Intact Transition Epitope Mapping-Force Interferences by Variable Extensions (ITEM-FIVE).

Investigations on binding strength differences of non-covalent protein complex components were performed by mass spectrometry. T4 fibritin foldon (T4Ff) is a well-studied miniprotein, which together with its biotinylated version served as model system to represent a compactly folded protein to which an Intrinsically Disordered Region (IDR) was attached. The apparent enthalpies of the gas phase dissociation reactions of the homo-trimeric foldon F-F-F and of the homo-trimeric triply biotinylated foldon bF-bF-bF have been determined to be rather similar (3.32 kJ/mol and 3.85 kJ/mol) but quite distinct from those of the singly and doubly biotinylated hetero-trimers F-F-bF and F-bF-bF (1.86 kJ/mol and 1.08 kJ/mol). Molecular dynamics simulations suggest that the ground states of the (biotinylated) T4Ff trimers are highly symmetric and well comparable to each other, indicating that the energy levels of all four (biotinylated) T4Ff trimer ground states are nearly indistinguishable. The experimentally determined differences and/or similarities in enthalpies of the complex dissociation reactions are explained by entropic spring effects, which are noticeable in the T4Ff hetero-trimers but not in the T4Ff homo-trimers. A lowering of the transition state energy levels of the T4Ff hetero-trimers seems likely because the biotin moieties, mimicking intrinsically disordered regions (IDRs), induced asymmetries in the transition states of the biotinylated T4Ff hetero-trimers. This transition state energy level lowering effect is absent in the T4Ff homo-trimer, as well as in the triply biotinylated T4Ff homo-trimer. In the latter, the IDR-associated entropic spring effects on complex stability cancel each other out. ITEM-FIVE enabled semi-quantitative determination of energy differences of complex dissociation reactions, whose differences were modulated by IDRs attached to compactly folded proteins.

Dried serum spots on pre-punched filter paper discs are ready-to-use storage and shipping devices for blood-borne antigens and antibodies.

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 µl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 µl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.

Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A - pepsin complex binding strength differences.

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (Ca and Cb) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.

Intact Transition Epitope Mapping-Serological Inspection by Epitope EXtraction (ITEM-SIX).

Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism's serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer's gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping-Serological Inspections by Epitope EXtraction (ITEM-SIX).

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis.

With Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody.

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

Cell wall channels of Rhodococcus species: identification and characterization of the cell wall channels of Rhodococcus corynebacteroides and Rhodococcus ruber.

The cell wall of Rhodococcus corynebacteroides formerly known as Nocardia corynebacteroides contains cell wall channels that are responsible for the cell wall permeability of this bacterium. Based on partial sequencing of the polypeptide subunits and a BLAST search, we identified one polypeptide of R. corynebacteroides (PorARc) and two polypeptides (PorARr and PorBRr) from the closely related bacterium Rhodococcus ruber. The corresponding genes, porARc (606 bp), porARr (702 bp), and porBRr (540 bp) are constituents of the known genome of R. corynebacteroides DSM-20151 and R. ruber DSM-43338, respectively. porARr and porBRr of R. ruber are possibly forming a common operon coding for the polypeptide subunits of the cell wall channel. The genes coding for PorARc and for PorARr and PorBRr without signal peptide were separately expressed in the porin-deficient Escherichia coli BL21DE3Omp8 strain and the proteins were purified to homogeneity. All proteins were checked for channel formation in lipid bilayers. PorARc formed channels with characteristics that were very similar to those of a previous study. The proteins PorARr and PorBRr expressed in E. coli could alone create channels in lipid bilayer membranes, despite the possibility that the two corresponding genes form a porin operon and that both subunits possibly form the cell wall channels in vivo. Based on amino acid sequence comparison of a variety of proteins forming cell wall channels in bacteria of the suborder Corynebacterineae, it seems very likely that PorARc, PorARr, and PorBRr are members of a huge family of proteins (PF09203) that form MspA-like cell wall channels.

Clostridium perfringens Beta2 toxin forms highly cation-selective channels in lipid bilayers.

Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A-G) of C. perfringens. Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens. Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.

Mass Spectrometric and Bio-Computational Binding Strength Analysis of Multiply Charged RNAse S Gas-Phase Complexes Obtained by Electrospray Ionization from Varying In-Solution Equilibrium Conditions.

We investigated the influence of a solvent's composition on the stability of desorbed and multiply charged RNAse S ions by analyzing the non-covalent complex's gas-phase dissociation processes. RNAse S was dissolved in electrospray ionization-compatible buffers with either increasing organic co-solvent content or different pHs. The direct transition of all the ions and the evaporation of the solvent from all the in-solution components of RNAse S under the respective in-solution conditions by electrospray ionization was followed by a collision-induced dissociation of the surviving non-covalent RNAse S complex ions. Both types of changes of solvent conditions yielded in mass spectrometrically observable differences of the in-solution complexation equilibria. Through quantitative analysis of the dissociation products, i.e., from normalized ion abundances of RNAse S, S-protein, and S-peptide, the apparent kinetic and apparent thermodynamic gas-phase complex properties were deduced. From the experimental data, it is concluded that the stability of RNAse S in the gas phase is independent of its in-solution equilibrium but is sensitive to the complexes' gas-phase charge states. Bio-computational in-silico studies showed that after desolvation and ionization by electrospray, the remaining binding forces kept the S-peptide and S-protein together in the gas phase predominantly by polar interactions, which indirectly stabilized the in-bulk solution predominating non-polar intermolecular interactions. As polar interactions are sensitive to in-solution protonation, bio-computational results provide an explanation of quantitative experimental data with single amino acid residue resolution.

Profiling of intact blood proteins by matrix-assisted laser desorption/ionization mass spectrometry without the need for freezing - Dried serum spots as future clinical tools for patient screening.

RATIONALE: To open up new ways for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based patient screening, blood serum is the most preferred specimen because of its richness in patho-physiological information and due to ease of collection. To overcome deleterious freeze/thaw cycles and to reduce high costs for shipping and storage, we sought to develop a procedure which enables MALDI-MS protein profiling of blood serum proteins without the need for serum freezing. METHODS: Blood sera from patients/donors were divided into portions which after pre-incubation were fast frozen. Thawed aliquots were deposited on filter paper discs and air-dried at room temperature. Intact serum proteins were eluted with acid-labile detergent-containing solutions and were desalted by employing a reversed-phase bead system. Purified protein solutions were screened by MALDI-MS using standardized instrument settings. RESULTS: MALDI mass spectra from protein solutions which were eluted from filter paper discs and desalted showed on average 25 strong ion signals (mass range m/z 6000 to 10,000) from intact serum proteins (apolipoproteins, complement proteins, transthyretin and hemoglobin) and from proteolytic processing products. Semi-quantitative analysis of three ion pairs: m/z 6433 and 6631, m/z 8205 and 8916, as well as m/z 9275 and 9422, indicated that the mass spectra from either pre-incubated fast-frozen serum or pre-incubated dried serum spot eluted serum contained the same information on protein composition. CONCLUSIONS: A workflow that avoids the conventional cold-chain and yet enables the investigation of intact serum proteins and/or serum proteolysis products by MALDI-MS profiling was developed. The presented protocol tremendously broadens the clinical application of MALDI-MS and simultaneously allows a reduction in the costs for storage and shipping of serum samples. This will pave the way for clinical screening of patients also in areas with limited access to health care systems, and/or specialized laboratories.

Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization.

Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10-12 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10-12 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10-12 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10-12 was calculated for the troponin I epitope peptide-antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.

ITEM-THREE analysis of a monoclonal anti-malaria antibody reveals its assembled epitope on the pfMSP119 antigen.

Rapid diagnostic tests are first-line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population-screening assays, high-quality reagents and well-characterized antigens and antibodies are needed. An important property of antigen-antibody binding is recognition specificity, which best can be estimated by mapping an antibody's epitope on the respective antigen. We have cloned a malarial antigen-containing fusion protein, MBP-pfMSP119, in Escherichia coli, which then was structurally and functionally characterized before and after high pressure-assisted enzymatic digestion. We then used our previously developed method, intact transition epitope mapping-targeted high-energy rupture of extracted epitopes (ITEM-THREE), to map the area on the MBP-pfMSP119 antigen surface that is recognized by the anti-pfMSP119 antibody G17.12. We identified three epitope-carrying peptides, 386GRNISQHQCVKKQCPQNSGCFRHLDE411, 386GRNISQHQCVKKQCPQNSGCFRHLDEREE414, and 415CKCLLNYKQE424, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP-pfMSP119 antigen whose identification was substantiated by positive and negative control experiments. In conclusion, our data help to establish a workflow to obtain high-quality control data for diagnostic assays, including the use of ITEM-THREE as a powerful analytical tool. Data are available via ProteomeXchange: PXD019717.

Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO).

We have developed an electrospray mass spectrometry method which is capable to determine antibody affinity in a gas phase experiment. A solution with the immune complex is electrosprayed and multiply charged ions are translated into the gas phase. Then, the intact immune-complex ions are separated from unbound peptide ions. Increasing the voltage difference in a collision cell results in collision induced dissociation of the immune-complex by which bound peptide ions are released. When analyzing a peptide mixture, measuring the mass of the complex-released peptide ions identifies which of the peptides contains the epitope. A step-wise increase in the collision cell voltage difference changes the intensity ratios of the surviving immune complex ions, the released peptide ions, and the antibody ions. From all the ions´ normalized intensity ratios are deduced the thermodynamic quasi equilibrium dissociation constants (KDm0g#) from which are calculated the apparent gas phase Gibbs energies of activation over temperature (ΔGm0g#T). The order of the apparent gas phase dissociation constants of four antibody - epitope peptide pairs matched well with those obtained from in-solution measurements. The determined gas phase values for antibody affinities are independent from the source of the investigated peptides and from the applied instrument. Data are available via ProteomeXchange with identifier PXD016024. SIGNIFICANCE: ITEM - TWO enables rapid epitope mapping and determination of apparent dissociation energies of immune complexes with minimal in-solution handling. Mixing of antibody and antigen peptide solutions initiates immune complex formation in solution. Epitope binding strengths are determined in the gas phase after electrospraying the antibody / antigen peptide mixtures and mass spectrometric analysis of immune complexes under different collision induced dissociation conditions. Since the order of binding strengths in the gas phase is the same as that in solution, ITEM - TWO characterizes two most important antibody properties, specificity and affinity.

Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE).

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human ß-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.

Mass spectrometric epitope mapping.

Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229-241, 2018.

Mass spectrometric characterization of protein structures and protein complexes in condensed and gas phase.

Proteins are essential for almost all physiological processes of life. They serve a myriad of functions which are as varied as their unique amino acid sequences and their corresponding three-dimensional structures. To fulfill their tasks, most proteins depend on stable physical associations, in the form of protein complexes that evolved between themselves and other proteins. In solution (condensed phase), proteins and/or protein complexes are in constant energy exchange with the surrounding solvent. Albeit methods to describe in-solution thermodynamic properties of proteins and of protein complexes are well established and broadly applied, they do not provide a broad enough access to life-science experimentalists to study all their proteins' properties at leisure. This leaves great desire to add novel methods to the analytical biochemist's toolbox. The development of electrospray ionization created the opportunity to characterize protein higher order structures and protein complexes rather elegantly by simultaneously lessening the need of sophisticated sample preparation steps. Electrospray mass spectrometry enabled us to translate proteins and protein complexes very efficiently into the gas phase under mild conditions, retaining both, intact protein complexes, and gross protein structures upon phase transition. Moreover, in the environment of the mass spectrometer (gas phase, in vacuo), analyte molecules are free of interactions with surrounding solvent molecules and, therefore, the energy of inter- and intramolecular forces can be studied independently from interference of the solvating environment. Provided that gas phase methods can give information which is relevant for understanding in-solution processes, gas phase protein structure studies and/or investigations on the characterization of protein complexes has rapidly gained more and more attention from the bioanalytical scientific community. Recent reports have shown that electrospray mass spectrometry provides direct access to six prime protein complex properties: stabilities, compositions, binding surfaces (epitopes), disassembly processes, stoichiometries, and thermodynamic parameters.

Apparent activation energies of protein-protein complex dissociation in the gas-phase determined by electrospray mass spectrometry.

We have developed a method to determine apparent activation energies of dissociation for ionized protein-protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein-protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ([Formula: see text]) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein-protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants. Graphical abstract Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.

Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phosphopeptide analysis with a solidified ionic liquid matrix.

A solidified ionic liquid matrix (SILM) consisting of 3-aminoquinoline, α-cyano-4- hydroxycinnamic acid and ammonium dihydrogen phosphate combines the benefits of liquid and solid MALDI matrices and proves to be well suitable for phosphopeptide analysis using MALDI-MS in the low femtomole range. Desalting and buffer exchange that typically follow after phosphopeptide elution from metal oxide affinity chromatography (MOAC) materials can be omitted. Shifting the pH from acidic to basic during target preparation causes slow matrix crystallization and homogeneous embedding of the analyte molecules, forming a uniform preparation from which (phospho)peptides can be ionized in high yields over long periods of time. The novel combination of MOAC-based phosphopeptide enrichment with SILM preparation has been developed with commercially available standard phosphopeptides and with α-casein as phosphorylated standard protein. The applicability of the streamlined phosphopeptide analysis procedure to cell biological and clinical samples has been tested (i) using affinity-enriched endogenous TRIM28 from cell cultures and (ii) by analysis of a two-dimensional gel-separated protein spot from a bladder cancer sample.

Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.

We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions as a function of incubation time. For proving that a provoked proteolytic activity in mild acidic solution caused the appearance of the observed peptides, control measurements were performed (i) with pepstatin as protease inhibitor, (ii) with heat-denatured samples, (iii) at pH 1.7, and (iv) at pH 7.5. Mass spectrometric fragmentation analysis showed that the observed peptides encompass the amino acid sequences 1-24 and 1-26 from the N-terminus of human serum albumin. Investigations on peptidase specificities suggest that the two best candidates for the observed serum albumin cleavages are cathepsin D and E. Reproducibility, robustness, and sensitivity prove the potential of the developed limited proteolysis assay to become of clinical importance for estimating dynamics of plasma-borne proteases with respect to associated pathophysiological tissue conditions.