This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein-protein interactions (PPIs) with a focus on their function within cells, organs and organisms. The significance of post-translational modifications within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis-based and mass spectrometry (MS)-based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.
Protein-protein interactions; electrophoresis; mass spectrometry; proteomics; bioinformatics
Single-seeded fruit of the sacred lotus Nelumbo nucifera Gaertn var. China Antique from NE China have viability as long as ~1300 years determined by direct radiocarbon-dating, having a germination rate of 84%. The pericarp, a fruit tissue that encloses the single seeds of Nelumbo, is considered one of the major factors that contribute to fruit longevity. Proteins that are heat stable and have protective function may be equally important to seed viability. We show proteins of Nelumbo fruit that are able to withstand heating, 31% of which remained soluble in the 110°C-treated embryo-axis of a 549-yr-old fruit and 76% retained fluidity in its cotyledons. Genome of Nelumbo is published. The amino-acid sequences of 11 “thermal proteins” (soluble at 100°C) of modern Nelumbo embryo-axes and cotyledons, identified by mass spectrometry, Western blot and bioassay, are assembled and aligned with those of an archaeal-hyperthermophile Methancaldococcus jannaschii (Mj; an anaerobic methanogen having a growth optimum of 85°C) and with five mesophile angiosperms. These thermal proteins have roles in protection and repair under stress. More than half of the Nelumbo thermal proteins (55%) are present in the archaean Mj, indicating their long-term durability and history. One Nelumbo protein-repair enzyme exhibits activity at 100°C, having a higher heat-tolerance than that of Arabidopsis. A list of 30 sequenced but unassembled thermal proteins of Nelumbo is supplemented.
Nelumbo nucifera China Antique; Eleven 100°C-Heat-Soluble Proteins; Hyperthermophile-Mesophile Protein-Alignments; Stress-and-Repair Proteins; 30 Additional Thermal-Proteins
The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.
Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
Basic fibroblast growth factor (bFGF) plays a crucial role in diverse cellular functions from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Herein, we describe stabilization of bFGF by covalent conjugation of a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors such as heat, mild and harsh acidic conditions, storage, and proteolytic degradation, compared to native bFGF. After applied stress, the conjugate was also significantly more active than the control conjugate system where the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for stabilization of heparin-binding growth factors in general.
We used affinity-purification mass spectrometry to identify 747 candidate proteins that are complexed with Huntingtin (Htt) in distinct brain regions and ages in Huntington’s disease (HD) and wildtype mouse brains. To gain a systems-level view of the Htt interactome, we applied Weighted Gene Correlation Network Analysis (WGCNA) to the entire proteomic dataset to unveil a verifiable rank of Htt-correlated proteins and a network of Htt-interacting protein modules, with each module highlighting distinct aspects of Htt biology. Importantly, the Htt-containing module is highly enriched with proteins involved in 14-3-3 signaling, microtubule-based transport, and proteostasis. Top-ranked proteins in this module were validated as novel Htt interactors and genetic modifiers in an HD Drosophila model. Together, our study provides a compendium of spatiotemporal Htt-interacting proteins in the mammalian brain, and presents a conceptually novel approach to analyze proteomic interactome datasets to build in vivo protein networks in complex tissues such as the brain.
We report the development of novel reagents for cell-level protein quantification, referred to as Caltech Isobaric Tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side-chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d5-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cut-off problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.
Native protein mass spectrometry (MS), the measurement of proteins and protein complexes from non-denaturing solutions, with electrospray ionization (ESI) has utility in the biological sciences. Protein complexes exceeding 1 MDa have been measured by MS and ion mobility spectrometry (IMS), and the data yields information not only regarding size, but structural details can be revealed also. ESI-IMS allows the relative stability of protein–ligand binding to be measured. Top-down MS, the direct dissociation of the intact gas phase biomolecule, can generate sequence and identity information for monomeric (denatured) proteins, and topology information for noncovalent protein complexes. For protein complexes with small molecule ligands, i.e., drugs, cofactors, metals, etc., top-down MS with electron capture dissociation can be used to elucidate the site(s) of ligand binding. Increasing protein ESI charging, e.g., supercharging, enhances the efficiency for dissociation of protein complexes.
electrospray; protein; top-down; supercharging; hemoglobin; enolase; adenylate kinase
Aggregation of α-synuclein (α-syn) is implicated as being causative in the pathogenesis of Parkinson’s disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel “molecular tweezer” (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity of α-syn. In vitro, CLR01 inhibited the assembly of α-syn into β-sheet-rich fibrils and caused disaggregation of pre-formed fibrils, as determined by thioflavin T fluorescence and electron microscopy. α-Syn toxicity was studied in cell cultures and was completely mitigated by CLR01 when α-syn was expressed endogenously or added exogenously. To determine if CLR01 was also protective in vivo, we used a novel zebrafish model of α-syn toxicity (α-syn-ZF), which expresses human, wild-type α-syn in neurons. α-Syn-ZF embryos developed severe deformities due to neuronal apoptosis and most of them died within 48 to 72 h. CLR01 added to the water significantly improved zebrafish phenotype and survival, suppressed α-syn aggregation in neurons, and reduced α-syn-induced apoptosis. α-Syn expression was found to inhibit the ubiquitin proteasome system in α-syn-ZF neurons, resulting in further accumulation of α-syn. Treatment with CLR01 almost completely mitigated the proteasome inhibition. The data suggest that CLR01 is a promising therapeutic agent for the treatment of Parkinson’s disease and other synucleinopathies.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-012-0105-1) contains supplementary material, which is available to authorized users.
Parkinson’s disease; zebrafish; synucleinopathy; amyloid; neuroprotection
We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability.
Chemical Cross-linkers; Cross-linked Peptide; Protein Structure; Protein-Protein Interaction; Mass Spectrometry; Copper-Catalyzed Azide-Alkyne Cycloaddition; Reporter Ion
Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.
Green tea; human pancreatic adenocarcinoma HPAF-II cells; Hsp90; Trap1; Hsp27
Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly therefore is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass-spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were non-toxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of non-toxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.
Inhibitor; Protein aggregation; Alzheimer’s disease; Amyloid β-protein; Tau; Islet amyloid polypeptide; Insulin; Calcitonin; β2-microglobulin; Transthyretin; Toxicity
Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid sample, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG), and thus represents the largest protein successfully ionized by DESI to date.
desorption electrospray ionization; mass spectrometry; noncovalent protein complexes; ion mobility
A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. Toward understanding the antibody-induced inhibition mechanism, characterization of morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates was pursued. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine-3-glucuronide NHS-ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially, the cause of activity loss due to the coupling reaction.
Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.
Tandem mass spectrometry (MS/MS) of intact, noncovalently-bound protein-ligand complexes can yield structural information on the site of ligand binding. Fourier transform ion cyclotron resonance (FT-ICR) top-down MS of the 29 kDa carbonic anhydrase-zinc complex and adenylate kinase bound to adenosine triphosphate (ATP) with collisionally activated dissociation (CAD) and/or electron capture dissociation (ECD) generates product ions that retain the ligand and their identities are consistent with the solution phase structure. Increasing gas phase protein charging from electrospray ionization (ESI) by the addition of supercharging reagents, such as m-nitrobenzyl alcohol and sulfolane, to the protein analyte solution improves the capability of MS/MS to generate holo-product ions. Top-down proteomics for protein sequencing can be enhanced by increasing analyte charging.
electrospray ionization; noncovalent complexes; supercharging; protein-ligand binding; adenylate kinase; carbonic anhydrase
Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based techniques by developing a multifunctional electrode with closed-loop temperature control, while minimizing overall system complexity, and addressing challenges associated with rapid evaporation. For the EWOD chip design, we discuss the performance of multifunctional surface electrodes for actuation, localized Joule heating, and thermistic temperature sensing. Furthermore, a hydrophilic pattern is formed in the multifunctional electrode to control the location of an evaporating droplet on the electrode. To demonstrate the capabilities and limitations of this technique, we performed three experiments and measured the results using MALDI-MS: (i) insulin disulfide reductions in DTT over a range of heater temperatures (22 to 70 °C) to show how reaction rates can be affected by thermal control, (ii) insulin disulfide reductions at 130 °C in DMSO to demonstrate a reaction in a high boiling point solvent, and (iii) tryptic digestions of cytochrome c at 22 and 40 °C to show that heated droplets can yield reasonably higher peptide sequence coverage than unheated droplets. Although they do not decouple the effects of changing temperatures and concentrations, these experiments verified that thermal cycling by EWOD electrodes accelerates reaction rates in liquid droplets in air.
Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids.
Materials and Methods
We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography–tandem mass spectrometry was used to identify the salivary N-glycoproteins.
A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions.
Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.
Proteomics; Mass spectrometry; Isoelectric focusing; N-linked glycoproteins; Whole saliva; Parotid saliva; Submandibular saliva; Sublingual saliva; Disease biomarker
A hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer was used for top-down characterization of the abundant human salivary Cystatins, including S, S1, S2, SA, SN, C, and D, using collisionally activated dissociation (CAD) after chromatographic purification of the native, disulfide intact proteins from saliva. Post-translational modifications and protein sequence polymorphisms arising from single nucleotide polymorphisms (SNPs) were assigned from precursor and product ion masses at a tolerance of 10 ppm allowing confident identification of individual intact mass tags. Cystatins S, S1, S2, SA and SN were cleaved of a N-terminal 20 amino-acid signal peptide, and Cystatin C a 26-residue peptide, to yield a generally conserved N-terminus. In contrast, Cystatin D isoforms with 24 and 28 amino-acid residue N-terminal truncations were found such that their N-termini were not conserved. Cystatin S1 was phosphorylated at Ser3, while S2 was phosphorylated at Ser1 and Ser3 of the mature protein, in agreement with previous work. Both Cystatin D isoforms carried the polymorphism C46R (SNP: rs1799841). The 14328 Da isoform of Cystatin SN previously assigned with polymorphism P31L due to a SNP (rs2070856) was found only in whole saliva. Parotid secretions contained no detectable Cystatins while whole saliva largely mirrored the contents of submandibular/sublingual (SMSL) secretions. Top-down high-resolution mass spectrometry is a powerful tool for the identification and characterization of potential protein biomarkers in saliva.
Canonical and noncanonical nuclear factor κB (NF-κB) signaling are the two basic pathways responsible for the release of NF-κB dimers from their inhibitors. Enhanced NF-κB signaling leads to inflammatory and proliferative diseases; thus, inhibitory pathways that limit its activity are critical. Whereas multiple negative feedback mechanisms control canonical NF-κB signaling, none has been identified for the noncanonical pathway. Here, we describe a mechanism of negative feedback control of noncanonical NF-κB signaling that attenuated the stabilization of NF-κB–inducing kinase (NIK), the central regulatory kinase of the non-canonical pathway, induced by B cell–activating factor receptor (BAFF-R) and lymphotoxin β receptor (LTβR). Inhibitor of κB (IκB) kinase α (IKKα) was previously thought to lie downstream of NIK in the non-canonical NF-κB pathway; we showed that phosphorylation of NIK by IKKα destabilized NIK. In the absence of IKKα-mediated negative feedback, the abundance of NIK increased after receptor ligation. A form of NIK with mutations in the IKKα-targeted serine residues was more stable than wild-type NIK and resulted in increased noncanonical NF-κB signaling. Thus, in addition to the regulation of the basal abundance of NIK in unstimulated cells by a complex containing tumor necrosis factor receptor–associated factor (TRAF) and cellular inhibitor of apoptosis (cIAP) proteins, IKKα-dependent destabilization of NIK prevents the uncontrolled activity of the noncanonical NF-κB pathway after receptor ligation.
Drebrin is a filament binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full length protein. Site directed mutagenesis, electron microscopic (EM) reconstruction and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and 1,1-methanediyl bis(methanethiosulfonate) (MTS1). Site directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1, and drebrin's native cysteine 308 is in close proximity to C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), can link the N-terminal G-S extension of the recombinant DrABD, to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different length (5.4 – 19 Å). These results suggest that the ‘core’ DrABD is centered on actin's subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of EM reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin binding proteins.
actin; drebrin; electron microscopy; mass spectrometry; cross-linking; mapping
Protein-polymer conjugates exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Multimeric conjugates are predicted to surpass the activity of monomeric conjugates. Herein, we report a straightforward method to synthesize multimeric polymer-conjugates. Four armed poly(N-isopropylacrylamide) (pNIPAAm) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of a tetra-functionalized trithiocarbonate chain transfer agent (CTA). The polymer molecular weight, architecture and polydispersity index (PDI) were verified by gel permeation chromatography (GPC), dynamic light scattering gel permeation chromatography (DLS-GPC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This approach afforded well-defined polymers (PDI's < 1.06) and the ability to target various molecular weights. Maleimide functional groups were introduced at the chain ends by heating the polymers in the presence of a furan-protected azo-initiator. This allowed for site-specific conjugation of V131C T4 lysozyme to the polymers to generate multimeric protein-polymer conjugates. MALDI-TOF mass spectrometry, electrospray ionization gas-phase electrophoretic-mobility macromolecule analysis (ESI-GEMMA), gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) of the trypsin digests demonstrated that multimeric protein-polymer conjugates had formed. This simple strategy provides ready access to star protein-polymer conjugates for application in the fields of drug discovery, drug delivery, and nanotechnology.
RAFT; N-isopropylacrylamide; star polymer; protein-polymer conjugate; multimeric
Iodination of tyrosine residues in proteins has many uses in chemistry, biology, and medicine. Site specific identification of the sites of iodination is important for many of these uses. Reported herein is a facile method employing photodissociation and mass spectrometry to localize sites of iodination in whole proteins. Absorption of ultraviolet photons by iodotyrosine results in loss of iodine via homolytic bond dissociation. The resulting protein radical fragments in the vicinity of the iodotyrosine upon collisional activation. Analysis of the fragments within the vicinity of each tyrosine residue in the protein enables quantitative evaluation of the likelihood for iodination at each site. The results are compared with both traditional bottom up and top down mass spectrometric methods. Radical directed dissociation yields results in agreement with traditional approaches but requires significantly less effort and is inherently more sensitive. One limitation occurs when multiple tyrosine residues are in close proximity, in which case the extent of iodination at each residue may be difficult to determine. This limitation is frequently problematic for traditional approaches as well.
An electrospray-assisted laser desorption/ionization source with an infrared OPO laser (IR-ELDI) was constructed and optimized for peptide and protein mass spectrometry analysis. Similar to ELDI with an ultraviolet laser, IR-ELDI generates multiply charged molecules for peptides and proteins measured under ambient sampling conditions. Both samples in the dried state and analyte solutions can be directly measured by IR-ELDI without the presence of a conventional MALDI matrix. However, the analysis of sample solutions is shown to greatly enhance the sensitivity of the mass spectrometry measurement, as a 100-fold sensitivity gain for peptide measurements was measured. The limit of detection of IR-ELDI was determined to be 250 fmol for bradykinin (1.1 kDa), 100 fmol for ubiquitin (8.6 kDa), and 500 fmol for carbonic anhydrase (29 kDa). IR-ELDI is amenable for MS and MSn analysis for proteins up to 80 kDa transferrin. IR-ELDI-MS may be a useful tool for protein sequencing analysis from complex biological matrices, with minimal sample preparation required.