Homobifunctional Cross-linker Research Articles

A grafting approach for nisin-chitosan bio-based antibacterial films: preparation and characterization.

Nisin is a bacteriocin produced by Gram-positive lactic acid bacterium,Lactococcus lactisand currently recognized in the Generally Recognızed as Safe (GRAS) category due to its non-toxicity. Herein, nisin has been grafted to chitosan structure to obtain natural bio-active films with enhanced antibacterial activity. Grafting was performed using ethyl ester lysine diisocyanate and dimer fatty acid-based diisocyanate (DDI); two different close to fully bio-based diisocyanates and Disuccinimidyl suberate; a homo-bifunctional molecule acting as a crosslinker between amino groups. The grafting process allowed the chemical immobilization of nisin to chitosan structure. Physicochemical characterization studies showed the successful grafting of nisin. The antibacterial activity againstStaphylococcus aureuswas evident for all nisin modified chitosan films and best pronounced when DDI was used as a crosslinker with a maximum zone of inhibition of ∼13 mm. All nisin grafted chitosan films were cytocompatible and the cell viability of L929 fibroblasts were >80% pointing out the non-toxic structure. Considering the results of the presented study, bio-based diisocyanates and homo-bifunctional crosslinkers are effective molecules in synthesis of nisin grafted chitosan structures and the new chitosan based antibacterial biopolymers obtained after nisin modification come forward as promising non-toxic and bioactive candidates to be applied in medical devices, implants, and various food coating products.

Easy and Versatile Technique for the Preparation of Stable and Active Lipase-Based CLEA-like Copolymers by Using Two Homofunctional Cross-Linking Agents: Application to the Preparation of Enantiopure Ibuprofen.

An easy and versatile method was designed and applied successfully to obtain access to lipase-based cross-linked-enzyme aggregate-like copolymers (CLEA-LCs) using one-pot, consecutive cross-linking steps using two types of homobifunctional cross-linkers (glutaraldehyde and putrescine), mediated with amine activation through pH alteration (pH jump) as a key step in the process. Six lipases were utilised in order to assess the effectiveness of the technique, in terms of immobilization yields, hydrolytic activities, thermal stability and application in kinetic resolution. A good retention of catalytic properties was found for all cases, together with an important thermal and storage stability improvement. Particularly, the CLEA-LCs derived from Candida rugosa lipase showed an outstanding behaviour in terms of thermostability and capability for catalysing the enantioselective hydrolysis of racemic ibuprofen ethyl ester, furnishing the eutomer (S)-ibuprofen with very high conversion and enantioselectivity.

Synthesis and preliminary immunologic properties of di-/trisaccharide-conjugates related to Bacillus anthracis

Herein, the di- and trisaccharide mimics of the hexasaccharide antigen related to Bacillus anthracis were synthesized and covalently coupled with carrier proteins, such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), to form the corresponding glycoconjugates 1–6. 2,3,4,6-Tetra-O-benzyl thioglycoside and 2-deoxyl-2-phthalylamino-3,4,6-tri-O-benzyl thioglycoside were applied as glycosyl donors to guarantee α or β-configuration of the newly formed glycosidic bonds. Glutaraldehyde was used as a homobifunctional cross-linker for high-efficiency coupling. The synthetic KLH-glycoconjugates 2, 4 and 6 were also used to vaccinate female Balb/c mice and the preliminary results of ELISA uncovered that all three KLH-conjugates could induce immune responses and generate oligosaccharide-specific total IgG antibodies. The trisaccharide 8, the glycosyl part of glycoconjugate 4, is of great immunogenicity.

Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

Since its discovery in circulating blood seven decades ago, cell-free DNA (cfDNA) has become a highly focused subject in cancer management using liquid biopsy. Despite its clinical utility, the extraction of cfDNA from blood has many technical difficulties, including a low efficiency of recovery and long processing times. We introduced a magnetic bead-based cfDNA extraction method using homobifunctional crosslinkers, including dimethyl suberimidate dihydrochloride (DMS). Owing to its bifunctional nature, DMS can bind to DNA through either covalent or electrostatic bonding. By adopting amine-conjugated magnetic beads, DMS–DNA complexes can be rapidly isolated from blood plasma. Using standard washing and eluting processes, we successfully extracted cfDNA from plasma within 10 min. This method yielded a 56% higher extraction efficiency than that of a commercial product (QIAamp kit). Furthermore, the instant binding mechanism of amine coupling between the microbeads and DMS–DNA complexes significantly reduced the processing time. These results highlight the potential of this magnetic bead-based homobifunctional crosslinker platform for extraction of cfDNA from blood plasma.

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC).

The folate receptor alpha (FR), which is overexpressed in solid tumors including NSCLC, can be utilized for active tumor targeting to afford more effective cancer therapies. In this context, cytochrome c (Cyt c) has drawn attention to cancer research because it is non-toxic, yet, when delivered to the cytoplasm of cancer cells, can kill them by inducing apoptosis. Cyt c nanoparticles (NPs, 169 ± 9 nm) were obtained by solvent precipitation with acetonitrile, and stabilized by reversible homo-bifunctional crosslinking to accomplish a Cyt-c-based drug delivery system that combines stimulus-responsive release and active targeting. Cyt c was released under intracellular redox conditions, due to an S–S bond in the NPs linker, while NPs remained intact without any release under extracellular conditions. The NP surface was decorated with a hydrophilic folic acid–polyethylene glycol (FA–PEG) polymer for active targeting. The FA-decorated NPs specifically recognized and killed cancer cells (IC50 = 47.46 µg/mL) that overexpressed FR, but showed no toxicity against FR-negative cells. Confocal microscopy confirmed the preferential uptake and apoptosis induction of our NPs by FR-positive cancer cells. In vivo experiments using a Lewis lung carcinoma (LLC) mouse model showed visible NP accumulation within the tumor and inhibited the growth of LLC tumors.

Fast cross-linking by DOPA2 promotes the capturing of a stereospecific protein complex over nonspecific encounter complexes.

Transient and weak protein-protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes EIN/HPr and EIIAGlc/EIIBGlc as our model systems, we evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities. We showed previously that DOPA2 (di-ortho-phthalaldehyde with a di-ethylene glycol spacer arm) cross-links proteins 60-120 times faster than DSS (disuccinimidyl suberate). We found that though most of the intermolecular cross-links of either cross-linker are consistent with the encounter complexes (ECs), an ensemble of short-lived binding intermediates, more DOPA2 intermolecular cross-links could be assigned to the stereospecific complex (SC), the final lowest-energy conformational state for the two interacting proteins. Our finding suggests that faster cross-linking captures the SC more effectively and cross-linkers of different reactivities potentially probe protein-protein interaction dynamics across multiple timescales.

High generation dendrimer decorated poly-Ɛ-caprolactone/polyacrylic acid electrospun nanofibers for the design of a bioelectrochemical sensing surface

In this study, poly-Ɛ-caprolactone (PCL) and poly(acrylic) acid (PAA)-based electrospun nanofibers were prepared for the immobilization of pyranose oxidase (PyOx) to design a bioelectrochemical detection system. Different amounts of PAA were used to increase hydrophilicity (decreased contact angle) of the PCL electrospun nanofibers. To provide a multipoint attachment side to bind PyOx by covalent bonds, various amounts of high generation (G5) poly(amidoamine) (PAMAM) dendrimer with amino groups were added to the PCL:PAA backbone. To attach PyOx onto the PCL:PAA/PAMAM electrospun nanofibers, glutaraldehyde was used as a homobifunctional crosslinker. Firstly, PCL:PAA ratio was optimized to obtain the best electrospun nanofibers without beads and with decreased contact angle. Then, the effect of the PAMAM amount on the morphology of PCL:PAA and contact angle was tested. The obtained PCL:PAA/PAMAM electrospun nanofiber was characterized by scanning electron microscopy (SEM). Then, the presence of PAMAM in the structure and the success of PyOx immobilization onto PCL:PAA/PAMAM were proven by SEM and an energy dispersive X-ray analyzer (SEM-EDX). Finally, PCL:PAA/PAMAM/PyOx was characterized, calibrated, and applied to analyze glucose in samples without any interfering effect of some chemicals. Briefly, the nanofibers modification with dendrimers and the conjugation of PyOx onto formed nanostructures was successfully performed, and a novel electrospun nanofiber-based sensor system was developed for target detection.

Efficient Portable Urea Biosensor Based on Urease Immobilized Membrane for Monitoring of Physiological Fluids.

Numerous studies have addressed the utilization of glutaraldehyde (GA) as a homobifunctional cross-linker. However, its applicability has been impeded due to several issues, including the tendency of GA molecules to undergo polymerization. Herein, a portable urea biosensor was developed for the real-time monitoring of the flow of physiological fluids; this was achieved by using disuccinimidyl cross-linker-based urease immobilization. Urease was immobilized on a porous polytetrafluoroethylene (PTFE) solid support using different disuccinimidyl cross-linkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and bis-N-succinimidyl-(pentaethylene glycol) ester (BS(PEG)5). A urease activity test revealed that DSS exhibited the highest urease immobilizing efficiency, whereas FT-IR analysis confirmed that urease was immobilized on the PTFE membrane via DSS cross-linking. The membrane was inserted in a polydimethylsiloxane (PDMS) fluidic chamber that generated an electrochemical signal in the presence of a flowing fluid containing urea. Urea samples were allowed to flow into the urea biosensor (1.0 mL/min) and the signal was measured using chronoamperometry. The sensitivity of the DSS urea biosensor was the highest of all the trialed biosensors and was found to be superior to the more commonly used GA cross-linker. To simulate real-time monitoring in a human patient, flowing urea-spiked human serum was measured and the effective urease immobilization of the DSS urea biosensor was confirmed. The repeatability and interference of the urea biosensor were suitable for monitoring urea concentrations typically found in human patients.

Detection of Total Bacterial Load in Water Samples Using a Disposable Impedimetric Sensor

Detection of bacterial colonies in water is a pressing issue in recent times owing to its implications on human health, leading to an urgent need for low cost, disposable sensors. In this work, we report a disposable impedimetric sensor for detection of total bacterial colonies present in water samples. The sensor consists of a polyaniline coated filter paper functionalized with a homo-bi functional crosslinker, glutaraldehyde. Prior to the functionalization step, two Ag electrodes are deposited on the filter paper using conductive silver paste. The aldehyde groups of glutaraldehyde bind with the amine functional groups present on bacterial cell wall resulting in a change in dielectric constant, and consequently, the capacitive effect between the two Ag electrodes. Also, bacterial metabolism affects the medium conductivity and in turn, the medium resistance changes. The combined effect of changes in capacitive effect as well as resistance is reflected as a change in AC impedance measured using a frequency response analyzer (FRA). E. coli and Pseudomonas aeruginosa bacteria are detected in PBS buffer up to ~ 500 cfu/mL. Testing is also carried out by spiking tap water and grey water with known concentrations of bacteria samples. The detection limits obtained are 500 – 1000 cfu/mL with a response time of ~ 20 minutes. The developed sensor provides rapid, moderately sensitive, cost-effective solution for detecting total bacterial load in water samples.

Versatile Reversible Cross-Linking Strategy to Stabilize CCMV Virus Like Particles for Efficient siRNA Delivery.

Virus like particles obtained from the Cowpea Chlorotic Mottle Virus (CCMV) represent an innovative platform for drug delivery applications. Their unique reversible self-assembly properties as well as their suitability for both cargo loading and functionalization make them a versatile scaffold for numerous purposes. One of the main drawbacks of this platform is however its limited stability at physiological conditions. Herein, we report the development of a general reversible cross-linking strategy involving the homobifunctional cross-linker DTSSP (3,3′-dithiobis (sulfosuccinimidylpropionate)) which is suitable for particle stabilization. This methodology is adaptable to different CCMV variants in the presence or absence of a stabilizing cargo without varying neither particle shape nor size thus extending the potential use of these protein cages in nanomedical applications. Cross-linked particles are stable at neutral pH and 37 °C and they are capable of protecting loaded cargo against enzymatic digestion. Furthermore, the reversible nature of the cross-linking ensures particle disassembly when they are taken up by cells. This was demonstrated via the highly effective delivery of active siRNA into cells.

Cross-linker-Modulated Nanogel Flexibility Correlates with Tunable Targeting to a Sterically Impeded Endothelial Marker.

Deformability of injectable nanocarriers impacts rheological behavior, drug loading, and affinity target adhesion. Here, we present atomic force microscopy (AFM) and spectroscopy measurements of nanocarrier Young's moduli, tune the moduli of deformable nanocarriers with cross-linkers, and demonstrate vascular targeting behavior that correlates with Young's modulus. Homobifunctional cross-linkers were introduced into lysozyme-dextran nanogels (NGs). Single particle-scale AFM measurements determined NG moduli varying from ∼50-150 kPa for unmodified NGs or NGs with a short hydrophilic cross-linker (2,2'-(ethylenedioxy)bis(ethylamine), EOD) to ∼350 kPa for NGs modified with a longer hydrophilic cross-linker (4,9-dioxa-1,12-dodecanediamine, DODD) to ∼10 MPa for NGs modified with a longer hydrophobic cross-linker (1,12-diaminododecane, DAD). Cross-linked NGs were conjugated to antibodies for plasmalemma vesicle associated protein (PLVAP), a caveolar endothelial marker that cannot be accessed by rigid particles larger than ∼100 nm. In previous work, 150 nm NGs effectively targeted PLVAP, where rigid particles of similar diameter did not. EOD-modified NGs targeted PLVAP less effectively than unmodified NGs, but more effectively than DODD or DAD modified NGs, which both yielded low levels of targeting, resembling results previously obtained with polystyrene particles. Cross-linked NGs were also conjugated to antibodies against intracellular adhesion molecule-1 (ICAM-1), an endothelial marker accessible to large rigid particles. Cross-linked NGs and unmodified NGs targeted uniformly to ICAM-1. We thus demonstrate cross-linker modification of NGs, AFM determination of NG mechanical properties varying with cross-linker, and tuning of specific sterically constrained vascular targeting behavior in correlation with cross-linker-modified NG mechanical properties.

The bacterial lipid II flippase MurJ functions by an alternating-access mechanism

The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology. Bacteria use multiprotein machineries for the synthesis of the PG cell wall during cell division and elongation that can be targeted by antibiotics such as the β-lactams. Lipid II, the lipid-linked precursor for PG biogenesis, is synthesized in the inner leaflet of the cytoplasmic membrane and then translocated across the bilayer, where it is ultimately polymerized into PG. In Escherichia coli, MurJ, a member of the MOP exporter superfamily, has been recently shown to have lipid II flippase activity that depends on membrane potential. Because of its essentiality, MurJ could potentially be targeted by much needed novel antibiotics. Recent structural information suggests that a central cavity in MurJ alternates between inward- and outward-open conformations to flip lipid II, but how these conformational changes occur are unknown. Here, we utilized structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe the conformational changes of MurJ in E. coli cells. We found that paired cysteine substitutions in transmembrane domains 2 and 8 and periplasmic loops of MurJ could be cross-linked with homobifunctional cysteine cross-linkers, indicating that MurJ can adopt both inward- and outward-facing conformations in vivo Furthermore, we show that dissipating the membrane potential with an ionophore decreases the prevalence of the inward-facing, but not the outward-facing state. Our study provides in vivo evidence that MurJ uses an alternating-access mechanism during the lipid II transport cycle.

A novel method for site-specific chemical SUMOylation: SUMOylation of Hsp90 modulates co-chaperone binding in vitro

SUMO is covalently attached to lysine side chains in target proteins by the action of a cascade of E1, E2, and E3 ligases. Unlike ubiquitin, SUMO does not target proteins for degradation but rather plays a regulatory role in activating target proteins or directing them to multiprotein complexes. Isolating SUMOylated proteins from native sources is challenging because of the low stoichiometry of SUMOylation that occurs for any given target protein in cells. Here we report a novel strategy to couple SUMO to the site of a target lysine for the purpose of in vitro study. Introduction of a single cysteine after the C terminal diglycine motif and a cysteine in place of a target lysine in a substrate protein allows for efficient and specific crosslinking of SUMO using a homo-bifunctional maleimide crosslinker. We demonstrate that SUMO can be crosslinked in this manner to amino acid position 178 in the dimeric molecular chaperone, Hsp90. Chemically SUMOylated Hsp90 has very similar ATPase activity compared to unmodified Hsp90 but displays preferential co-chaperone binding in vivo. Our novel strategy can easily be applied to other SUMOylated or ubiquitinated target protein in vitro.

Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein-Protein Interactions.

Cross-linking mass spectrometry (XL-MS) has become an emerging technology for defining protein-protein interactions (PPIs) and elucidating architectures of large protein complexes. Up to now, the most widely used cross-linking reagents target lysines. Although such reagents have been successfully applied to map PPIs at the proteome-wide scale, comprehensive PPI profiling would require additional cross-linking chemistries. Cysteine is one of the most reactive amino acids and an attractive target for cross-linking owing to its unique role in protein structures. Although sulfhydryl-reactive cross-linkers are commercially available, their applications in XL-MS studies remain sparse, likely due to the difficulty in identifying cysteine cross-linked peptides. Previously, we developed a new class of sulfoxide-containing MS-cleavable cross-linkers to enable fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS n). Here, we present the development of a new sulfoxide-containing MS-cleavable homobifunctional cysteine-reactive cross-linker, bismaleimide sulfoxide (BMSO). We demonstrate that BMSO-cross-linked peptides display the same characteristic fragmentation pattern during collision-induced dissociation (CID) as other sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS n. Additionally, we show that BMSO can complement amine- and acidic-residue-reactive reagents for mapping protein-interaction regions. Collectively, this work not only enlarges the toolbox of MS-cleavable cross-linkers with diverse chemistries, but more importantly expands our capacity and capability of studying PPIs in general.

False discovery rate estimation and heterobifunctional cross-linkers.

False discovery rate (FDR) estimation is a cornerstone of proteomics that has recently been adapted to cross-linking/mass spectrometry. Here we demonstrate that heterobifunctional cross-linkers, while theoretically different from homobifunctional cross-linkers, need not be considered separately in practice. We develop and then evaluate the impact of applying a correct FDR formula for use of heterobifunctional cross-linkers and conclude that there are minimal practical advantages. Hence a single formula can be applied to data generated from the many different non-cleavable cross-linkers.

Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody–urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MSE peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester (SM(PEG)2), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM(PEG)2), which targets the cysteine added to the antibody C-terminus. V21H4-DOS47 was determined to be the superior conjugate as the antibody is easily produced and purified at high levels, and the conjugate can be efficiently generated and purified using methods easily transferrable for cGMP production. In addition, V21H4-DOS47 retains higher binding activity than V21H1-DOS47, as the native lysine residues are unmodified.

Impedance Spectroscopy-Based Detection of Cardiac Biomarkers on Polyaniline Coated Filter Paper

Cardiac biomarker detection helps in early diagnosis of cardiac syndromes. However, a rapid, cost-effective, reliable, and easy-to-use sensor is still unavailable. Herein, we report a disposable, label free, impedance-based biosensor for the detection of two common cardiac biomarkers, Myoglobin (Myo), and Myeloperoxidase (MPO). The sensor is fabricated on Whatman filter paper coated with a conducting polymer, polyaniline (PAni). The PAni-coated paper is functionalized with glutaraldehyde, a homo-bi functional crosslinker, prior to the covalent attachment of antibodies specific to each biomarker protein. Non-specific active regions of the sensor are blocked by treatment with bovine serum albumin. Conductive silver paste is used to define electrodes for AC impedance measurement on the PAni coated filter paper. Binding of the target protein (Myo or MPO) to the specific antibody changes the impedance between the electrodes; this is detected using AC impedance spectroscopy. The impedance is measured using a frequency response analyzer with an AC excitation voltage of 100 mV in the frequency range of 100 Hz-100 kHz. We can successfully detect Myo and MPO in buffer solution within the concentration range of 100 ng/mL-50 μg/mL. Detection limit obtained for these cardiac biomarkers spiked in human serum is 1 μg/mL. Further blocking using human serum improves the lower detection limit to 500 ng/mL. The developed biosensor uses inexpensive materials and fabrication techniques, detects cardiac biomarkers in clinically relevant concentrations rapidly (i.e., within 20 mins), and can be disposed in an environment friendly manner, thus making the sensor suitable for diagnostic applications.

Polyfluorophenyl Ester-Terminated Homobifunctional Cross-Linkers for Protein Conjugation

Along with N-hydroxysuccinimidyl, p-nitrophenyl, and phenylseleno esters, tetra- and penta-fluorophenyl esters were comparatively evaluated in term of their reactivity and hydrolytic stability. Their homobifunctional cross-linkers were prepared to conjugate proteins with small molecules, including carbohydrates, fluorescent dyes, and poly(ethylene glycol) monomethyl ether. The conjugations proceeded under mild conditions, affording the corresponding protein conjugates with good efficiency.

A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS).

The chemical cross-linking/mass spectrometry (MS) approach is a growing research field in structural proteomics that allows gaining insights into protein conformations. It relies on creating distance constraints between cross-linked amino acid side chains that can further be used to derive protein structures. Currently, the most urgent task for designing novel cross-linking principles is an unambiguous and automated assignment of the created cross-linked products. Here, we introduce the homobifunctional, amine-reactive, and water soluble cross-linker azobisimidoester (ABI) as a prototype of a novel class of cross-linkers. The ABI-linker possesses an innovative modular scaffold combining the benefits of collisional activation lability with open shell chemistry. This MS-cleavable cross-linker can be efficiently operated via free radical initiated peptide sequencing (FRIPS) in positive ionization mode. Our proof-of-principle study challenges the gas phase behavior of the ABI-linker for the three amino acids, lysine, leucine, and isoleucine, as well as the model peptide thymopentin. The isomeric amino acids leucine and isoleucine could be discriminated by their characteristic side chain fragments. Collisional activation experiments were conducted via positive electrospray ionization (ESI) on two Orbitrap mass spectrometers. The ABI-mediated formation of odd electron product ions in MS/MS and MS3 experiments was evaluated and compared with a previously described azo-based cross-linker. All cross-linked products were amenable to automated analysis by the MeroX software, underlining the future potential of the ABI-linker for structural proteomics studies. Graphical Abstract ᅟ.

Novel Concepts of MS-Cleavable Cross-linkers for Improved Peptide Structure Analysis.

The chemical cross-linking/mass spectrometry (MS) approach is gaining increasing importance as an alternative method for studying protein conformation and for deciphering protein interaction networks. This study is part of our ongoing efforts to develop innovative cross-linking principles for a facile and efficient assignment of cross-linked products. We evaluate two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for automated analysis of cross-linked products. We introduce the bromine phenylurea (BrPU) linker that possesses a unique structure yielding a distinctive fragmentation pattern on collisional activation. Moreover, BrPU delivers the characteristic bromine isotope pattern and mass defect for all cross-linker-decorated fragments. We compare the fragmentation behavior of the BrPU linker with that of our previously described MS-cleavable TEMPO-Bz linker (which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy moiety connected to a benzyl group) that was developed to perform free-radical-initiated peptide sequencing. Comparative collisional activation experiments (collision-induced dissociation and higher-energy collision-induced dissociation) with both cross-linkers were conducted in negative electrospray ionization mode with an Orbitrap Fusion mass spectrometer using five model peptides. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the loss of an intact peptide from the cross-linked products. As the BrPU linker combines a characteristic mass shift with an isotope signature, it presents a more favorable combination for automated assignment of cross-linked products compared with the TEMPO-Bz linker. ᅟ.