations (IMS) have increasingly been coupled with mass spectrometry to increase peak capacity and deconvolute complex mass spectra in proteomics workflows. IMS separations can be integrated prior to or following the collisional activation step. Post-activation IMS separations have demonstrated many advantages, yet few instrument platforms are capable of this feat. Here, we present the fragmentation of peptide ions within a commercially available trapped-ion mobility spectrometry device. Fragmentation is initiated prior to mobility analysis enabling the separation of generated product ions. The added separation step deconvolutes product ion spectra and permits improved annotation of product ions. Furthermore, we demonstrate the isolation and fragmentation of mobility separated product ions with the downstream quadrupole and collisional cell. When applied to melittin and ubiquitin, this ion mobility assisted pseudo-MS3 fragmentation approach generates sequence coverage ∼50% greater than that of typical MS2 analyses. We envision this ion-mobility-assisted fragmentation technique as the foundation of a powerful new pseudo-MS3 workflow for application toward middle- or top-down proteomics.

Measuring the Energy Barrier of the Structural Change That Initiates Amyloid Formation.

Obtaining kinetic and thermodynamic information for protein amyloid formation can yield new insight into the mechanistic details of this biomedically important process. The kinetics of the structural change that initiates the amyloid pathway, however, has been challenging to access for any amyloid protein system. Here, using the protein ß-2-microglobulin (ß2m) as a model, we measure the kinetics and energy barrier associated with an initial amyloidogenic structural change. Using covalent labeling and mass spectrometry, we measure the decrease in solvent accessibility of one of ß2m's Trp residues, which is buried during the initial structural change, as a way to probe the kinetics of this structural change at different temperatures and under different amyloid forming conditions. Our results provide the first-ever measure of the activation barrier for a structural change that initiates the amyloid formation pathway. The results also yield new mechanistic insight into ß2m's amyloidogenic structural change, especially the role of Pro32 isomerization in this reaction.

Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry.

Tandem mass spectrometry (MS/MS) is the primary method for discovering, identifying, and localizing post-translational modifications (PTMs) in proteins. However, conventional positive ion mode collision induced dissociation (CID)-based MS/MS often fails to yield site-specific information for labile and acidic modifications due to low ionization efficiency in positive ion mode and/or preferential PTM loss. While a number of alternative methods have been developed to address this issue, most require specialized instrumentation or indirect detection. In this work, we present an amine-reactive TEMPO-based free radical initiated peptide sequencing (FRIPS) approach for negative ion mode analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation generates sequence informative ions for both phosphorylated and sulfated peptides with no significant PTM loss. Furthermore, FRIPS is compared to positive ion mode CID, electron transfer dissociation (ETD), as well as negative ion mode electron capture dissociation (niECD) and CID, both in terms of sequence coverage and fragmentation efficiency for phospho- and sulfo-peptides. Because FRIPS-based fragmentation has no particular instrumentation requirements and shows limited PTM loss, we propose this approach as a promising alternative to current techniques for analysis of labile and acidic PTMs.

Increased ß-Sheet Dynamics and D-E Loop Repositioning Are Necessary for Cu(II)-Induced Amyloid Formation by ß-2-Microglobulin.

ß-2-Microglobulin (ß2m) forms amyloid fibrils in the joints of patients undergoing dialysis treatment as a result of kidney failure. One of the ways in which ß2m can be induced to form amyloid fibrils in vitro is via incubation with stoichiometric amounts of Cu(II). To better understand the structural changes caused by Cu(II) binding that allow ß2m to form amyloid fibrils, we compared the effect of Ni(II) and Zn(II) binding, which are two similarly sized divalent metal ions that do not induce ß2m amyloid formation. Using hydrogen/deuterium exchange mass spectrometry (HDX/MS) and covalent labeling MS, we find that Ni(II) has little effect on ß2m structure, despite binding in the same region of the protein as Cu(II). This observation indicates that subtle differences in the organization of residues around Cu(II) cause distant changes that are necessary for oligomerization and eventual amyloid formation. One key difference that we find is that only Cu(II), not Ni(II) or Zn(II), is able to cause the cis-trans isomerization of Pro32 that is an important conformational switch that initiates ß2m amyloid formation. By comparing HDX/MS data from the three metal-ß2m complexes, we also discover that increased dynamics in the ß-sheet formed by the A, B, D, and E ß strands of the protein and repositioning of residues in the D-E loop are necessary aspects of ß2m forming an amyloid-competent dimer. Altogether, our results reveal new structural insights into the unique effect of Cu(II) in the metal-induced amyloid formation of ß2m.

Targeted Annotation of S-Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry.

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 µm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.

Profiling Protein S-Sulfination with Maleimide-Linked Probes.

Cysteine residues are susceptible to oxidation to form S-sulfinyl (R-SO2 H) and S-sulfonyl (R-SO3 H) post-translational modifications. Here we present a simple bioconjugation strategy to label S-sulfinated proteins by using reporter-linked maleimides. After alkylation of free thiols with iodoacetamide, S-sulfinated cysteines react with maleimide to form a sulfone Michael adduct that remains stable under acidic conditions. Using this sequential alkylation strategy, we demonstrate differential S-sulfination across mouse tissue homogenates, as well as enhanced S-sulfination following pharmacological induction of endoplasmic reticulum stress, lipopolysaccharide stimulation, and inhibitors of the electron transport chain. Overall, this study reveals a broadened profile of maleimide reactivity across cysteine modifications, and outlines a simple method for profiling the physiological role of cysteine S-sulfination in disease.

Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry.

Protein therapeutics are rapidly transforming the pharmaceutical industry. Unlike for small molecule therapeutics, current technologies are challenged to provide the rapid, high-resolution analyses of protein higher order structures needed to ensure drug efficacy and safety. Consequently, significant attention has turned to developing new methods that can quickly, accurately, and reproducibly characterize the three-dimensional structure of protein therapeutics. In this work, we describe a method that uses diethylpyrocarbonate (DEPC) labeling and mass spectrometry to detect three-dimensional structural changes in therapeutic proteins that have been exposed to degrading conditions. Using ß2-microglobulin, immunoglobulin G1, and human growth hormone as model systems, we demonstrate that DEPC labeling can identify both specific protein regions that mediate aggregation and those regions that undergo more subtle structural changes upon mishandling of these proteins. Importantly, DEPC labeling is able to provide information for up to 30% of the surface residues in a given protein, thereby providing excellent structural resolution. Given the simplicity of the DEPC labeling chemistry and the relatively straightforward mass spectral analysis of DEPC-labeled proteins, we expect this method should be amenable to a wide range of protein therapeutics and their different formulations.

Unique effect of Cu(II) in the metal-induced amyloid formation of ß-2-microglobulin.

ß-2-Microglobulin (ß2m) forms amyloid fibrils in the joints of patients undergoing hemodialysis treatment as a result of kidney failure. In the presence of stoichiometric amounts of Cu(II), ß2m self-associates into discrete oligomeric species, including dimers, tetramers, and hexamers, before ultimately forming amyloid fibrils that contain no copper. To improve our understanding of whether Cu(II) is unique in its ability to induce ß2m amyloid formation and to delineate the coordinative interactions that allow Cu(II) to exert its effect, we have examined the binding of Ni(II) and Zn(II) to ß2m and the resulting influence that these metals have on ß2m aggregation. We find that, in contrast to Cu(II), Ni(II) does not induce the oligomerization or aggregation of ß2m, while Zn(II) promotes oligomerization but not amyloid fibril formation. Using X-ray absorption spectroscopy and new mass spectrometry-related techniques, we find that different binding modes are responsible for the different effects of Ni(II) and Zn(II). By comparing the binding modes of Cu(II) with Ni(II), we find that Cu(II) binding to Asp59 and the backbone amide between the first two residues of ß2m are important for allowing the formation of amyloid-competent oligomers, as Ni(II) appears not to bind these sites on the protein. The oligomers formed in the presence of Zn(II) are permitted by this metal's ability to bridge two ß2m units via His51. These oligomers, however, are not able to progress to form amyloid fibrils because Zn(II) does not induce the required structural changes near the N-terminus and His31.

Identifying Zn-bound histidine residues in metalloproteins using hydrogen-deuterium exchange mass spectrometry.

In this work, we have developed a method that uses hydrogen-deuterium exchange (HDX) of C2-hydrogens of histidines coupled with mass spectrometry (MS) to identify Zn-bound histidines in metalloproteins. This method relies on differences in HDX reaction rates of Zn-bound and Zn-free His residues. Using several model peptides and proteins, we find that all Zn-bound His residues have substantially lower HDX reaction rates in the presence of the metal. The vast majority of non-Zn-binding His residues undergo no significant changes in HDX reaction rates when their reactivity is compared in the presence and absence of Zn. Using this new approach, we then determined the Zn binding site of ß-2-microglobulin, a protein associated with metal-induced amyloidosis. Together, these results suggest that HDX-MS of His C2-hydrogens is a promising new method for identifying Zn-bound histidines in metalloproteins.

The path forward for protein footprinting, covalent labeling, and mass spectrometry-based protein conformational analyses.

Mass spectrometry-based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ "bottom-up" workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal-in primary structure-concurrent post-translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom-up proteomics report the average PTM status and higher-order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low-copy number proteins below signal-to-noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top-down proteomics as an alternative.

Mobility-Assisted Pseudo-MS3 Sequencing of Protein Ions.

The sequencing of intact proteins within a mass spectrometer has many benefits but is frequently limited by the fact that tandem mass spectrometry (MS/MS) techniques often generate poor sequence coverages when applied to protein ions. To overcome this limitation, exotic MS/MS techniques that rely on lasers and radical chemistry have been developed. These techniques generate high sequence coverages, but they require specialized instrumentation, create products through multiple dissociation mechanisms, and often require long acquisition times. Recently, we demonstrated that protein ions can be dissociated in a trapped ion mobility spectrometry (TIMS) device prior to mobility separation in a commercial timsTOF. All generated product ions were distributed throughout the mobility dimension, and this separation enabled deconvolution of complex tandem mass spectra and could enable facile pseudo-MS3 interrogation of generated product ions with the downstream quadrupole and collision cell. A second activation step improves sequence coverage because the most labile bonds have been depleted during the first dissociation and subsequent dissociation events are more evenly distributed throughout the product ion backbone. In this work, we explore the potential of this mobility-assisted pseudo-MS3 (MAP) method on a commercial timsTOF and timsTOF Pro 2. We demonstrate that while MAP only generates 92% of the sequence coverage of the most effective MS/MS technique, it accomplished this feat in 1.5 min and could be facilely integrated with liquid chromatographic separations.

Improved Performance of Positive-Ion Mode Free Radical-Initiated Peptide Sequencing with p-TEMPO-Bz.

Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry technique that generates sequence informative ions via collisionally initiated radical chemistry. Collision activation homolytically cleaves an installed radical precursor and initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. While the FRIPS technique shows great promise, when applied to multiply charged derivatized peptide ions, a series of high-abundance mass losses are observed which siphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were assisted by the ortho-orientation of the radical precursor undergoing facile conversion into five- or six-membered intermediates or products and that when combined with the lower bond dissociation energy of the para-precursor, conjugated peptides would not undergo this chemistry. To test this assertion, we synthesized p-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. Indeed, we see dramatic attenuation of these undesired collisional processes and a significant increase in radical precursor ion abundance. The increase in ion abundance leads to a significant increase in the sequence coverage generated. These results demonstrate that p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a compelling alternative to the currently utilized o-TEMPO-Bz-based FRIPS.

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation.

Mass spectrometry-based analyses of protein conformation continue to grow in utilization due their speed, low sample requirements, and applicability to most protein systems. These techniques typically rely on chemical derivatization of proteins and as with all label-based analyses must ensure the integrity of the protein conformation throughout the duration of the labeling reaction. Hydroxyl radical footprinting of proteins and the recently developed fast fluoroalkylation of proteins attempt to bypass this consideration via rapid reactions that occur on time scales faster than protein folding, but they often require microfluidic setups or electromagnetic radiation sources. In this work, we demonstrate that ozonation of proteins and peptides, which normally occurs in the second to minute time scales, can be accelerated to the submillisecond to millisecond time scale with an electrospray ionization source. This rapid ozonation results in selective labeling of tryptophan and methionine residues. When applied to cytochrome C and carbonic anhydrase, this labeling technique is sensitive to solution conditions and correlates with solution-phase analyses of conformation. While significant work is still needed to characterize this fast chemical labeling strategy, it requires no complicated sample handling, electromagnetic radiation sources, or microfluidic systems outside of the electrospray source and may represent a facile alternative to other rapid labeling technologies that are utilized today.

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device.

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that has been increasingly adopted due to its inherently high resolution and reduced instrumental footprint. In currently deployed commercial instruments, however, typical modes of collisional activation do not precede IMS analysis, and thus, the instruments are incapable of performing CIU. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of native-like protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, ß-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.

Gas-Phase Hydrogen/Deuterium Scrambling in Negative-Ion Mode Tandem Mass Spectrometry.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a powerful method to characterize protein conformational dynamics. Workflows typically utilize pepsin digestion prior to MS analysis to yield peptide level structural resolution. Tandem mass spectrometry (MS/MS) can potentially facilitate determination of site-specific deuteration to single-residue resolution. However, to be effective, MS/MS activation must minimize the occurrence of gas-phase intramolecular randomization of solution-generated deuterium labels. While significant work has focused on understanding this process in positive-ion mode, little is known about hydrogen/deuterium (H/D) scrambling processes in negative-ion mode. Here, we utilize selectively deuterated model peptides to investigate the extent of intramolecular H/D scrambling upon several negative-ion mode MS/MS techniques, including negative-ion collision-induced dissociation (nCID), electron detachment dissociation (EDD), negative-ion free radical-initiated peptide sequencing (nFRIPS), and negative-ion electron capture dissociation (niECD). H/D scrambling was extensive in deprotonated peptides upon nCID and nFRIPS. In fact, the energetics required to induce dissociation in nCID are sufficient to allow histidine C-2 and Cß hydrogen atoms to participate in the scrambling process. EDD and niECD demonstrated moderate H/D scrambling with niECD being superior in terms of minimizing hydrogen migration, achieving ~ 30% scrambling levels for small c-type fragment ions. We believe the observed scrambling is likely due to activation during ionization and ion transport rather than during the niECD event itself.

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii.

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.

Label scrambling during CID of covalently labeled peptide ions.

Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification.