Reactive Residues Research Articles

Considerations for efficient surface functionalization of nanoparticles with a high molecular weight protein as targeting ligand

Functionalization of nanoparticles with ligands is a powerful tool to achieve efficient targeting of receptors expressed on specific cell types. For optimal ligand-receptor interactions, the ligands should be attached on the nanoparticle surface in a predictable manner with specific orientations and density that preserve their bioactivity. While there are many publications on nanoparticles functionalized with small ligands that meet these requirements, achieving these conditions is particularly challenging for protein-based ligands of higher molecular weight. Proteins have complex and often fragile structures with numerous reactive residues, and they generally do not withstand harsh reaction conditions well. They are also prone to non-specific adsorption. Thus, conjugation strategies have to be considered carefully and optimized for each individual protein-based ligand as well as for the particle platform. In this study, we present a comprehensive approach for site-selective conjugation between aminated silica nanoparticles (SiNPs) and the single accessible thiol in human serum albumin (HSA) (66.5 kDa). We varied several reaction parameters including the density of amino groups on the particle surface, protein to amino group molar ratios, and linker length and evaluated their effect on colloidal stability, mode of protein attachment, protein density, and binding capacity of the tethered protein. We demonstrated that particle surface properties strongly impact covalent conjugation. For SiNPs with low amino group density (5,000 NH2/particle), only 25% of the available surface was covered with protein, and up to 90% of HSA was non-specifically adsorbed. Adjusting the molar ratio of HSA and lengthening the linker did not substantially increase the amount of covalently-attached ligand. In contrast, SiNPs with high amino group density (20,000 NH2/particle) showed high protein loading accompanied by low levels of non-specific adsorption. Using a short linker and 1:1 HSA to NH2 molar ratio resulted in 70% surface coverage with HSA molecules. The mode of attachment and protein density strongly impacted the functionality of the immobilized HSA. High non-specific adsorption resulted in the loss of its binding capacity, whereas predominately covalently-conjugated HSA showed binding affinities higher than that of soluble HSA and had a Kd value in the range of about 6 to 12 nM. Our findings indicate that reaction parameters should be carefully assessed to obtain site-selective and specifically oriented conjugation that maintains the protein's binding capacity. The approach presented here may serve as general instruction for the immobilization of high molecular weight targeting proteins to the surfaces of nanoparticles.

Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed βH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound N1-N8-bis(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward βH-crystallins and in particular to the βB2- and mostly in βB3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the N8-terminal reactive portion of N1-mono(γ-glutamyl) SPD, the protein-bound N8-mono(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of βB3-crystallins cross-links by protein-bound N1-N8-bis(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the N8-terminal reactive residue of the crystallins bound N1-mono(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces N1-N8-bis(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

Molecular investigation of Coleopteran specific α-Amylase inhibitors from Amaranthaceae members

α-Amylase inhibitors (α-AIs) target α-amylases and interfere with the carbohydrate digestion of insects. Among different classes of α-AIs, a knottin-type inhibitor from Amaranthus hypochondriacus (AhAI) was found to be specific against coleopteran storage pests. In this report, we have characterized three previously unidentified knottin-type α-AIs from various Amaranthaceae plants namely, Amaranthus hypochondriacus (AhAI2), Alternanthera sessilis (AsAI) and Chenopodium quinoa (CqAI). They contain a signal peptide, pro-peptide, and mature peptide. The mature peptides of the new α-AIs shared 68 to 78% identity with AhAI and have highly variable pro-peptide regions. Along with the cystine-knot fold, they showed conservation of reactive site residues. All recombinant α-AIs were successfully expressed in their active form and native state using an oxidative cytoplasmic environment. Inhibition studies against various amylases revealed that these inhibitors showed selective inhibition of coleopteran recombinant insect α-amylases viz., Tribolium castaneum, and Callosobruchus chinensis. Tribolium castaneum α-amylase inhibition potency was highest for AhAI2 (Ki ~ 15 μM) followed by AsAI (Ki ~ 43 μM) and CqAI (Ki ~ 61 μM). Interaction analysis of these inhibitors illustrated that the reactive site of inhibitors make several non-covalent interactions with the substrate-binding pocket of coleopteran α-amylases. The selectivity of these inhibitors against coleopteran α-amylases highlights their potential in storage grain pest control.

Isobutyl acetate by reactive distillation. Part III. Conceptual design, simulation and optimization

This work studies the conceptual design of a Reactive Distillation (RD) process for isobutyl acetate production by esterification of acetic acid and isobutanol. Static and reactive residue curve map analyses were used to assess RD feasibility, and to identify suitable operating conditions for industrial implementation. The maps were obtained using validated phase equilibria and reaction kinetic models by coupling Aspen plus V.10 and Python. Afterwards, conceptual design outputs were used for a rigorous simulation and optimization of the process. The obtained results indicate that pure isobutyl acetate cannot be obtained as pure product from the RD column as it is a saddle node. However, isobutyl acetate can be obtained as bottom product at enough purity to fulfil urethane grade specifications. A final reactive distillation configuration was developed enabling to obtain almost complete reactants conversion, with a molar composition of isobutyl acetate > 99 % wt. at the bottoms of the column. According to the optimal operating conditions, isobutyl acetate via RD can be produced under an energy intensity of 0.77 kW/kgIbAC, a mass intensity of 1.16 kgreactants/kgIbAC, a productivity of 328.7 kgIbAC/m3h, and a production cost of 0.98 USD/ kgIbAC.

Transition metal catalyzed site-selective cysteine diversification of proteins

Abstract Site-specific protein conjugation is a critical step in the generation of unique protein analogs for a range of basic research and therapeutic developments. Protein transformations must target a precise residue in the presence of a plethora of functional groups to obtain a well-characterized homogeneous product. Competing reactive residues on natural proteins render rapid and selective conjugation a challenging task. Organometallic reagents have recently emerged as a powerful strategy to achieve site-specific labeling of a diverse set of biopolymers, due to advances in water-soluble ligand design, high reaction rate, and selectivity. The thiophilic nature of various transition metals, especially soft metals, makes cysteine an ideal target for these reagents. The distinctive reactivity and selectivity of organometallic-based reactions, along with the unique reactivity and abundancy of cysteine within the human proteome, provide a powerful platform to modify native proteins in aqueous media. These reactions often provide the modified proteins with a stable linkage made from irreversible cross-coupling steps. Additionally, transition metal reagents have recently been applied for the decaging of cysteine residues in the context of chemical protein synthesis. Orthogonal cysteine protecting groups and functional tags are often necessary for the synthesis of challenging proteins, and organometallic reagents are powerful tools for selective, rapid, and water-compatible removal of those moieties. This review examines transition metal-based reactions of cysteine residues for the synthesis and modification of natural peptides and proteins.

Site-Specific Antibody-Drug Conjugates in Triple Variable Domain Fab Format.

The interest in replacing the conventional immunoglobulin G (IgG) format of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) with alternative antibody and antibody-like scaffolds reflects a need to expand their therapeutic utility and potency while retaining their exquisite specificity, affinity, and low intrinsic toxicity. For example, in the therapy of solid malignancies, the limited tumor tissue penetration and distribution of ADCs in IgG format mitigates a uniform distribution of the cytotoxic payload. Here, we report triple variable domain Fab (TVD–Fab) as a new format that affords the site-specific and stable generation of monovalent ADCs without the Fc domain and a drug-to-antibody ratio (DAR) of 2. TVD–Fabs harbor three variable fragment (Fv) domains: one for tumor targeting and two for the fast, efficient, precise, and stable conjugation of two cargos via uniquely reactive lysine residues. The biochemical and in vitro cytotoxicity properties of a HER2-targeting TVD–Fab before and after conjugation to a tubulin inhibitor were validated. In vivo, the TVD–Fab antibody carrier revealed a circulatory half-life of 13.3 ± 2.5 h and deeper tumor tissue distribution compared to our previously reported dual variable domain (DVD)–IgG1 format. Taken together, the TVD–Fab format merits further investigations as an antibody carrier of site-specific ADCs targeting solid malignancies.

The Conformational Preference of Chemical Cross-linkers Determines the Cross-linking Probability of Reactive Protein Residues.

Chemical cross-linking mass spectrometry (XLMS) is an emerging technique in structural biology. Providing the cross-linked peptides are identified by mass spectrometry with high confidence, a distance restraint can be applied between the two reactive protein residues, with the upper bound corresponding to the maximal span of the cross-linker. However, as the upper bound is typically over 20 Å, cross-link distance restraints are unrestrictive and provide a marginal improvement in protein structural refinement. Here we analyze the experimental cross-links for lysine or acidic residues and show that the distribution of Cβ-Cβ' distances can be described with two overlapping Gaussian species. In addition to the pairwise occurrence probability of the reactive protein residues, we show that the distribution profile of the cross-link distances is determined by the intrinsic conformational propensity of the cross-linker. The cross-linker prefers either a compact or extended conformation and, once attached to a reactive protein residue, predominantly an extended conformation. Consequently, the long-distance Gaussian species occurs at a much higher probability than the short-distance species in the observed cross-links. Together, the probabilistic distribution of the cross-link distance allows the construction of a more restrictive restraint for structural modeling and better use of the XLMS data.

Generation and validation of structurally defined antibody-siRNA conjugates.

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody–siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.

Isobutyl acetate by reactive distillation. Non-reactive phase equilibrium and topological analysis

This work studies the non-reactive phase equilibria for the quaternary system Acetic acid - Isobutanol - Isobutyl acetate – Water. Initially, a comprehensive revision and collection of reported VLE, VLLE and LLE data of binary and ternary mixtures involving the studied components was carried out. Afterwards, an experimental evaluation of the ternary LLE for the mixture isobutanol, acetic acid and water, was done at 283, 293 and 303 K. The complete set of experimental and reported data were used to correlate the corresponding binary parameters of the NRTL-HOC thermodynamic model. The adjusted model agrees well with experiments, and it is able to fit the different reported binary azeotropes as verified in a topological diagram. The model also predicts one binary and two ternary azeotropes non-previously reported which are consistent with the azeotropic rule but are yet to be experimentally verified. Finally, a reactive residue curve map was constructed using the transformed compositions diagram. From the topological analysis of the map, it is concluded that pure Isobutyl acetate cannot be obtained as pure product from a reactive distillation column as it is saddle node.

2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

Dissolving Peptides in 0.1% Formic Acid Brings Risk of Artificial Formylation.

The ability of concentrated formic acid to formylate reactive amino acid residues is known from previous reports. In contrast, solvents containing a low concentration of formic acid are generally recognized as a safe environment for proteomic applications. The primary objective of this study was to explain the excessive formylation rate in tryptic peptides that did not come into contact with concentrated formic acid. We found out that the peptide formylation was associated with dissolving the peptides in a solvent containing mere 0.1% formic acid. Similar conclusions were drawn after analyzing publicly available proteomic data. We further demonstrated that these unwanted modifications can be averted via handling the samples at a low temperature or, obviously, via replacing formic acid in the solvent with trifluoroacetic acid. These simple countermeasures can contribute to a reduction in the part of the MS/MS spectra that remain unassigned to a peptide sequence.

Chemical Targeting of Membrane Transporters: Insights into Structure/Function Relationships.

Chemical modification of proteins is a vintage strategy that is still fashionable due to the information that can be obtained from this approach. An interesting application of chemical modification is linked with membrane transporters. These proteins have peculiar features such as the presence of hydrophobic and hydrophilic domains, which show different degree of accessibility to chemicals. The presence of reactive residues in the membrane transporters is at the basis of the chemical targeting strategy devoted to investigating structure/function relationships; in particular, information on the substrate binding site, regulatory domains, dimerization domains, and the interface between hydrophilic loops and transmembrane domains has been obtained over the years by chemical targeting. Given the difficulty in handling membrane transporters, their study experienced a great delay, particularly concerning structural information. Chemical targeting has been applied with reasonable success to some membrane transporters belonging to the families SLC1, SLC6, SLC7, and SLC22. Furthermore, some data on the potential application of chemical targeting in pharmacology are also discussed.

Mitochondria-Immobilized Fluorescent Probe for the Detection of Hypochlorite in Living Cells, Tissues, and Zebrafishes.

A mitochondria targeting and immobilized fluorescent probe (Rd1) using triphenylphosphonium as the targeting group and methoxymaleimide as the fixed site is designed for the detection of ClO-. The methoxymaleimide fixed group can react with nucleophiles, such as the reactive thiol groups present in mitochondrial polypeptides and proteins, and form covalent bonds to immobilize the probe within mitochondria. The immobilization of Rd1 enhances its ability to withstand the risk of leakage from mitochondria. Methoxymaleimide shows better reactivity toward Cys than glutathione (GSH), which decreases the ineffective labeling of GSH when it covalently bonds with the reactive thiol residues of mitochondrial proteins; furthermore, it can resist hydrolysis during a long-term storage in water, compared with the classic benzyl chloride fixed unit. The imaging results indicate that Rd1 displays enhanced retention within the mitochondria of cells and tissues upon the decrease of mitochondrial membrane potential (MMP) caused by different stimulations. Furthermore, it possesses the ability to visualize exogenous and endogenous ClO- in living cells, tissues, and zebrafishes.

An Activity-Based Methionine Bioconjugation Approach To Developing Proximity-Activated Imaging Reporters.

Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.

Textile Wastewater Treatment using Advanced Oxidation Process (AOP)

Wastewater from printing and dyeing industries is often colorful, contains reactive dye residues and chemicals, and requires proper treatment before being released into the environment. The treatment of textile wastes with biotechnology through the general use of high inorganic and organic concentrations is difficult for further process. Therefore, many industries make use of the advanced oxidation process (AOP) because of its ability to eliminate non-degradable organic components and avoid residual deposits removal. a new design of synthetic waste treatment was developed by installing the AOP on the existing wastewater treatment process (WWTP). However, the textile industry faces significant environmental problems because of its vast water consumption. This makes the resulting wastewater from their activities characterized by intense color, a large number of suspended solids, very unpredictable pH and high COD concentrations. Therefore, there is a need to implement some control measures in the future in order to minimize these waste problems. Experiments show that the pollutants can be physically removed by using the AOP process and COD decrease 51.7 % from 16.167 mg/L into 8.364 mg/L.

2-Hydroxy-(4-methylseleno)butanoic Acid Is Used by Intestinal Caco-2 Cells as a Source of Selenium and Protects against Oxidative Stress

Selenium (Se) participates in different functions in humans and other animals through its incorporation into selenoproteins as selenocysteine. Inadequate dietary Se is considered a risk factor for several chronic diseases associated with oxidative stress. The role of 2-hydroxy-(4-methylseleno)butanoic acid (HMSeBA), an organic form of Se used in animal nutrition, in supporting selenoprotein synthesis and protecting against oxidative stress was investigated in an in vitro model of intestinal Caco-2 cells. Glutathione peroxidase (GPX) and thioredoxin reductase (TXNRD) activities, selenoprotein P1 protein (SELENOP) and gene (SELENOP) expression, and GPX1 and GPX2 gene expression were studied in Se-deprived (FBS removal) and further HMSeBA-supplemented (0.1-625 μM, 72 h) cultures. The effect of HMSeBA supplementation (12.5 and 625 μM, 24 h) on oxidative stress induced by H2O2 (1 mM) was evaluated by the production of reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE) adducts, and protein carbonyl residues compared with a sodium selenite control (SS, 5 μM). Se deprivation induced a reduction (P<0.05) in GPX activity (62%), GPX1 expression, and both SELENOP (33%) and SELENOP expression. In contrast, an increase (P<0.05) in GPX2 expression and no effect in TXNRD activity (P=0.09) were observed. HMSeBA supplementation increased (P<0.05) GPX activity (12.5-625 μM, 1.68-1.82-fold) and SELENOP protein expression (250 and 625 μM, 1.87- and 2.04-fold). Moreover, HMSeBA supplementation increased (P<0.05) GPX1 (12.5 and 625 μM), GPX2 (625 μM), and SELENOP (12.5 and 625 μM) expression. HMSeBA (625 μM) was capable of decreasing (P<0.05) ROS (32%), 4-HNE adduct (49%), and protein carbonyl residue (75%) production after H2O2 treatment. Caco-2 cells can use HMSeBA as an Se source for selenoprotein synthesis, resulting in protection against oxidative stress.

Site-Specific Lysine Arylation as an Alternative Bioconjugation Strategy for Chemically Programmed Antibodies and Antibody–Drug Conjugates

By exploiting a uniquely reactive lysine residue (Lys99) for site-specific attachment of small molecules, the humanized catalytic antibody h38C2 has been used as bioconjugation module in the assembly of chemically programmed antibodies and antibody-drug conjugates. Treatment of h38C2 with β-lactam-functionalized small molecules has been previously shown to result in covalent conjugation by selective formation of a stable amide bond with the ε-amino group of the Lys99 residue. Here we report that heteroaryl methylsulfonyl (MS-PODA)-functionalized small molecules represent an alternative bioconjugation strategy through highly efficient, site-specific, and stable arylation of the Lys99 residue. A set of chemically programmed antibodies and antibody-drug conjugates assembled by Lys99 arylation provided proof-of-concept for the therapeutic utility of this alternative bioconjugation strategy. While being equally effective as β-lactam-functionalized ligands for bioconjugation with catalytic antibody h38C2, the MS-PODA moiety offers distinct synthetic advantages, making it highly attractive.

Conjugation Site Analysis of Lysine-Conjugated ADCs.

Lysine-conjugated antibody-drug conjugates (ADCs) are formed by attaching cytotoxic drugs to reactive lysine residues of monoclonal antibodies (mAbs) through chemical linkers. During production, the payloads are conjugated nonspecifically to lysine residues in mAbs, resulting in a heterogeneous mixture of ADCs with both different number and conjugation sites of drug payloads per mAb. On account of the drug conjugation sites and levels that both have significant influences on physical and pharmaceutical properties of ADCs, a reliable and straightforward approach for conjugation site analysis for ADCs is highly demanded. Herein, we used a lysine-conjugated ADC, Trastuzumab-MCC-DM1 (T-DM1), as a model ADC, and described an integrative strategy that combines the signature ion fingerprinting method for rapid and reliable filtering of DM1-conjugated peptides, and the normalized area quantitation approach for accurately gauging the conjugation levels for each identified site. This approach is believed to be readily applicable to other maytansinoid derivatives-modified ADCs, and more importantly, universally applicable to lysine-conjugated ADCs for both the recognition of conjugation sites and the measurement of conjugation levels.

Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.