Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.     

Delineation of in vivo assembled multiprotein complexes via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.     

Revisited BIA-MS combination: entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.     

Combination of a SAW-biosensor with MALDI mass spectrometric analysis

A S-sens K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor alpha-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin-antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.     

Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays

The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.     

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.     

Measurement of antigen-antibody interactions with biosensors

The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has revolutionized the measurement of antigen-antibody binding interactions. In this technique, one of the interacting partners is immobilized on a sensor chip and the binding of the other is followed by the increase in refractive index caused by the mass of bound species. The following immunochemical applications of this new technology will be described: (1) functional mapping of epitopes and paratopes by mutagenesis; (2) analysis of the thermodynamic parameters of the interaction; (3) measurement of the concentration of biologically active molecules; (4) selection of diagnostic probes.     

Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry

Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.     

Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.     

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins

Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.     

Kinetic Titration Series with Biolayer Interferometry

Biolayer interferometry is a method to analyze protein interactions in real-time. In this study, we illustrate the usefulness to quantitatively analyze high affinity protein ligand interactions employing a kinetic titration series for characterizing the interactions between two pairs of interaction patterns, in particular immunoglobulin G and protein G B1 as well as scFv IC16 and amyloid beta (1–42). Kinetic titration series are commonly used in surface plasmon resonance and involve sequential injections of analyte over a desired concentration range on a single ligand coated sensor chip without waiting for complete dissociation between the injections. We show that applying this method to biolayer interferometry is straightforward and i) circumvents problems in data evaluation caused by unavoidable sensor differences, ii) saves resources and iii) increases throughput if screening a multitude of different analyte/ligand combinations.     

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification

The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors.     

Binding hotspots on K‐ras: Consensus ligand binding sites and other reactive regions from probe‐based molecular dynamics analysis

We have used probe‐based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K‐Ras G12D. Combining the probe‐based query with an ensemble‐based pocket identification scheme and an analysis of existing Ras‐ligand complexes, we show that (i) pMD is a robust and cost‐effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure‐based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in nonpocket‐like regions with known protein‐ and membrane‐interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein‐biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu. Proteins 2015; 83:898–909. © 2015 Wiley Periodicals, Inc.     

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

The integration of SPR biosensors with mass spectrometry: possible applications for proteome analysis

The successful integration of biospecific interaction analysis based on surface plasmon resonance and mass spectrometry produces a powerful technique that couples the benefits of sensitive affinity capture and characterization of binding events with the ability to characterize interacting molecules. A variety of biosensors has been used to capture proteins and peptides biospecifically on sensor surfaces, with subsequent analysis using mass spectrometry. Applying this type of analysis to proteomic studies could lead to ligand and protein-complex identification, and might provide clues leading to the identification of pathways.     

A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement

The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.     

Identification of the ligands of protein interaction domains through a functional approach

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.