Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

A General Approach Towards Triazole‐Linked Adenosine Diphosphate Ribosylated Peptides and Proteins

Current methods to prepare adenosine diphosphate ribosylated (ADPr) peptides are not generally applicable due to the labile nature of this post‐translational modification and its incompatibility with strong acidic conditions used in standard solid‐phase peptide synthesis. A general strategy is presented to prepare ADPr peptide analogues based on a copper‐catalyzed click reaction between an azide‐modified peptide and an alkyne‐modified ADPr counterpart. The scope of this approach was expanded to proteins by preparing two ubiquitin ADPr analogues carrying the biological relevant α‐glycosidic linkage. Biochemical validation using Legionella effector enzyme SdeA shows that clicked ubiquitin ADPr is well‐tolerated and highlights the potential of this strategy to prepare ADPr proteins.     

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBM^GABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBM^GABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBM^GABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.     

Photosynthetic Antenna‐Reaction Center Mimicry with a Covalently Linked Monostyryl Boron‐Dipyrromethene–Aza‐Boron‐Dipyrromethene–C60 Triad

An efficient functional mimic of the photosynthetic antenna‐reaction center has been designed and synthesized. The model contains a near‐infrared‐absorbing aza‐boron‐dipyrromethene (ADP) that is connected to a monostyryl boron‐dipyrromethene (BDP) by a click reaction and to a fullerene (C₆₀) using the Prato reaction. The intramolecular photoinduced energy and electron‐transfer processes of this triad as well as the corresponding dyads BDP‐ADP and ADP‐C₆₀ have been studied with steady‐state and time‐resolved absorption and fluorescence spectroscopic methods in benzonitrile. Upon excitation, the BDP moiety of the triad is significantly quenched due to energy transfer to the ADP core, which subsequently transfers an electron to the fullerene unit. Cyclic and differential pulse voltammetric studies have revealed the redox states of the components, which allow estimation of the energies of the charge‐separated states. Such calculations show that electron transfer from the singlet excited ADP (¹ADP*) to C₆₀ yielding ADP^.+‐C₆₀^.? is energetically favorable. By using femtosecond laser flash photolysis, concrete evidence has been obtained for the occurrence of energy transfer from ¹BDP* to ADP in the dyad BDP‐ADP and electron transfer from ¹ADP* to C₆₀ in the dyad ADP‐C₆₀. Sequential energy and electron transfer have also been clearly observed in the triad BDP‐ADP‐C₆₀. By monitoring the rise of ADP emission, it has been found that the rate of energy transfer is fast (≈10¹¹ s^?1). The dynamics of electron transfer through ¹ADP* has also been studied by monitoring the formation of C₆₀ radical anion at 1000 nm. A fast charge‐separation process from ¹ADP* to C₆₀ has been detected, which gives the relatively long‐lived BDP‐ADP^.+C₆₀^.? with a lifetime of 1.47 ns. As shown by nanosecond transient absorption measurements, the charge‐separated state decays slowly to populate mainly the triplet state of ADP before returning to the ground state. These findings show that the dyads BDP‐ADP and ADP‐C₆₀, and the triad BDP‐ADP‐C₆₀ are interesting artificial analogues that can mimic the antenna and reaction center of the natural photosynthetic systems.     

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine‐rich snakin‐1 and ‐2 antimicrobial peptides by using a combination of solid‐phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40–50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin‐2 compared to natural snakin‐2, we demonstrated that synthetic snakin‐2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin‐2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.     

Semi‐Rationally Designed Short Peptides Self‐Assemble and Bind Hemin to Promote Cyclopropanation

The self‐assembly of short peptides gives rise to versatile nanoassemblies capable of promoting efficient catalysis. We have semi‐rationally designed a series of seven‐residue peptides that form hemin‐binding catalytic amyloids to facilitate enantioselective cyclopropanation with efficiencies that rival those of engineered hemin proteins. These results demonstrate that: 1) Catalytic amyloids can bind complex metallocofactors to promote practically important multisubstrate transformations. 2) Even essentially flat surfaces of amyloid assemblies can impart a substantial degree of enantioselectivity without the need for extensive optimization. 3) The ease of peptide preparation allows for straightforward incorporation of unnatural amino acids and the preparation of peptides made from d ‐amino acids with complete reversal of enantioselectivity.     

High‐throughput method for on‐target performic acid oxidation of MALDI‐deposited samples

An information‐rich on‐target performic acid oxidation method, which is compatible with alkylation for differentiation of free cysteine versus disulfide‐containing peptides, is described. On‐target oxidation is achieved using performic acid vapor to oxidize disulfide‐containing peptides and/or small proteins on the matrix‐assisted laser desorption/ionization (MALDI) sample deposits. The on‐target oxidation method is preferred over solution‐phase oxidation methods because (1) less sample handing is required, (2) oxidation throughput is drastically increased and (3) ion suppression effects are reduced because performic acid is not added directly to the MALDI spot. The utility of this method is demonstrated by simultaneous oxidation of multiple MALDI sample deposits containing model disulfide‐linked peptides, intact bovine insulin and a bovine ribonuclease A proteolytic digest. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

Histidine‐Rich Oligopeptides To Lessen Copper‐Mediated Amyloid‐β Toxicity

Brain copper imbalance plays an important role in amyloid‐β aggregation, tau hyperphosphorylation, and neurotoxicity observed in Alzheimer's disease (AD). Therefore, the administration of biocompatible metal‐binding agents may offer a potential therapeutic solution to target mislocalized copper ions and restore metallostasis. Histidine‐containing peptides and proteins are excellent metal binders and are found in many natural systems. The design of short peptides showing optimal binding properties represents a promising approach to capture and redistribute mislocalized metal ions, mainly due to their biocompatibility, ease of synthesis, and the possibility of fine‐tuning their metal‐binding affinities in order to suppress unwanted competitive binding with copper‐containing proteins. In the present study, three peptides, namely HWH , HK^CH , and HAH , have been designed with the objective of reducing copper toxicity in AD. These tripeptides form highly stable albumin‐like complexes, showing higher affinity for Cu^II than that of Aβ(1‐40). Furthermore, HWH , HK^CH , and HAH act as very efficient inhibitors of copper‐mediated reactive oxygen species (ROS) generation and prevent the copper‐induced overproduction of toxic oligomers in the initial steps of amyloid aggregation in the presence of Cu^II ions. These tripeptides, and more generally small peptides including the sequence His‐Xaa‐His at the N‐terminus, may therefore be considered as promising motifs for the future development of new and efficient anti‐Alzheimer drugs.     

Synthesis of Well‐Defined Adenosine Diphosphate Ribose Oligomers

The post‐translational modification of proteins that is known as adenosine diphosphate ribosylation (ADPr) regulates a wide variety of important biological processes, such as DNA‐damage repair and cellular metabolism. This modification is also involved in carcinogenesis and the process of aging. Therefore, a better understanding of the function of ADP‐ribosylation is crucial for the development of novel therapeutics. To facilitate the elucidation of the biology of ADPr, the availability of well‐defined fragments of poly(ADP‐ribose) is essential. Herein we report a solid‐phase synthetic approach for the preparation of ADP‐ribose oligomers of exactly defined length. The methodology is exemplified by the first reported synthesis of an ADP‐ribose dimer and trimer.     

The Two‐Step Assemblies of Basic‐Amino‐Acid‐Rich Peptide with a Highly Charged Polyoxometalate

Two‐step assembly of a peptide from HPV16 L1 with a highly charged europium‐substituted polyoxometalate (POM) cluster, accompanying a great luminescence enhancement of the inorganic polyanions, is reported. The mechanism is discussed in detail by analyzing the thermodynamic parameters from isothermal titration calorimetry (ITC), time‐resolved fluorescent and NMR spectra. By comparing the actions of the peptide analogues, a binding process and model are proposed accordingly. The driving forces in each binding step are clarified, and the initial POM aggregation, basic‐sequence and hydrophobic C termini of peptide are revealed to contribute essentially to the two‐step assembly. The present study demonstrates both a meaningful preparation for bioinorganic materials and a strategy using POMs to modulate the assembly of peptides and even proteins, which could be extended to other proteins and/or viruses by using peptides and POMs with similar properties.     

Identification of isomeric 5‐hydroxytryptophan‐ and oxindolylalanine‐containing peptides by mass spectrometry

Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5‐hydroxytryptophan (5‐HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2‐nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5‐HTP residues. The mass shift of 152.98 m/z units allowed identifying 5‐HTP‐ besides Trp‐containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia‐ and 5‐HTP‐derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y₁ ions at m/z 175.12 derived from peptides with C‐terminal arginine residues. The pseudo MS³ spectra acquired on a quadrupole time‐of‐flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia‐containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5‐HTP‐cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification. Copyright © 2012 John Wiley & Sons, Ltd.     

Near‐IR Excitation Transfer and Electron Transfer in a BF2‐Chelated Dipyrromethane–Azadipyrromethane Dyad and Triad

A molecular dyad and triad, comprised of a known photosensitizer, BF₂‐chelated dipyrromethane (BDP), covalently linked to its structural analog and near‐IR emitting sensitizer, BF₂‐chelated tetraarylazadipyrromethane (ADP), have been newly synthesized and the photoinduced energy and electron transfer were examined by femtosecond and nanosecond laser flash photolysis. The structural integrity of the newly synthesized compounds has been established by spectroscopic, electrochemical, and computational methods. The DFT calculations revealed a molecular‐clip‐type structure for the triad, in which the BDP and ADP entities are separated by about 14 Å with a dihedral angle between the fluorophores of around 70°. Differential pulse voltammetry studies have revealed the redox states, allowing estimation of the energies of the charge‐separated states. Such calculations revealed a charge separation from the singlet excited BDP (¹BDP*) to ADP (BDP^.+‐ADP^.?) to be energetically favorable in nonpolar toluene and in polar benzonitrile. In addition, the excitation transfer from the singlet BDP to ADP is also envisioned due to good spectral overlap of the BDP emission and ADP absorption spectra. Femtosecond laser flash photolysis studies provided concrete evidence for the occurrence of energy transfer from ¹BDP* to ADP (in benzonitrile and toluene) and electron transfer from BDP to ¹ADP* (in benzonitrile, but not in toluene). The kinetic study of energy transfer was measured by monitoring the rise of the ADP emission and revealed fast energy transfer (ca. 10¹¹ s^?1) in these molecular systems. The kinetics of electron transfer via ¹ADP*, measured by monitoring the decay of the singlet ADP at λ=820 nm, revealed a relatively fast charge‐separation process from BDP to ¹ADP*. These findings suggest the potential of the examined ADP–BDP molecules to be efficient photosynthetic antenna and reaction center models.     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.     

Easy‐to‐Attach/Detach Solubilizing‐Tag‐Aided Chemical Synthesis of an Aggregative Capsid Protein

A solubilizing Trt‐K₁₀ tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt‐K₁₀ tag comprises a hydrophilic oligo‐Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol‐type introducing reagent Trt(OH)‐K₁₀ under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol‐additive‐free native chemical ligation can be modified directly in a one‐pot manner to facilitate the isolation of the product by high‐performance liquid chromatography. Finally, the Trt‐K₁₀ tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy‐to‐attach/detach tag‐aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.     

NMR‐Solution Structures in Methanol of an α‐Heptapeptide,of a β3/β2‐Nonapeptide,and of an all‐β3‐Icosapeptide Carrying the 20 Proteinogenic Side Chains

The NMR‐solution structure of an α‐heptapeptide with a central Aib residue was investigated in order to verify that, in contrast to β‐peptides, short α‐peptides do not form a helical structures in MeOH. Although the central Aib residue was found to induce a bend in the experimentally determined structure, no secondary structure typical for longer α‐peptides or proteins was found. A β²/β³‐nonapeptide with polar, positively charged side chains was subjected to NMR analysis in MeOH and H₂O. Whereas, in MeOH, it folds into a 10/12‐helix very similar to the structure determined for a corresponding β²/β³‐nonapeptide with only aliphatic side chains, no dominant conformation could be determined in H₂O. Finally, the NMR analysis of a β³‐icosapeptide containing the side chains of all 20 proteinogenic amino acids in MeOH is described. It revealed that this 20mer folds into a 3₁₄‐helix over its whole length forming six full turns, the longest 3₁₄‐helix found so far. Together, our findings confirm that, in contrast to α‐peptides, β‐peptides not only form helices with just six residues, but also form helices that are longer than helical sections usually observed in proteins or natural peptides. The higher helix‐forming propensity of long β‐peptides is attributed to the conformation‐stabilizing effect of the staggered ethane sections in β‐peptides which outweighs the detrimental effect of the increasing macrodipole.     

Chain‐Terminating and Clickable NAD+ Analogues for Labeling the Target Proteins of ADP‐Ribosyltransferases

ADP‐ribosyltransferases (ARTs) use NAD⁺ as a substrate and play important roles in numerous biological processes, such as the DNA damage response and cell cycle regulation, by transferring multiple ADP‐ribose units onto target proteins to form poly(ADP‐ribose) (PAR) chains of variable sizes. Efforts to identify direct targets of PARylation, as well as the specific ADP‐ribose acceptor sites, must all tackle the complexity of PAR. Herein, we report new NAD⁺ analogues that are efficiently processed by wild‐type ARTs and lead to chain termination owing to a lack of the required hydroxy group, thereby significantly reducing the complexity of the protein modification. Due to the presence of an alkyne group, these NAD⁺ analogues allow subsequent manipulations by click chemistry for labeling with dyes or affinity markers. This study provides insight into the substrate scope of ARTs and might pave the way for the further developments of chemical tools for investigating PAR metabolism.