Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding,Stability, and Structure of the ShK Toxin

ShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.     

Molecular mechanism of the sea anemone toxin ShK recognizing the Kv1.3 channel explored by docking and molecular dynamic simulations

Computational methods are employed to simulate the interaction of the sea anemone toxin ShK in complex with the voltage-gated potassium channel Kv1.3 from mice. All of the available 20 structures of ShK in the Protein Data Bank were considered for improving the performance of the rigid protein docking of ZDOCK. The traditional and novel binding modes were obtained among a large number of predicted complexes by using clustering analysis, screening with expert knowledge, energy minimization, and molecular dynamic simulations. The quality and validity of the resulting complexes were further evaluated to identify a favorable complex structure by 500 ps molecular dynamic simulations and the change of binding free energies with a computational alanine scanning technique. The novel and reasonable ShK-Kv1.3 complex structure was found to be different from the traditional model by using the Lys22 residue to block the channel pore. From the resulting structure of the ShK-Kv1.3 complex, ShK mainly associates the channel outer vestibule with its second helical segment. Structural analysis first revealed that the Lys22 residue side chain of the ShK peptide just hangs between C and D chains of the Kv1.3 channel instead of physically blocking the channel pore. The obvious loss of the ShK Ser20Ala and Tyr23Ala mutant binding ability to the Kv1.3 channel is caused by the conformational change. The five hydrogen bonds between Arg24 in ShK and H404(A) and D402(D) in Kv1.3 make Arg24 the most crucial for its binding to the Kv1.3 channel. Besides the detailed interaction between ShK and Kv1.3 at the atom level, the significant conformational change induced by the interaction between the ShK peptide and the Kv1.3 channel, accompanied by the gradual decrease of binding free energies, strongly implies that the binding of the ShK peptide toward the Kv1.3 channel is a dynamic process of conformational rearrangement and energy stabilization. All of these can accelerate the development of ShK structure-based immunosuppressants.     

Solid‐Phase Combinatorial Synthesis and Biological Evaluation of Destruxin E Analogues

The solid‐phase combinatorial synthesis of cyclodepsipeptide destruxin E has been demonstrated. The combinatorial synthesis of cyclization precursors 8 was achieved by using a split and pool method on SynPhase Lanterns. The products were successfully macrolactonized in parallel in the solution phase by using 2‐methyl‐6‐nitrobenzoic anhydride and 4‐(dimethylamino)pyridine N‐oxide to afford macrolactones 9 , and the subsequent formation of an epoxide in the side chain gave 18 member destruxin E analogues 6 . Biological evaluation of analogues 6 indicated that the N‐MeAla residue was crucial to the induction of morphological changes in osteoclast‐like multinuclear cells (OCLs). Based on structure–activity relationships, azido‐containing analogues 15 were then designed for use as a molecular probe. The synthesis and biological evaluation of analogues 15 revealed that 15 b , in which the Ile residue was replaced with a Lys(N₃) residue, induced morphological changes in OCLs at a sufficient concentration, and modification around the Ile residue would be tolerated for attachment of a chemical tag toward the target identification of destruxin E ( 1 ).     

A stereochemical test of a proposed structural feature of the nicotinic acetylcholine receptor

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the alphaVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the alphaVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism.     

Tuning the Binding Affinity and Selectivity of Perfluoroaryl-Stapled Peptides by Cysteine-Editing

A growing number of approaches to “staple” α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here, the replacement of l -cysteine with “cysteine analogues” in combinations of different stereochemistry, side chain length and beta-carbon substitution, is explored to examine the influence that the thiol-containing residue(s) has on target protein binding affinity in a well-explored model system, p53–MDM2/MDMX, which is constituted by the interaction of the tumour suppressor protein p53 and proteins MDM2 and MDMX, which regulate p53 activity. In some cases, replacement of one or more l -cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two d -cysteine residues, favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured surface plasmon resonance data.     

Total Synthesis and Functional Evaluation of Fourteen Derivatives of Lysocin E: Importance of Cationic,Hydrophobic, and Aromatic Moieties for Antibacterial Activity

Lysocin E ( 1 ) is a structurally complex 37‐membered depsipeptide comprising 12 amino‐acid residues with an N‐methylated amide and an ester linkage. Compound 1 binds to menaquinone (MK) in the bacterial membrane to exert its potent bactericidal activity. To decipher the biologically important functionalities within this unique antibiotic, we performed a comprehensive structure‐activity relationship (SAR) study by systematically changing the side‐chain structures of l ‐Thr‐1, d ‐Arg‐2, N‐Me‐d ‐Phe‐5, d ‐Arg‐7, l ‐Glu‐8, and d ‐Trp‐10. First, we achieved total synthesis of the 14 new side‐chain analogues of 1 by employing a solid‐phase strategy. We then evaluated the MK‐dependent liposomal disruption and antimicrobial activity against Staphylococcus aureus by 1 and its analogues. Correlating data between the liposome and bacteria experiments revealed that membrane lysis was mainly responsible for the antibacterial functions. Altering the cationic guanidine moiety of d ‐Arg‐2/7 to a neutral amide, and the C7‐acyl group of l ‐Thr‐1 to the C2 or C11 counterpart decreased the antimicrobial activities four‐ or eight‐fold. More drastically, chemical mutation of d ‐Trp‐10 to d ‐Ala‐10 totally abolished the bioactivities. These important findings led us to propose the biological roles of the side‐chain functionalities.     

The dehydroalanine effect in the fragmentation of ions derived from polypeptides

The fragmentation of peptides and proteins upon collision‐induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non‐proteinogenic amino acid with an unsaturated side‐chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c‐ and z‐ions. Because these fragment ion types are not commonly observed upon activation of positively charged even‐electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S‐alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas‐phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S‐alkyl cysteine residues and conversion of multiply‐protonated peptides to radical cations. In the latter case, loss of radical side‐chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue‐specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.     

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.     

Mutation of isoleucine 705 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae affects lanosterol's C/D-ring cyclization and 17α/β-exocyclic side chain stereochemistry

Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/β-exocyclic side chain stereochemistry.     

Synthesis and conformational analysis of 17alpha,21-cyclo-22-unsaturated analogues of calcitriol

Six new calcitriol analogues, conformationally restricted at their side chain by the introduction of both a cyclopropane ring at C17-C20 and a double or triple bond at C22, were synthesized using the Wittig-Horner approach to construct the triene system. The six CD-ring and side-chain bearing fragments were prepared from ketone 14 by a divergent route to generate both series of epimers at C20, followed by stereoselective cyclopropanation. The (E)-alkenyl side chain was synthesized by means of a Wittig reaction. The alkynyl side chain was prepared by Corey-Fuchs homologation, followed by alkylation. The (Z)-alkenyl side chain was prepared from the previous alkyne by partial hydrogenation. The 20-epi analogues bind more strongly to VDR than the corresponding analogues with the C20 natural stereochemistry. These results can be reasoned by conformational analysis and hydrophobic interactions with the VDR ligand-binding domain.     

Influence of variation of a side chain on the folding equilibrium of a β‐peptide: Limitations of one‐step perturbation

In a recent study (Lin et al., Helv. Chim. Acta 2011, 94 , 597), the one‐step perturbation method was applied to tackle a challenging computational problem, that is, the calculation of the folding free enthalpies ΔG_F,U of six hepta‐β‐peptides with different, Ala, Val, Leu, Ile, Ser, or Thr, side chains in the fifth residue. The ΔG_F,U values obtained using one‐step perturbation based on a single molecular dynamics simulation of a judiciously chosen reference state with soft‐core atoms in the side chain of the fifth residue showed an overall accuracy of about k_BT for the four peptides with nonpolar side chains, but twice as large deviations were observed for the peptides with polar side chains. Here, alternative reference‐state Hamiltonians that better cover the conformational space relevant to these peptides are investigated, and post simulation rotational sampling of the χ₁ and χ₂ torsional angles of the fifth residue is carried out to sample different orientations of the side chain. A reference state with rather soft atoms yields accurate ΔG_F,U values for the peptides with the Ser and Thr side chains, but it failed to correctly predict the folding free enthalpy for one peptide with a nonpolar side chain, that is, Leu. Based on the results and those of earlier studies, possible ways to improve the accuracy of the efficient one‐step perturbation technique to compute free enthalpies of folding are discussed. © 2013 Wiley Periodicals, Inc.     

Synthesis and Analysis of the Conformational Preferences of 5‐Aminomethyloxazolidine‐2,4‐dione Scaffolds: First Examples of β2‐ and β2, 2‐Homo‐Freidinger Lactam Analogues

Constrained peptidomimetic scaffolds are of considerable interest for the design of therapeutically useful analogues of bioactive peptides. We present the single‐step cyclization of (S)‐ or (R)‐α‐hydroxy‐β²‐ or α‐substituted‐α‐hydroxy‐β^{2, 2}‐amino acids already incorporated within oligopeptides to 5‐aminomethyl‐oxazolidine‐2,4‐dione (Amo) rings. These scaffolds can be regarded as unprecedented β²‐ or β^{2, 2}‐homo‐Freidinger lactam analogues, and can be equipped with a proteinogenic side chain at each residue. In a biomimetic environment, Amo rings act as inducers of extended, semi‐bent or folded geometries, depending on the relative stereochemistry and the presence of α‐substituents.     

Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high‐resolution multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

Glycopeptidolipids (GPLs) are abundant in the cell walls of different species of mycobacteria and consist of tripeptide‐amino‐alcohol core of D‐Phe‐D‐allo‐Thr‐D‐Ala‐L‐alaninol linked to 3‐hydroxy or 3‐methoxy C_26–34 fatty acyl chain at the N‐terminal of D‐Phe via amide linkage, and a 6‐deoxytalose (6‐dTal) and an O‐methyl rhamnose residues, respectively, attach to D‐allo‐Thr and the terminal L‐alaninol. They are important cell‐surface antigens that are implicated in the pathogenesis of opportunistic mycobacteria belonging to the Mycobacterium avium complex. In this contribution, we described multiple‐stage linear ion trap in conjunction with high‐resolution mass spectrometry towards structural characterization of complex GPLs as [M + Na]⁺ ions isolated from Mycobacterium smegmatis, a fast‐growing and non‐pathogenic mycobacterial species. Following resonance excitation in an ion trap, MSⁿ spectra of the [M + Na]⁺ ions of GPLs contained mainly b and y series ions that readily determine the peptide sequence. Fragment ions from MSⁿ also afford locating the 6‐dTal and O‐methyl rhamnose residues linked to the D‐allo‐Thr and terminal L‐alaninol of the peptide core, respectively, as well as recognizing the modifications of the glycosides, including their acetylation and methylation states and the presence of succinyl group. The GPL families consisting of 3‐hydroxy fatty acyl and of 3‐methoxy fatty acyl substituents are readily distinguishable. The MS profiles of the GPLs from cells are dependant on the conditions they were grown, and several isobaric isomers were identified for many of the molecular species. These multiple‐stage mass spectrometric approaches give detailed structures of GPL in complex mixtures of which the isomeric structures are difficult to define using other analytical methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Gramicidin S Derivatives Containing cis‐ and trans‐Morpholine Amino Acids (MAAs) as Turn Mimetics

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D ‐Phe‐Pro two‐residue turn motifs in the rigid cyclic β‐hairpin structure of GS was replaced with morpholine amino acids (MAA 2 – 5 ), differing in stereochemistry and length of the side‐chain. The conformational properties of the thus obtained GS analogues ( 6 – 9 ) was assessed by using NMR spectroscopy and X‐ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8 , containing the dipeptide isostere trans‐MAA 4 , has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and ‐negative bacterial strains is better than the derivatives 6 , 7 and 9 .     

Characteristic neutral loss of CH3CHO from Thr‐containing sodium‐associated peptides

A characteristic neutral loss of 44 Da is observed in the MS/MS spectra of Thr‐containing sodiated peptides. A combination of tandem mass spectrometry and quantum chemical calculations calculated at the B3LYP/6‐311G (d, p) level of ab initio theory is used to elucidate this fragmentation pathway. The high resolution mass spectrometry data indicate this neutral loss is acetaldehyde lost from the side chain of Thr rather than CO₂. The intensity of this neutral loss can be enhanced when Thr residue is far from the C‐terminus and when the C‐terminus is esterified as well. The mechanism of the acetaldehyde loss is proposed to adopt a McLafferty‐type rearrangement reaction, which involves a proton transfer from the hydroxyl of Thr side chain to its C‐terminal neighboring carbonyl oxygen inducing the cleavage of the C_a–C_β bond. This mechanism is further supported by examining the fragmentation of a [GT(tBu)G + Na]⁺ peptide derivative and by comparing the product ion spectra of [M + Na‐44]⁺ of [GTGA + Na]⁺ with [M + Na]⁺ of [GGGA + Na]⁺. A similar neutral loss of HCHO can also be detected in Ser‐containing peptides. Our computational results reveal that the most stable [GTG + Na]⁺ ion is present as a tridentate charge‐solvated structure and the dissociation leading to the 44 loss is dynamically and energetically favorable. Copyright © 2015 John Wiley & Sons, Ltd.     

A molecular model of a point mutation (Val297Met) in the serine protease domain of protein C

A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.     

Protein side chain modeling with orientation-dependent atomic force fields derived by series expansions

We describe the development of new force fields for protein side chain modeling called optimized side chain atomic energy (OSCAR). The distance‐dependent energy functions (OSCAR‐d) and side‐chain dihedral angle potential energy functions were represented as power and Fourier series, respectively. The resulting 802 adjustable parameters were optimized by discriminating the native side chain conformations from non‐native conformations, using a training set of 12,000 side chains for each residue type. In the course of optimization, for every residue, its side chain was replaced by varying rotamers, whereas conformations for all other residues were kept as they appeared in the crystal structure. Then, the OSCAR‐d were multiplied by an orientation‐dependent function to yield OSCAR‐o. A total of 1087 parameters of the orientation‐dependent energy functions (OSCAR‐o) were optimized by maximizing the energy gap between the native conformation and subrotamers calculated as low energy by OSCAR‐d. When OSCAR‐o with optimized parameters were used to model side chain conformations simultaneously for 218 recently released protein structures, the prediction accuracies were 88.8% for χ₁, 79.7% for χ_{1 + 2}, 1.24 Å overall root mean square deviation (RMSD), and 0.62 Å RMSD for core residues, respectively, compared with the next‐best performing side‐chain modeling program which achieved 86.6% for χ₁, 75.7% for χ_{1 + 2}, 1.40 Å overall RMSD, and 0.86 Å RMSD for core residues, respectively. The continuous energy functions obtained in this study are suitable for gradient‐based optimization techniques for protein structure refinement. A program with built‐in OSCAR for protein side chain prediction is available for download at http://sysimm.ifrec.osaka‐u.ac.jp/OSCAR/ . © 2011 Wiley Periodicals, Inc. J Comput Chem 2011     

Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of delta131delta

NMR relaxation data on disordered proteins can provide insight into both structural and dynamic properties of these molecules. Because of chemical shift degeneracy in correlation spectra, detailed site-specific analyses of side chain dynamics have not been possible. Here, we present new experiments for the measurement of side chain dynamics in methyl-containing residues in unfolded protein states. The pulse schemes are similar to recently proposed methods for measuring deuterium spin relaxation rates in (13)CH(2)D methyl groups in folded proteins.(1) However, because resolution in (1)H-(13)C correlation maps of unfolded proteins is limiting, relaxation data are recorded as a series of (1)H-(15)N spectra. The methodology is illustrated with an application to the study of side chain dynamics in delta131delta, a large disordered fragment of staphylococcal nuclease containing residues 1-3 and 13-140 of the wide-type protein. A good correlation between the order parameters of the symmetry axes of the methyl groups and the backbone (1)H-(15)N bond vectors of the same residue is observed. Simulations establish that such a correlation is only possible if the unfolded state is comprised of an ensemble of structures which are not equiprobable. A motional model, which combines wobbling-in-a-cone and Gaussian axial fluctuations, is proposed to estimate chi(1) torsion angle fluctuations, sigma(chi)()1, of Val and Thr residues on the basis of the backbone and side chain order parameters. Values of sigma(chi)()1 are approximately 10 degrees larger than what has previously been observed in folded proteins. Of interest, the value of sigma(chi)()1 for Val 104 is considerably smaller than for other Val or Thr residues, suggesting that it may be part of a hydrophobic cluster. Notably large (15)N transverse relaxation rates are observed in this region. To our knowledge, this is the first time that side chain dynamics in an unfolded state have been studied in detail by NMR.     

Effects of acidic peptide size and sequence on trivalent praseodymium adduction and electron transfer dissociation mass spectrometry

Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr ‐ H]²⁺; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues generate [M + Pr]³⁺, which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H]⁴⁺; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non‐metallated c‐ and z‐ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N‐terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal‐peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III). Copyright © 2017 John Wiley & Sons, Ltd.