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The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG  recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N -linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Mei et al.)

Numerous biological activities have been reported for peptides or peptide-rich fractions from hydrolyzed food proteins. Some of the properties of peptides include antioxidant, antimicrobial, anti-inflammation, antihypertensive, and immune system modulation. To evaluate the efficacy of peptides in vivo, foods have been used as carrier vehicles. However, there are many molecules in foods that can react or interact with peptides, thereby reducing the bioavailability or bioactivity of these peptides. The Schiff base reactions of peptides with reducing sugars are well established. Peptides can also react with oxidized lipids or reactive oxygen species. Secondary metabolites such as quinones in foods, can react as well with amine or thiol groups of peptides. All these reactions affect the concentration of peptides. This review summarizes and discusses some of the implication of those reactions on the availability of bioactive peptides within the food matrix. PRACTICAL APPLICATIONS: Bioactive peptides possess specific effects on foods like preventing oxidative rancidity. In human, they may lower blood pressure or reduce inflammation. The knowledge on the type of reactions these peptides may encountered when foods are used as transport vehicles is essential. This will prevent variability in intake and ensure proper dosage and reproducible results., (© 2017 Wiley Periodicals, Inc.)

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis. Chromatographic data provide a set of sequence-specific information complementary to mass spectrometry. In order to compare measured retention times (RTs) with those calculated from the sequences derived from protein databases, it is necessary to develop chromatographic models and tools allowing the prediction of RT and elution order for peptides with modified residues. In this work we extended recently introduced critical liquid chromatography of biomacromolecule model for prediction of RTs for peptides containing asparagines, aspartic acid, and isoaspartic acid residues., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Acids are often used for deamidating proteins, but the literature on acetic acid deamidation of proteins is sparse. Previous research on acetic acid-induced modification of proteins has focused on peptide proteolysis by relatively high concentrations of acetic acid (>1.5 mol L(-1)) rather than on the accompanying effect of deamidation. Therefore the objective of this study was to determine the deamidation effect of acetic acid with as little peptide proteolysis as possible by employing low-concentration acetic acid (<0.05 mol L(-1)) to deamidate wheat gluten. Changes in surface hydrophobicity, conformation, functional properties and nutritional characteristics of acetic acid-modified samples were determined and compared with those of hydrochloric acid (HCl)-modified samples., Results: At similar degree of deamidation and nitrogen solubility index, samples deamidated with acetic acid showed less destruction of peptides bandings, better foaming properties and a more decompacted form (lower S--S content in protein as determined by Raman spectroscopy) than those deamidated with HCl and also exhibited improved emulsification capacity and emulsion stability compared with native wheat gluten. Acetic acid deamidation led to fewer changes in peptide molecular size and secondary structure of wheat gluten compared with HCl deamidation according to the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and Fourier transform infrared spectroscopy respectively. Amino acid analysis revealed that the nutritional characteristics of wheat gluten were well maintained after deamidation with acetic acid., Conclusion: The results show that low-concentration acetic acid can modify wheat gluten mainly by deamidation, resulting in deamidated wheat gluten with good functional and nutritional properties.

Capillary electrophoresis (CE) was compared with reversed-phase liquid chromatography for its ability to separate native and deamidated peptides. CE is shown to provide superior resolution of these peptides due to its charge-based separation mechanism. Fraction collection performed using a standard CE instrument equipped with a 96-well plate permits subsequent characterization by nanospray mass spectrometric (MS) analysis. Additional in-depth analysis by MS/MS is able to provide the location of the deamidation site based on y-ion mass shifts of 1Da.

Activation of small intestinal gluten-reactive CD4(+) T-cells is a critical event in celiac disease. Deamidation of specific glutamine residues by tissue transglutaminase enhances the binding of T-cell activating gliadin epitopes to DQ2, increasing T-cell recognition. Our purpose was to investigate whether deamidated gliadin epitopes can be generated in the small intestinal mucosa by tissue transglutaminase and to characterize the location of the process. Intestinal explants from pig intestine and frozen biopsy slices from human and rat intestine were incubated with alpha-gliadin peptides containing the immunodominant motif. Monoclonal antibodies specifically recognizing the non-deamidated and/or the deamidated epitope were used for immunofluorescence studies. We conclude that endogenous tissue transglutaminase can mediate extracellular deamidation of gliadin peptides in the lamina propria. Gliadin peptides with more than one recognition site can be simultaneously cross-linked and deamidated extracellularly in the lamina propria, and might be of importance for the antibody response seen in untreated celiac disease patients.

The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4+ T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 alpha-I (PQPELPYPQ) and DQ8 alpha-I (EGSFQPSQE), respectively, are reported.

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z(n)-57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments.

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n = 0, 1, 3, 5, 7, 9, 11), (3) QLnR (n = 0, 1, 3, 5, 7, 9), and (4) QLn (n = 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability.

In the present study, reactions of sodium nitrite with proteins/peptides were characterized with mass spectrometry. The reaction generates two major products: replacement of the amino group by a hydroxyl group and formation of an alkene derivative by loss of a NH3 group at the N-terminus and the side chain of lysine residues of proteins/peptides. The reaction proceeds rapidly in weak acidic solution and at 37 degrees C in the presence of a millimolar concentration of nitrite, demonstrating that nitrite induces nitrosative deamination in proteins and peptides. The facile nitrite-induced modification of amino groups of protein/peptides changes the chemical nature of proteins and may have various applications in peptide synthesis, analytical chemistry, and protein engineering. It also provides information to enhance our understanding of functions of nitrite ions in biology and food preservation., (Copyright 2006 John Wiley & Sons, Ltd.)

The utility of peptide self-assembly can be extended by covalent capture of these supramolecular materials. Disulfide bond formation, native chemical ligation, olefin metathesis, radical capture and oxidative lysine cross-linking have been used recently to help stabilize and characterize a variety of self-assembled peptides. These include natural peptides, proteins and protein mimics such as alpha-helical coiled coils, amyloid-like beta-sheet fibres, portions of p53, glutathione S-transferase and elastin as well as unnatural peptide constructs such as cyclic peptide nanotubes and cylindrical micelles of peptide amphiphiles.

Processes involving self-assembly of monomeric units into organized polymeric arrays are currently the subject of much attention, particularly in the areas of nanotechnology and biomaterials. One biological example of a protein polymer with potential for self-organization is elastin. Elastin is the extracellular matrix protein that imparts the properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Tropoelastin, the approximately 70 kDa soluble monomeric form of elastin, is highly nonpolar in character, consisting essentially of 34 alternating hydrophobic and crosslinking domains. Crosslinking domains contain the lysine residues destined to form the covalent intermolecular crosslinks that stabilize the polymer. We and others have suggested that the hydrophobic domains are sites of interactions that contribute to juxtaposition of lysine residues in preparation for crosslink formation. Here, using recombinant polypeptides based on sequences in human elastin, we demonstrate that as few as three hydrophobic domains flanking two crosslinking domains are sufficient to support a self-assembly process that aligns lysines for zero-length crosslinking, resulting in formation of the crosslinks of native elastin. This process allows fabrication of a polymeric matrix with solubility and mechanical properties similar to those of native elastin., (Copyright 2003 Wiley Periodicals, Inc. Biopolymers 70: 445-455, 2003)

Eptifibatide, a synthetic peptide inhibitor of the platelet glycoprotein IIb/IIIa receptor, has been studied as an antithrombotic agent in a variety of acute ischemic coronary syndromes. The purpose of the present study was to characterize the disposition of 14C-eptifibatide in man after a single intravenous (i.v.) bolus dose. 14C-Eptifibatide (approximately 50 microCi) was administered to eight healthy men as a single 135-microgram/kg i.v. bolus. Blood, breath carbon dioxide, urine, and fecal samples were collected for up to 72 hours postdose and analyzed for radioactivity by liquid scintillation spectrometry. Plasma and urine samples were also assayed by liquid chromatography with mass spectrometry for eptifibatide and deamidated eptifibatide (DE). Mean (+/- SD) peak plasma eptifibatide concentrations of 879 +/- 251 ng/mL were achieved at the first sampling time (5 minutes), and concentrations then generally declined biexponentially, with a mean distribution half-life of 5 +/- 2.5 minutes and a mean terminal elimination half-life of 1.13 +/- 0.17 hours. Plasma eptifibatide concentrations and radioactivity declined in parallel, with most of the radioactivity (82.4%) attributed to eptifibatide. A total of approximately 73% of administered radioactivity was recovered in the 72-hour period following 14C-eptifibatide dosing. The primary route of elimination was urinary (98% of the total recovered radioactivity), whereas fecal (1.5%) and breath (0.8%) excretion was small. Eptifibatide is cleared by both renal and nonrenal mechanisms, with renal clearance accounting for approximately 40% of total body clearance. Within the first 24 hours, the drug is primarily excreted in the urine as unmodified eptifibatide (34%), DE (19%), and more polar metabolites (13%).

Polyclonal antisera made against synthetic peptides corresponding to expected tryptic fragments of gamma crystallin have been used to screen tryptic digests of total proteins from cataractous versus normal human lenses. One of these antisera recognizes two peptides that were found in greater amounts from digests of cataractous lenses. These two peptides shared a common sequence that contained an aspartate residue in place of an expected asparagine, suggesting that increased deamidation of this residue had occurred in the human cataractous lens in vivo.

1. Protease and deaminase activities and the metabolism of peptides were measured in rumen fluid from ciliate-free sheep and from sheep with a limited population of small entodinia. The same measurements were repeated following inoculation of the latter group with a more typical mixed ciliate population. 2. Protease and dialanine uptake activities of mixed rumen micro-organisms were not significantly influenced by protozoa. Trialanine uptake, leucine aminopeptidase (EC 3.4.11.1), deaminase and trypsin-like protease activities were 70, 107, 73 and 91% higher with the limited population, and 72, 58, 64 and 55% higher when mixed protozoa were present, indicating a major role for the protozoa in these activities.

The radiolytic deamination and dephosphorylation of peptides and phosphate esters in oxygenated solutions are OH-induced and involve the formation and subsequent degradation of the peroxyl radicals RCONHC(O2)R2 and POC(O2)R2 respectively. Reaction analogues in the degradation chemistry of these peroxyl intermediates from various peptides and phosphate esters including protein and DNA are evaluated in detail. The evidence is that the reaction mechanisms recently proposed for removal of POC(O2)R2 radicals in the radiolytic scission of the DNA strand are identical in form with the mechanisms identified in earlier studies of RCONHC(O2)R2 radicals in the radiolytic cleavage of the peptide chain. The analogy includes the chemical forms of the final products and their specific yields.

The 220 MHz spectra of tocinamide and deaminotocinamide, the ring moieties of oxytocin and of deamino-oxytocin, respectively, were investigated in [U-(2)H]dimethylsulfoxide solution. Extensive decoupling and exchange experiments allow complete spectral assignment and determination of many coupling constants. Circular dichroism spectra assigned a right-hand screw sense to the C-S-S-C group. The conformations of tocinamide and deaminotocinamide are different from each other and from those suggested for the ring portions of oxytocin and deamino-oxytocin. The relationship of this difference to biological activity is commented upon. The importance of the interaction of the side chain with the ring in oxytocin and deamino-oxytocin is emphasized.

Charge variants, as an important quality attribute of mAbs, must be comprehensively characterized and monitored during development. However, due to their complex structure, the characterization of charge variants is challenging, labor-intensive, and time-consuming when using traditional approaches. This work combines on-line and off-line 2D-LC-MS to comprehensively characterize mAb charge variants and quickly offer precise instructions for process development. Six charge variant peaks of mAb 1 were identified using the developed platform. Off-line 2D-LC-MS analysis at the peptide level showed that the acidic peak P1 and the basic peaks P4 and P5 were caused by the deamidation of asparagine, the oxidation of methionine, and incomplete C-terminal K loss, respectively. On-line 2D-LC-MS at the intact protein level was used to identify the root causes, and it was found that the acidic peak P2 and the basic peak P6 were due to the glutathionylation of cysteine and succinimidation of aspartic acid, respectively, which were not found in off-line 2D-LC-MS because of the loss occurring during pre-treatment. These results suggest that process development could focus on cell culture for adjustment of glutathionylation. In this paper, we propose the concept of precision process development based on on-line 2D-LC-MS, which could quickly offer useful data with only 0.6 mg mAb within 6 h for precise instructions for process development. Overall, the combination of on-line and off-line 2D-LC-MS can characterize mAb charge variants more comprehensively, precisely, and quickly than other approaches. This is a very effective platform with routine operations that provides precise instructions for process development within hours, and will help to accelerate the development of innovative therapeutics. [ABSTRACT FROM AUTHOR]

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity. [ABSTRACT FROM AUTHOR]

The structure and function of human leucocyte antigen (HLA-A) is well known and is an extremely variable protein. From the public HLA-A database, we chose 26 high frequency HLA-A alleles (45% of sequenced alleles). Using five arbitrary references from these alleles, we analyzed synonymous mutations at the third codon position (sSNP3) and non-synonymous mutations (NSM). Both mutation types showed non-random locations of 29 sSNP3 codons and 71 NSM codons in the five reference lists. Most sSNP3 codons show identical mutation types with many mutations resulting from cytosine deamination. We proposed 23 ancestral parents of sSNP3 in five reference sequences using conserved parents in five unidirectional codons and 18 majority parents in reciprocal codons. These 23 proposed ancestral parents show exclusive codon usage of G3 or C3 parents located on both DNA strands that mutate to A3 or T3 variants mostly (76%) by cytosine deamination The sSNP3 and NSM show clear separation of the two variant types with most sSNP3 located in conserved areas in exons 2, 3 and 4, compared to most NSM appearing in two Variable Areas with no sSNP3 in the latter parts of exons 2 (α1) and 3 (α2). The Variable Areas contain NSM (polymorphic) residues at the center of the groove that bind the foreign peptide. We find distinctly different mutation patterns in NSM codons from those of sSNP3. Namely, G-C to A-T mutation frequency was much smaller, suggesting that evolutional pressures of deamination and other mechanisms applied to the two areas are significantly different. [ABSTRACT FROM AUTHOR]

Antigens incorporated in subunit vaccines are typically poorly immunogenic, so a strong immunostimulant (adjuvant) and/or delivery system is required to boost immunogenicity. In this work, the various functional polymer nanostructures, that is, rods, worms, spheres, and tadpoles are used to develop potent peptide antigen delivery systems. The antigen PADRE-J8 (PJ8), derived from Group A Streptococcus (GAS) M-protein, is either physically mixed or chemically conjugated to polymeric nanoparticles of different shapes. The physical mixture of polymeric nanoparticles and antigen is more effective in inducing antibody production than their chemical conjugates. Moreover, rod-shaped polymeric nanoparticles in physical mixture with PJ8 elicited higher and more opsonic antibody titers than powerful complete Freund's adjuvant (CFA)-adjuvanted antigen. Herein, for the first time it is demonstrated that a) the block copolymer, in nanoparticle form, can act as an immune adjuvant, b) nanoparticle shape plays a crucial role in their immunogenicity, and c) antigen conjugation is not required, nor is antigen encapsulation or absorption. [ABSTRACT FROM AUTHOR]

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved 18O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the 18O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the 18O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations. [ABSTRACT FROM AUTHOR]

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Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu2+. The optimum PG substrate was Cbz-Gln-Gly, and the Km and Vmax values were 1.68 mM and 1.41 μM mg protein-1 min-1, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins. [ABSTRACT FROM AUTHOR]

Proteins are the main N compounds and are important sources of both C and N for plants and microbes. Triple-labeled dissolved organic N obtained from white clover shoots was separated into five different molecular weight (Mw) size fractions (> 1 kDa). In this study, we investigated the mineralization of and the presence of C and N remaining in soil solution over 14 days across different soil pH values. The evolved CO2 decreased by increasing Mw, suggesting an apparent bottleneck in the decomposition of organic N above Mw 30 kDa. The initial parallel loss of 13C and 15 N from soil was followed by an increase in dissolved 15 N describing a two-phase process of organic N turnover. The first-phase concerns the removal of organic N whereas the second-phase concerns the release of inorganic N. Microbial N and C use efficiency was increased with increasing Mw size suggesting the importance of organic N quality in predicting the impact on soil C and N cycling. [ABSTRACT FROM AUTHOR]

Celiac disease (CeD) is a T-cell-dependent enteropathy with autoimmune features where tissue transglutaminase (TG2)-mediated posttranslational modification of gliadin peptides has a decisive role in the pathomechanism. The humoral immune response is reported to target mainly TG2-deamidated γ-gliadin peptides. However, α-gliadin peptides, like p57-68, playing a crucial role in the T-cell response, and p31-43, a major trigger of innate responses, also contain B-cell gliadin epitopes and γ-gliadin like motifs. We aimed to identify if there are anti-gliadin-specific antibodies in CeD patients targeting the p31-43 and p57-68 peptides and to examine whether deamidation of these peptides could increase their antigenicity. We explored TG2-mediated deamidation of the p31-43 and p57-68 peptides, and investigated serum antibody reactivity toward the native and deamidated α and γ-gliadin peptides in children with confirmed CeD and in prospectively followed infants at increased risk for developing CeD. We affinity-purified antibody populations utilizing different single peptide gliadin antigens and tested their binding preferences for cross-reactivity in real-time interaction assays based on bio-layer interferometry. Our results demonstrate that there is serum reactivity toward p31-43 and p57-68 peptides, which is due to cross-reactive γ-gliadin specific antibodies. These γ-gliadin specific antibodies represent the first appearing antibody population in infancy and they dominate the serum reactivity of CeD patients even later on and without preference for deamidation. However, for the homologous epitope sequences in α-gliadins shorter than the core QPEQPFP heptapeptide, deamidation facilitates antibody recognition. These findings reveal the presence of cross-reactive antibodies in CeD patients recognizing the disease-relevant α-gliadins. [ABSTRACT FROM AUTHOR]

A combination of mass spectrometry (MS) and 18O‐water labeling has been applied to absolute quantitative proteomics. We introduce a new method called quantitative analysis using unlabeled standards and isotope‐labeled analytes (QAUSIA). An experimental procedure with multiple‐step 18O labeling is proposed to produce such 18O‐enriched peptides for investigation of labeling efficiency, response factor, and calibration curve. Furthermore, calibration curves employing unlabeled peptide as internal standard showed good linearity and similar slopes calculated from the mass spectra and the extracted ion chromatograms. Subsequently, the QAUSIA strategy was implemented on the targeted peptide of ovalbumin, and the calibration slope obtained from QAUSIA using the unlabeled synthetic peptide as a standard showed very similar results to the AQUA‐based approach. In addition, the concentration of the QAUSIA strategy using the targeted peptide of ovalbumin was 5.07 ± 0.14 nmol with respect to the theoretical expected value of 5.02 nmol, showing excellent accuracy. [ABSTRACT FROM AUTHOR]

Specific tumor uptake of peptide radiopharmaceuticals depends on tumor binding affinity and their radiochemical purity. Several important parameters that influence the 99mTc‐labeling and consequently the radiochemical purity of 6‐hydrazinonicotinamide (HYNIC)‐conjugated peptide are radionuclide activity, the amount of peptide, the amount of coligands, and the amount of reducing agents (stannous ion). In this review article, we have attempted studying these parameters in the HYNIC‐conjugated peptides (somatostatin, cholecystokinin/gastrin, bombesin, and RGD analogs) from a new perspective to obtain most used and optimized radio‐stoichiometric relationships. One of the most important results in this review is that for 99mTc‐labeling of HYNIC‐conjugated peptides, it is better to consider the most calculated mole ratio between technetium‐99m and the peptide (mole ratio of technetium‐99m to the peptide 1:200–400). The statistical results also show that among these 99mTc‐labeled peptides, the most used and favorable coligand is tricine/EDDA with two to one (2:1) mole ratio. These optimized radio‐stoichiometric relationships, favorable coligand mole ratio, and applicable radiolabeling points can greatly improve the labeling process of the HYNIC‐conjugated peptides, by reducing trial and error, increasing specific activity, and saving materials. [ABSTRACT FROM AUTHOR]

Abstract Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis due to the potential negative effect of these modifications on drug efficacy and stability. Succinimide has long been known to be an intermediate product of asparagine deamidation and aspartic acid isomerization, but despite the key role of succinimide in these reactions, its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site. These results are then used to establish a hydrophobic interaction chromatography (HIC)–based method that can be used for release and stability studies. In this method, unmodified protein, deamidated products, and succinimide are well separated and quantified. Good correlation was obtained between the data from low-pH protein digestion–based peptide mapping and the HIC-based method. Method qualification showed that the HIC-based method is robust, accurate, and precise and has excellent linearity. Highlights • Succinimide can be accumulated to significant level in therapeutic protein. • Succinimide is preserved in low-pH peptide map, which can be used for succinimide characterization. • Succinimide, deamidated, and unmodified protein can be well separated by hydrophobic interaction chromatography. [ABSTRACT FROM AUTHOR]

Role of water in chemical (in)stability is revisited, with focus on deamidation in freeze-dried amorphous proteins and peptides. Two distinct patterns for deamidation versus water have been reported, that is, a consistent increase in rate constant with water, and a “hockey stick”–type behavior. For the latter, deamidation is essentially independent of water at lower water contents and accelerates when water content increases above a threshold value. Two simple kinetic models are developed to analyze literature-reported relationships between water content and deamidation rate constants. One model is based on catalytic role of water clusters in enabling proton transfer, which is a critical reaction step. Water clusters are formed when water content increases above a threshold value, while unclustered (and less catalytically-active) water molecules are predominant at lower water levels. The second model considers the dual role of water, as both a destabilizer via catalysis and a stabilizer of protein native structure. Considering that both models emphasize the importance of local structure and that local structure is intrinsically related to fast (and non-cooperative) relaxation modes, it is plausible to expect correlations between local mobility, such as beta-relaxation, and amorphous chemical instability. [ABSTRACT FROM AUTHOR]

Peptide mass fingerprinting of bone collagen (ZooMS) has previously been proposed as a method to calculate the extent of the non-enzymatic degradation of glutamine into glutamic acid (deamidation). Temporal and spatial variation of glutamine deamidation at a single site, however, has not been investigated. Here we apply ZooMS screening of Châtelperronian and Early Holocene bone specimens from Quinçay, France, to explore temporal and spatial variation in glutamine deamidation. Our results indicate that chronological resolution is low, while spatial variation is high. Nevertheless, our analysis allows the identification of bone specimens that have undergone diagenetic histories remarkably different (either in length or in type) from spatially related bone specimens. Therefore, ZooMS ammonium-bicarbonate screening is capable of testing bone assemblage homogeneity, which could guide subsequent analysis and interpretation. [ABSTRACT FROM AUTHOR]

Hypothesis The incorporation of a succinic acid-derived moiety in amino acid derivatives would favor an intramolecular catalysis of a deamidation reaction. Such reaction would permit controlled disassembly of molecular hydrogelators and the use of the hydrogels for controlled release of actives. Experimental Low molecular weight hydrogelators containing a succinic acid-derived moiety were prepared by conventional organic synthesis procedures. Hydrogels were examined by electron microscopy and 1 H NMR studies were carried out to evaluate the solubility in water of the hydrogelators and the deamidation reaction. Liberation of Rose Bengal entrapped in the hydrogels was monitored by UV–Vis spectroscopy. Findings Molecular hydrogels formed by pseudopeptidic derivatives of l -valine suffer a thermal deamidation reaction, leading to partial disassembly. The succinic acid-derived moiety present in the gelators is responsible of intramolecular catalysis of a deamidation reaction. Such neighboring group effect is reminiscent of biochemical processes such as protein deamidation and self-excision of inteins. It has been found that the thermodynamic equilibrium of the deamidation reaction is regulated by the efficiency of hydrogelation. As a proof of concept, the thermally promoted deamidation is applied to controlled release of Rose Bengal. [ABSTRACT FROM AUTHOR]

Site-specific mapping of multiple deamidations in peptides is a challenging analytical task. In this work, capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) is presented as a high-resolution tool for the detailed characterization of these subtle modifications in peptides. The 4.5-kDa peptide drug TRI-1144, which contains five closely-positioned potential deamidation sites, was selected as model compound. TRI-1144 was exposed to acidic conditions and/or elevated temperatures for 1–14 h. Stressed samples were analyzed using a background electrolyte (BGE) of 150 mM ammonium formate (pH 6.0) in combination with a capillary coated with a bilayer of Polybrene-dextran sulfate. Separation of deamidated and deacetylated TRI-1144 species, including several positional isomers, was greatly enhanced by adding up to 40 vol% of acetonitrile-isopropanol (87.5:12.5, v/v) to the BGE, allowing reliable determination of the number of deamidations/deacetylations per degradation product. Collision-induced dissociation MS/MS was conducted on the separated peptide components in order to reveal the exact position of deamidation on the peptide chain. Obtained fragment ions showed overlapping isotopic distributions in their MS/MS spectra resulting from the comigration of different isomeric deamidated species. Comparison of theoretical and measured isotope distributions for specific y ions of peptide fragments yielded the identity and relative abundance of isomeric deamidated products. The developed CE-MS/MS methodology was used for the highly selective evaluation of TRI-1144 stability under different stress conditions, providing detailed qualitative and semi-quantitative degradation maps of the peptide drug. [ABSTRACT FROM AUTHOR]

A large number of peptides are generated during the processing of dry-cured ham that can be affected by post-translational modifications (PTM). One of the most studied PTMs is the oxidation of susceptible residues but other modifications such as deamidation have been scarcely reported in the literature. This work has been focused on the impact of deamidation in those peptides generated in 12-months dry-cured hams. The results obtained in Biceps femoris and Semimembranosus muscles showed that 52% and 48% of the identified peptides, respectively, were affected by PTMs, with a total of 277 and 282 sequences showing deamidation in both muscles, respectively. It appears that deamidation is not influenced by the type of muscle but is probably favored by characteristic processing conditions like temperature, pH, and salting of hams. The knowledge of the affected sequences provides novel information for a better characterisation of proteolysis phenomena during the processing of dry-cured ham. [ABSTRACT FROM AUTHOR]

BACKGROUND Bitter taste is the main limiting factor for various applications of protein hydrolysates. Frequently used physicochemical methods for debittering protein hydrolysates come with some undesired side effects. Deamidation-induced modification would be a very promising technique to improve the flavor of wheat gluten hydrolysates ( WGHs). This study was designed to determine the effect of deamidation with certain enzymes or acid treatment on the chemical composition, bitterness and umami properties of WGHs. The difference between umami peptide and free glutamic acid on the suppression of bitterness is emphatically discussed. The optimal scheme is proposed based on the flavor of WGHs and the yield of peptides. RESULTS The generation of umami substances suppressed bitter signal transduction. When the content of umami substances was relatively high, the umami-enhancing properties of umami peptides were obviously effective. The intensity of umami taste was high enough to further suppress bitter taste in the course of neurocognitive functioning. CONCLUSION When WGHs were treated with Glutaminase for 180 min, the umami taste score increased from 1.62 to 4.27 and the bitter taste score decreased from 1.33 to 0.65. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications. [ABSTRACT FROM AUTHOR]

The utility of Meldrum's activated furan (MAF) for the colorimetric detection of sub ppm levels of amines in solution, on solid supports, and as vapors is reported. MAF is synthesized in one step from inexpensive and commercially available starting materials and exhibits high selectivity for primary and secondary amines in the presence of competing nucleophiles. The reaction between activated furans and amines results in a distinct color change, discernable by the naked eye. UV/Vis absorption spectroscopy was utilized to monitor reactions in solution and determine detection limits. Additionally, solutions of MAF were useful as stains for thin layer chromatography and for monitoring solid-phase synthesis of peptides and peptidomimetics. Finally, MAF was used to detect volatile amines released from fish samples, demonstrating potential for food spoilage applications. [ABSTRACT FROM AUTHOR]

Prostate specific membrane antigen (PSMA)-positive exosomes have the potential to serve as highly sensitive biomarkers for prostate cancer detection. Herein, a sensitive electrochemical biosensor for the ultrasensitive detection of PSMA-positive exosomes has been constructed based on a peptide-templated AgNPs nanoprobe. In this work, PSMA-specific binding peptides immobilized on a gold electrode were responsible for prostate cancer-derived exosomes capturing. Well-designed peptide (CCY- LWYIKC) serves a dual role: as a signal probe and as a recognizer in the exosomes-identification process. Specifically, LWYIKC bind to cholesterol at the exosome membranes, and CCY function as peptide templates to host a large number of silver nanoparticles, leading to a strong electrochemical signal. Thus, the concentration of exosomes can be quantified via electrochemical signal. This innovative method displayed a wide detection range of 102 to 108 particles/μL and a detection limit as low as 37 particles/μL. Notably, the method has shown outstanding performance when validated using clinical samples, suggesting its potential for clinical applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • A Random Forest model evaluated via a fivefold cross-validation was put forward. • The model was used for estimating the air–water interfacial tension of various surface-active species. • A database with the air–water interfacial tension of 691 surfactants was used for training the model. • Structure-performance relationships were obtained for surface active molecules. • Sequences of amino acids are proposed for screening peptides with high interfacial activity. The surface tension at the critical micelle concentration (ST CMC) is an important descriptor for surfactants in applications including cosmetics, pharmaceuticals and food. A predictive ST CMC Random Forest model was trained with 691 conventional surfactants and 9 amino acids. The model was evaluated by fivefold cross-validation, and the prediction power was tested for peptides by direct comparison with the experimental ST CMC values found in the literature or measured in this work. Predictions were also conducted for short peptide permutations. The estimated ST CMC approached the experimental values as the peptide length decreased, suggesting a strong influence of secondary structures for longer sequences where the developed algorithm fails to make robust predictions. Concerning the short peptide permutations, the model estimated lower ST CMC values as the carbon number increased within the hydrophobic portions. This highlights the importance of the hydrophobic amino acids leucine, isoleucine, and phenylalanine in peptides with attractive surfactant properties. [ABSTRACT FROM AUTHOR]

Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the resulting product ion structures using infrared ion spectroscopy with the free electron laser FELIX. The influence of the side chain length (Asn versus Gln) and of the amino acid sequence on the deamidation reaction has been examined. Molecular structures for the product ions are determined by comparison of experimental IR spectra with spectra predicted by density functional theory (DFT). The reaction mechanisms identified for the four dipeptides AlaAsn, AsnAla, AlaGln, and GlnAla are not the same. For all four dipeptides, primary deamidation takes place from the amide side chain (and not from the N-terminus) and, in most cases, resembles the mechanisms previously identified for the protonated amino acids asparagine and glutamine. Secondary fragmentation reactions of the deamidation products have also been characterized and provide further insight in - and confirmation of - the identified mechanisms. Overall, this study provides a comprehensive molecular structure map of the deamidation chemistry of this series of dipeptides. [ABSTRACT FROM AUTHOR]