Analyzing protein-protein interactions by quantitative mass spectrometry

Since most cellular processes depend on interactions between proteins, information about protein–protein interactions (PPIs) provide valuable insights into protein function. Over the last years, quantitative affinity purification followed by mass spectrometry (q-AP-MS) has become a powerful approach to investigate PPIs in an unbiased manner. In q-AP-MS the protein of interest is biochemically enriched together with its interaction partners. In parallel, a control experiment is performed to control for non-specific binding. Quantitative mass spectrometry is then employed to compare protein levels in both samples and to exclude non-specific contaminants. Here, we provide two detailed q-AP-MS protocols for pull-downs with immobilized bait proteins or transient transfection of tagged expression constructs. We discuss benefits and limitations of q-AP-MS and highlight critical parameters that need to be considered. The protocols and background information presented here allow the reader to adapt the generic q-AP-MS strategy for a wide range of biological questions.     

Isolation and affinity maturation of hapten-specific antibodies

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.     

Domain-level stability of an antibody monitored by reduction, differential alkylation, and mass spectrometry analysis

Human immunoglobulin G1 (IgG1) contains 12 domains, and each has an intrachain disulfide bond that connects the two layers of antiparallel β-sheets. These intrachain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with [¹²C]iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alkylation with [¹³C]iodoacetic acid. The percentage of modification by [¹²C]iodoacetic acid of each cysteine residue was calculated using mass spectra of the cysteine-containing tryptic peptides and used to follow the unfolding of each domain. It demonstrated that the CH2 domain was the least stable domain of the antibody, whereas the CH3 domain was the most stable domain of the antibody. Other domains showed intermediate resistance to the denaturant concentration, similar to the overall unfolding transition monitored by the intrinsic tryptophan fluorescence wavelength shift.     

Method for suppressing non-specific protein interactions observed with affinity resins

Previous high throughput data analysis from several different approaches to affinity purification of protein complexes have revealed catalogues of contaminating proteins that persistently co-purify. Some of these contaminating proteins appear to be specific to one particular affinity matrix used or even to the artificial affinity tags introduced into endogenous proteins for the purpose of purification.A recent approach to minimising non-specific protein interactions in high throughput screens utilises pre-equilibration of affinity surfaces with thiocyanate anions to reduce non-specific binding of proteins. This approach not only reduces the effect of contaminating proteins but also promotes the enrichment of the specific binding partners. Here, we have taken this method and adapted it in an attempt to reduce the abundance of common contaminants in affinity purification experiments. We found the effect varied depending on the bait used, most likely due to its endogenous abundance.     

Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody

X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11–24), a short second sub-epitope (residues 83–89) and a third at the C-terminus (residues 112–123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and β chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF.     

Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein

Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.     

Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe

Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin–TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin–TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein–protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.     

Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase

Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30min to purify BChE from 1ml plasma. The method uses a microfuge spin column to build a 0.2ml procainamide affinity column. The eluted BChE contains 3-4mug of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.     

Catching and separating protein ligands by functional affinity electrophoresis

A new kind of affinity electrophoresis called functional affinity electrophoresis (FAEP) is a technique used to separate and/or capture proteins according to their functions in a native polyacrylamide gel. Protein A:immunoglobulin G, avidin:biotin, antibody:antigen, and concanavalin A:glycoprotein interactions are used to demonstrate this technique. Protein A, avidin, monoclonal anti-bovine serum albumin (BSA) antibody, and concanavalin A are embedded in distinct regions of a 7.5% native polyacrylamide gel. Some of each of the embedded proteins get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these proteins do not show significant electrophoretic mobility or they migrate in a direction opposite to the protein analytes, as in avidin. We clearly observe that polyclonal anti-human myoglobin antibody, biotinylated insulin, BSA, and ovalbumin (glycoprotein) are captured and separated in distinct regions of a FAEP gel by protein A, avidin, monoclonal anti-BSA antibody, and concanavalin A, respectively.     

ELISA reagent coverage evaluation by affinity purification tandem mass spectrometry

Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been used to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput. There are, however, inherent difficulties reconciling the protein-centric results of MS characterization with ELISA, or providing assurance that ELISA has acceptable coverage against all process-specific HCP impurities that could pose safety or efficacy risks. Here, we describe efficient determination of ELISA reagent coverage by proteomic analysis following affinity purification with a polyclonal anti-HCP reagent (AP-MS). The resulting HCP identifications can be compared with the actual downstream process impurities for a given process to enable a highly focused assessment of ELISA reagent suitability. We illustrate the utility of this approach by performing coverage evaluation of an anti-HCP polyclonal against both an HCP immunogen and the downstream HCP impurities identified in a therapeutic monoclonal antibody after Protein A purification. The overall goal is to strategically implement affinity-based mass spectrometry as part of a holistic framework for evaluating HCP process clearance, ELISA reagent coverage, and process clearance risks. We envision coverage analysis by AP-MS will further enable a framework for HCP impurity analysis driven by characterization of actual product-specific process impurities, complimenting analytical methods centered on consideration of the total host cell proteome.     

High-throughput and multiplexed regeneration buffer scouting for affinity-based interactions

Affinity-based analyses on biosensors depend partly on regeneration between measurements. Regeneration is performed with a buffer that efficiently breaks all interactions between ligand and analyte while maintaining the active binding site of the ligand. We demonstrated a regeneration buffer scouting using the combination of a continuous flow microspotter with a surface plasmon resonance imaging platform to simultaneously test 48 different regeneration buffers on a single biosensor. Optimal regeneration conditions are found within hours and consume little amounts of buffers, analyte, and ligand. This workflow can be applied to any ligand that is coupled through amine, thiol, or streptavidin immobilization.     

Primary structure of equine myelin basic protein by mass spectrometry

Equine myelin basic protein (MBP) has been isolated from spinal cord and shown to consist of a number of components (charge isomers) by alkaline-urea gel electrophoresis. Mass analyses of several of these components showed that each was posttranslationally modified and some have been identified. Component 1, the most cationic charge isomer, was sequenced by a combination of liquid chromatography and mass spectrometry of peptides obtained by proteolytic digestion. At 172 residues it is slightly larger than the bovine (169) and the human (170). A major difference between bovine and equine sequences was the replacement of AQGH (bovine residues 76-79) by SRDG (equine). A number of other replacements involving single amino acids were also found. Methylated arginine (residue 108 equine) was found as both the mono- and the dimethylated derivative and represents the first MS/MS evidence for this modification in any MBP.     

Unraveling the dynamics of protein interactions with quantitative mass spectrometry

Knowledge of structure and dynamics of proteins and protein complexes is important to unveil the molecular basis and mechanisms involved in most biological processes. Protein complex dynamics can be defined as the changes in the composition of a protein complex during a cellular process. Protein dynamics can be defined as conformational changes in a protein during enzyme activation, for example, when a protein binds to a ligand or when a protein binds to another protein. Mass spectrometry (MS) combined with affinity purification has become the analytical tool of choice for mapping protein–protein interaction networks and the recent developments in the quantitative proteomics field has made it possible to identify dynamically interacting proteins. Furthermore, hydrogen/deuterium exchange MS is emerging as a powerful technique to study structure and conformational dynamics of proteins or protein assemblies in solution. Methods have been developed and applied for the identification of transient and/or weak dynamic interaction partners and for the analysis of conformational dynamics of proteins or protein complexes. This review is an overview of existing and recent developments in studying the overall dynamics of in vivo protein interaction networks and protein complexes using MS-based methods.     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

Optimization of surface plasmon resonance experiments: Case of high mobility group box 1 (HMGB1) interactions

Surface plasmon resonance (SPR) is a powerful technique for evaluating protein–protein interactions in real time. However, inappropriately optimized experiments can often lead to problems in the interpretation of data, leading to unreliable kinetic constants and binding models. Optimization of SPR experiments involving “sticky” proteins, or proteins that tend to aggregate, represents a typical scenario where it is important to minimize errors in the data and the kinetic analysis of those data. This is the case of High Mobility Group Box 1 and the receptor of advanced glycation end products. A number of improvements in protein purification, buffer composition, immobilization conditions, and the choice of flow rate are shown to result in substantial improvements in the accurate characterization of the interactions of these proteins and the derivation of the corresponding kinetic constants.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry

We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE–SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE–SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP–HPLC–TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains.