Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies.

Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing within a Symmetric Complex.

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with two catalytic A subunits and two carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B subunits, activated FXIII (FXIIIa) covalently cross links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamics simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly or activity to reduce bleeding and thrombotic complications.

TREM-1 multimerization is essential for its activation on monocytes and neutrophils.

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptor expressed on innate immune cells. By promoting the amplification of inflammatory signals that are initially triggered by Toll-like receptors (TLRs), TREM-1 has been characterized as a major player in the pathophysiology of acute and chronic inflammatory diseases, such as septic shock, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. However, the molecular events leading to the activation of TREM-1 in innate immune cells remain unknown. Here, we show that TREM-1 is activated by multimerization and that the levels of intracellular Ca2+ release, reactive oxygen species, and cytokine production correlate with the degree of TREM-1 aggregation. TREM-1 activation on primary human monocytes by LPS required a two-step process consisting of upregulation followed by clustering of TREM-1 at the cell surface, in contrast to primary human neutrophils, where LPS induced a rapid cell membrane reorganization of TREM-1, which confirmed that TREM-1 is regulated differently in primary human neutrophils and monocytes. In addition, we show that the ectodomain of TREM-1 is able to homooligomerize in a concentration-dependent manner, which suggests that the clustering of TREM-1 on the membrane promotes its oligomerization. We further show that the adapter protein DAP12 stabilizes TREM-1 surface expression and multimerization. TREM-1 multimerization at the cell surface is also mediated by its endogenous ligand, a conclusion supported by the ability of the TREM-1 inhibitor LR12 to limit TREM-1 multimerization. These results provide evidence for ligand-induced, receptor-mediated dimerization of TREM-1. Collectively, our findings uncover the mechanisms necessary for TREM-1 activation in monocytes and neutrophils.

Hsp90 oligomerization process: How can p23 drive the chaperone machineries?

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer-oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a "protein wedge" regarding the Hsp90 dimer closure and the Hsp90 oligomerization process.

Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries.

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries.

Optimized protocol for protein macrocomplexes stabilization using the EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, zero-length cross-linker.

Since noncovalent protein macrocomplexes are implicated in many cellular functions, their characterization is essential to understand how they drive several biological processes. Over the past 20 years, because of its high sensitivity, mass spectrometry has been described as a powerful tool for both the protein identification in macrocomplexes and the understanding of the macrocomplexes organization. Nonetheless, stabilizing these protein macrocomplexes, by introducing covalent bonds, is a prerequisite before their analysis by the denaturing mass spectrometry technique. In this study, using the Hsp90/Aha1 macrocomplex as a model (where Hsp denotes a heat shock protein), we optimized a double cross-linking protocol with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). This protocol takes place in a two-step process: initially, a cross-linking is performed according to a previously optimized protocol, and then a second cross-linking is performed by increasing the EDC concentration, counterbalanced by a high dilution of sample and, thus, protein macrocomplexes. Using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we verified the efficiency of our optimized protocol by submitting (or not submitting) samples to the K200 MALDI MS analysis kit containing N-succinimidyl iodo-acetate, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester), suberic acid bis(N-hydroxysuccinimide ester), disuccinimidyl tartrate, and dithiobis(succinimidyl) propionate, developed by the CovalX Company. Results obtained show that our optimized cross-linking protocol allows a complete stabilization of protein macrocomplexes and appears to be very accurate. Indeed, contrary to other cross-linkers, the "zero-length" feature of the EDC reagent prevents overdetermination of the mass of complexes, because EDC does not remain as part of the linkage.

Structural and functional role of INI1 and LEDGF in the HIV-1 preintegration complex.

Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3' processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.

Monitoring ligand modulation of protein-protein interactions by chemical cross-linking and High-Mass MALDI mass spectrometry.

Analyzing the effect of ligands on protein-protein interactions is important to better understand the cellular processes. In vitro characterization of these modulations remains challenging because of the drawbacks associated with the analysis of noncovalent interactions. To facilitate the analysis, stabilization of the protein complex by chemical cross-linking followed by High-Mass MALDI mass spectrometry is a recently developed method offering several advantages: No need for immobilization or special tags, the analysis is possible directly on wild-type protein complexes, no need for buffer exchange, large applicability range for any type of protein complex from 0 to 1,500 kDa. Using this method, we analyzed the effect of the inhibitors Nutlin-3a and Nutlin-3b on the protein complex MDM2-p53. Using this fast and sensitive method, the IC(50) values of these inhibitors have been determined.

A specific protein disorder catalyzer of HIV-1 Nef.

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ∼16 µM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.

Biochemical and biophysical characterization of the Mg2+-induced 90-kDa heat shock protein oligomers.

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.

Detection of nucleic acid-nuclear hormone receptor complexes with mass spectrometry.

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR x RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR x RXR x DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR.

Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full-length prion protein amyloid fibrils.

Understanding the structural basis that distinguishes the amyloid form of the prion protein from its monomeric homologue is of crucial importance to elucidate the mechanism of the lethal diseases related to this protein. Recently, an in vitro conversion system was established which reproduces the transition of recombinant prion protein PrP(23-230) from its native alpha-helical rich form into an aggregated amyloid beta-sheet rich form with physicochemical properties reminiscent to those of the disease-related isoform of the prion protein, PrPSc. To study the tertiary and quaternary structural organization within recombinant amyloid fibrils from mouse, mPrP(23-231)betaf; bovine, bPrP(23-230)betaf; and elk, ePrP(23-230)betaf; we utilized hydrogen/deuterium (H/D) exchange analyzed by matrix-assisted laser desorption/ionization (MALDI) and nano-electrospray (nano-ESI) mass spectrometry. No significant differences were found by measuring the deuterium exchange kinetics of the aggregated fibrillar forms for mPrP(23-231)betaf, bPrP(23-230)betaf and ePrP(23-230)betaf, indicating a similar overall structural organization of the fibrils from all three species. Next, we characterized the solvent accessibility for the soluble and fibrillar forms of the mouse prion protein by hydrogen exchange, pepsin proteolysis and nano-ESI ion trap mass spectrometry analysis. In its amyloid form, two highly protected regions of mPrP(23-231) comprising residues [24-98] and [182-212] were identified. The residues between the two highly protected stretches were found to be more solvent exposed, but less than in the soluble protein, and might therefore rather form part of a fibrillar interface.

Towards high-throughput identification of endocrine disrupting compounds with mass spectrometry.

High-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking has the ability to monitor the ligand-dependent dimerization of the human estrogen receptor alpha ligand binding domain (hERalpha LBD) in solution. Because only ER ligands enhance the homodimer abundance, we evaluated the ability of this label-free approach for identifying endocrine disrupting compounds (EDCs) in a high-throughput manner. This was achieved by combining an automated liquid handler with an automated MS acquisition procedure, which allowed a five-fold gain in operator time compared to a fully manual approach. To detect ligand binding with enough confidence, the receptor has to be incubated with at least a 10 microM concentration of the test compound. Based on the increase of the measured homodimer intensity, eight compounds with a relative binding affinity (RBA, relative to the natural hormone estradiol) >7% were identified as ER ligands among the 28 chemicals tested. Two other compounds, quercetin and 4-tert-amylphenol, were also identified as ER ligands, although their RBAs have been reported to be only 0.01% and 0.000055%, respectively. This suggests that these two ligands have a higher affinity for hERalpha LBD than reported in the literature. The high-mass MALDI approach thus allows identifying high affinity EDCs in an efficient way.

Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor.

Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3' processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

Monitoring ligand modulation of protein-protein interactions by mass spectrometry: estrogen receptor alpha-SRC1.

Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.

Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding.

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry.

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.

Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry.

An analytical strategy for the analysis of antigen epitopes by chemical cross-linking and mass spectrometry is demonstrated. The information of antigen peptides involved in the binding to an antibody can be obtained by monitoring the antigen peptides modified by a partially hydrolyzed cross-linker in the absence and in the presence of an antibody. This approach was shown to be efficient for characterization of the epitope on bovine prion protein bPrP(25-241) specifically recognized by a monoclonal antibody, 3E7 (mAb3E7), with only a small amount of sample (200 picomoles) needed. After cross-linking of the specific immuno complex, a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer equipped with an ion conversion dynode (ICD) high-mass detector was used to optimize the amount of cross-linked complex formed at 202 kDa before proteolytic digestion. To identify the cross-linked peptides after proteolysis without ambiguity, isotope-labeled cross-linkers, disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6), together with high-resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) were used. As a result, a complete fading of the peak intensities corresponding to the peptides representing the epitope was observed when bPrP/mAb3E7 complexes were formed.

Proteomic profiling of a snake venom using high mass detection MALDI-TOF mass spectrometry.

Proteomic profiling involves identification and quantification of protein components in complex biological systems. Most of the mass profiling studies performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been restricted to peptides and small proteins (<20 kDa) because the sensitivity of the standard ion detectors decreases with increasing ion mass. Here we perform a protein profiling study of the snake venom Sistrurus miliarius barbouri, comparing 2D gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) with a high mass cryodetector MALDI-TOF instrument (Macromizer), whose detector displays an uniform sensitivity with mass. Our results show that such MS approach can render superior analysis of protein complexity compared with that obtained with the electrophoretic and chromatographic approaches. The summation of ion impacts allows relative quantification of different proteins, and the number of ion counts correlates with the peak areas in the reversed-phase HPLC. Furthermore, the sensitivity reached with the high mass cryodetection MS technology clearly exceeds the detection limit of standard high-sensitivity staining methods.

Detection of noncovalent complexes in biological samples by intensity fading and high-mass detection MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has not yet contributed widely to the study of intact noncovalent biomolecular complexes, because MALDI is known to cause dissociation of the interaction partners and induce formation of nonspecific aggregates. Here, we present a new strategy to circumvent this problem. It is based on intensity fading (in the low m/z range) and high-mass detection MALDI mass spectrometry (MS), using a cryodetector (in the high m/z range), with and without chemical cross-linking of the interaction partners. The study focuses on noncovalent interactions between the human enzyme carboxypeptidase A (hCPA) and three protease inhibitors (PCI, TCI, and LCI) present in heterogeneous mixtures of other nonbinding molecules derived from a biological source, an extract from leech (Hirudo medicinalis). Another example involves an extract of the sea anemone Stichodactyla helianthus, which is used without previous fractionation to detect the specific complex between the enzyme trypsin and the endogenous SphI-1 inhibitor. The results give insight into the mechanism of intensity fading MS and demonstrate that the specificity of binding is greatly favored when the overall concentrations of the analytes (nonbinding molecules, protease inhibitor and target enzyme) present in a biological sample of interest are kept at low concentrations, in the sub-micromolar range. Higher concentrations may lead to unspecific interactions and the formation of aggregates both during the MALDI process and during reaction with the cross-linking reagents. This strategy is expected to advance the field of high-throughput affinity-based approaches, by taking advantage of a new generation of high mass detectors for MALDI-TOF instruments.