Gas-phase anionic complexes of alkali metal ions and peptides: Structure and collision activated decompositions

Alkali metal ions and anionic peptides can be desorbed into the gas phase to give metal-bound peptides and bis(peptide) complexes bearing a ? 1 charge. Although amide nitrogens of peptide bonds are deprotonated in the gas phase by alkali metal ions, this reacion does not occur in solution. Metal-bound dipeptide anions exist as a single structure, whereas those of tripeptide complexes have three structures as revealed by tandem mass spectrometric studies. Ions of bis(peptide) complexes of alkali metals decompose upon collisional activation principally to form deprotonated peptides, in contrast to bis(peptide) complexes of alkaline earth metal ions, which undergo elimination of a neutral peptide.     

Novel method for [M + Cu]+ ion formation by matrix-assisted laser desorption ionization

Direct deposition of a MALDI sample onto a copper sample stage and irradiation with UV light (337 nm) produces copper adduct ions of both the matrix and analyte molecules. This technique for introducing Cu⁺ into the gas-phase avoids suppression of ion signal that accompanies addition of metal salts to the sample solution. We observe good correlation between the number of basic residues in peptides and the number of Cu⁺ ions that add to the peptide. For example, the peptide KRQHPG contains three basic residues and forms ions with up to three Cu⁺ adducts. Postsource decay experiments demonstrate that for arginine containing peptides, arginine anchors the Cu⁺ ion. That is, all metastable ions contain the arginine complexed to Cu⁺ and the only immonium ion observed is that of arginine–Cu⁺. In addition, preliminary calculations indicate that guanidine has the highest Cu⁺ ion affinity followed by histidine.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

A molecular tweezer for lysine and arginine

Lysine and arginine play a key role in numerous biological recognition processes controlling, inter alia, gene regulation, glycoprotein targeting and vesicle transport. They are also found in signaling peptide sequences responsible, e.g. for bacterial cell wall biosynthesis, Alzheimer peptide aggregation and skin regeneration. Almost none of all artificial receptor structures reported to date are selective and efficient for lysine residues in peptides or proteins. An artificial molecular tweezer is introduced which displays an exceptionally high affinity for lysine (K(a) approximately 5000 in neutral phosphate buffer). It features an electron-rich torus-shaped cavity adorned with two peripheral anionic phosphonate groups. Exquisite selectivity for arginine and lysine is achieved by threading the whole amino acid side chain through the cavity and subsequent locking by formation of a phosphonate-ammonium/guanidinium salt bridge. This pseudorotaxane-like geometry is also formed in small basic signaling peptides, which can be bound with unprecedented affinity in buffered aqueous solution. NMR titrations, NOESY and VT experiments as well as ITC measurements and Monte Carlo simulations unanimously point to an enthalpy-driven process utilizing a combination of van der Waals interactions and substantial electrostatic contributions for a conformational lock. Since DMSO and acetonitrile compete with the amino acid guest inside the cavity, a simple change in the cosolvent composition renders the whole complexation process reversible.     

Charge state effect on the zwitterion influence on stability of non-covalent interaction of single-stranded DNA with peptides

Negative ion ESI mass spectrometry was used to study the gas-phase stability and dissociation pathways of peptide-DNA complexes. We show that bradykinin and three modified peptides containing the basic residue arginine or lysine form stable interactions with single-stranded oligonucleotides. ESI-MS/MS of complexes of T(8) with PPGFSPFRR resulted in a major dissociation pathway through cleavage of the peptide covalent bond. The stability of the complex is due to electrostatic interaction between the negatively charged phosphate group and the basic side chain of the arginine and lysine residues as demonstrated by Vertes et al. and Woods et al. In fact, the present work establishes the role played by zwitterions on complex stabilisation. The presence of protons in nucleobase and/or amino acid contributes in reinforcing the strength of the salt bridge (SB) interaction. The zwitterionic form of the most basic of amino acid residues, arginine, is assumed to form a strong SB interaction to the negatively charged phosphate groups of DNA. This non-covalent complex is stable enough to withstand disruption of the non-covalent interaction and to first break the covalent bond. Moreover, the dependence of fragmentation patterns upon the complex charge state is explained by the fact that the net number of negative charges modulates the number of zwitterionic sites, which stabilise the complexes. Finally, the weak influence of the nucleobase is assumed by the existence of competition for proton addition between the nucleobase and the R/K side chain leading to a decrease in the stabilisation of the SB interaction.     

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.     

Formation of Anionic Peptide Radicals In Vacuo

In this paper, we demonstrate for the first time the formation of radical anionic peptides [M - 2H]*- through a one-electron transfer mechanism upon low-energy collision-induced dissociation (CID) of gas-phase singly charged [Mn(III)(salen)(M - 2H)]*- complex ions [where salen is N,N'-ethylenebis(salicylideneiminato) and M is an angiotensin III derivative]. The types of fragment ions formed from [M - 2H]*- share some similarities with those from the cationic radical peptides M*+ and [M + H]*2+, but differ significantly from those of the corresponding deprotonated peptides [M - H]-. Fragmentation of [M - 2H]*- radical anionic angiotensin III derivatives leads preferentially to product ions of side-chain cleavage of amino acid residues, z-type and minor x-type fragment ions, most of which are types rarely observed in low-energy CID spectra of deprotonated analogs. The degree of competitive dissociation of the complexes is highly dependent on the nature of the substituted salen derivatives. The yields of anionic peptide radicals were enhanced to the greatest extent when electron withdrawing groups were positioned at the 5 and 5' positions, but the effect was rather modest when such groups resided at the 3 and 3' positions. Substituting a cyclohexyl unit of a salen with phenyl or naphthyl moieties at the 8 and 8' positions also facilitated electron-transfer pathways.     

Structure, Stability, and Lability of Copper(II) Complexes with Triglycine

Equilibrium constants of complex formation, rate constants of chemical exchange reactions, and characteristics of electronic absorption spectra for species detected in aqueous solution of copper(II) with triglycine were determined, and conclusions on the structure of the complexes were made. A possibility of H-bond formation between the ammonium group of the zwitter-ionic form of the ligand and the second peptide oxygen in the anionic form of an adjacent ligand was shown. Kinetics and mechanisms of ligand and proton exchanges in solutions of copper(II) bistripeptide complexes with the ligand containing a deprotonated peptide nitrogen atom were studied. A new mechanism was proposed for hydroxide-catalyzed substitution reactions in copper(II) complexes with tripeptides.     

“Best Match” Model and Effect of Na+/H+ Exchange on Anion Attachment to Peptides and Stability of Formed Adducts in Negative Ion Electrospray Mass Spectrometry

The “Best Match” model has been extended to account for the role that Na⁺/H⁺ exchange plays on anion attachment in negative ion electrospray. Without any Na⁺/H⁺ exchange on (Glu) fibrinopeptide B, the higher basicity anions F^? and CH₃COO^? can hardly form observable adducts; however, after multiple Na⁺/H⁺ exchanges, adduct formation is enabled. Moreover, dissociation pathways of CF₃COO^? adducts with singly deprotonated peptides that have undergone 0 to 3 Na⁺/H⁺ exchanges exhibit a shift in CID product ions from losing predominately CF₃COOH (case of 0 Na⁺/H⁺ exchanges) to losing predominately CF₃COO^? (case of 3 Na⁺/H⁺ exchanges). These phenomena can be rationalized by considering that Na⁺ cations exchange at, and serve to “block”, the most acidic sites, thereby forcing implicated anions to attach to lower acidity protons. In addition to forming ion pairs with carboxylate groups, Na⁺ also participates in formation of tri-atomic ions of the form ANaA^? during adduct dissociation. The fact that low gas-phase basicity (GB) anions preferentially form ANaA^? species, even though high GB anions form more stable tri-atomic species, indicates that the monatomic ions were not in close contact in the initial adduct. The propensity for formation of stable anionic adducts is dependent on the degree of matching between anion GBs and GB_app of deprotonated sites on the peptide. The GB_app is raised dramatically as the charge state of the peptide increases via a through-space effect. The presence of Na⁺ on carboxylate sites substantially decreases the GB_app by neutralizing these sites, while slightly increasing the intrinsic GBs by an inductive effect.

Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785–2793). The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens (¹H/²H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution.     

Covalent and non‐covalent binding in the ion/ion charge inversion of peptide cations with benzene‐disulfonic acid anions

Protonated angiotensin II and protonated leucine enkephalin‐based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4‐formyl‐1,3‐benzenedisulfonic acid (FBDSA) and 1,3‐benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M + FBDSA‐H]^? via collisional‐induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e. no covalent bond formation). A series of studies with model systems confirmed that strong non‐covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically bound complexes. The results reported here shed new light on the nature of non‐covalent interactions in gas phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Electron capture dissociation of peptides metalated with alkaline-earth metal ions

The possible use of divalent alkaline-earth metal ions, including Mg2+, Ca2+, Sr2+, and Ba2+, as charge carrier for electron capture dissociation of peptides was investigated. Model peptides of RGGGVGGGR and NGGGWGGGN were used to simplify the interpretation of spectral information. It was demonstrated that useful electron capture dissociation (ECD) tandem mass spectra of these metalated peptides could be generated. Interestingly, peptides metalated with different alkaline-earth metal ions generated very similar ECD tandem mass spectra. Metalated c-ions and z-ions were the predominant fragment ions. Only Mg2+-metalated peptides gave somewhat different results. Some nonmetalated c-ions were observed from ECD of [RGGGVGGGR + Mg]2+ but not from [NGGGWGGGN + Mg]2+. Together with some ab initio calculations, it was established that the bound metal ions might activate the acidity of the amide hydrogen. With the presence of high proton affinity moiety, such as N-terminal amino group and/or side chain of the arginine residues, the metalated peptide ions could exist predominantly in their zwitterion forms, in which one or two backbone amide group(s) was deprotonated and the high proton affinity functional group(s) was protonated. It was believed that electron capture leads primarily to the reduction of the mobile proton rather than the metal ions. With this zwitterion model, the formation of nonmetalated c-fragments and the generation of similar ECD spectra for peptides metalated with various alkaline-earth metal ions could readily to be explained. Another interesting observation in the ECD mass spectra of metalated peptides is related to the enhanced formation of the minor ECD products, i.e., (c - 1)(+*) and (z + 1)+ ions. Together with ab initio calculations using a truncated peptide model, various possible reaction mechanisms for the formation of these minor ECD products were evaluated. It was concluded that hydrogen transfer between the initiated formed c and z(.) species plays an important role in the formation (c - 1)(+*) and (z + 1)+ ions. Although peptides metalated with these metal ions do not have better ECD efficiency compared to the multiply-protonated peptides, it provides practical accessibility of ECD methods to analyze small peptides with no basic amino acid residues.     

Gas-phase investigation of Pd(II)-alanine complexes with small native and derivatized peptides containing histidine

We report here the generation of gas-phase complexes containing Pd(II), a ligand (deprotonated alanine, A-), and/or N-terminus derivatized peptides containing histidine as one of the amino acids. The species were produced by electrospray ionization, and their gas-phase reactions were investigated using ion-trap tandem mass spectrometry. Pd(II) forms a stable diaqua complex in the gas phase of the formula, [Pd(A-) (H(2)O)(2)]+, (where A- = deprotonated alanine) along with ternary complexes containing A- and peptide. The collision-induced dissociation (CID) patterns of the binary and ternary complexes were investigated, and the dissociation patterns for the ternary complexes suggest that: (a) the imidazole ring of the histidine side group may be the intrinsic binding site of the metal ion, and (b) the peptides fragment primarily by cleavage of the amide bond to the C-terminal side of the histidine residues. These observations are in accord with previous solution-state studies in which Pd(II) was shown to cause hydrolysis of an amide bond of a peptide at the same position.     

Protein structure information from mass spectrometry? Selective titration of arginine residues by sulfonates

Noncovalently bound complexes between basic sites of peptides/proteins and sulfonates are studied using Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Reactive sulfonate dyes such as Cibacron Blue F3G-A are known to bind to protonated amino groups on the exterior of a protein. In this work, we examine a wide range of other sulfonates with distinctly simpler structure and more predictable reactivity. Naphthalene-sulfonic acid derivatives were found to bind to arginine only, as opposed to expected binding to all basic sites (Arg, Lys and His). Detailed control experiments were designed to unambigously confirm this selectivity and to rule out nonspecific adduct formation in the gas phase. The data show that the number of complex adducts found equals the number of accessible arginine sites on the surface of folded peptides and proteins, plus the N-terminus. Lys and His are not complexed nor are buried residues with hindered access. MALDI-MS can therefore provide fast information related to the exposed surface of these biomolecules. Additional titration experiments with 1-anilino-naphthalene-8-sulfonic acid (ANS) revealed that this fluorescent dye, which was often hypothesized to bind to so-called molten globule states of proteins, behaved exactly like all other naphthalene-sulfonic acids. ANS binding thus occurs largely through the sulfonate group.     

Amyloid-β peptide structure in aqueous solution varies with fragment size

Various fragment sizes of the amyloid-β (Aβ) peptide have been utilized to mimic the properties of the full-length Aβ peptide in solution. Among these smaller fragments, Aβ16 and Aβ28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the Aβ16, Aβ28, and Aβ42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the Aβ16, Aβ28, and Aβ42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of Aβ42 cannot be completely captured by the Aβ16 and Aβ28 fragments. For example, the β-sheet structures in the N-terminal region of Aβ16 and Aβ28 are either not present or the abundance is significantly decreased in Aβ42. The α-helix and β-sheet abundances in Aβ28 and Aβ42 show trends--to some extent--with the potential of mean forces but no such trend could be obtained for Aβ16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in Aβ28 but this trend is vice versa for Aβ42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length Aβ42 peptide cannot be completely mimicked by studying the Aβ16 and Aβ28 peptides.     

Effect of peptide length on the interaction between consensus peptides and DOPC/DOPA bilayers

The effect of peptide length and electrostatics on the interaction between Cardin motif peptides and lipid membranes was investigated for (AKKARA)(n) (n = 1-4) and (ARKAAKKA)(n) (n = 1-3) peptides (A, K, and R refer to alanine, lysine, and arginine, respectively) by fluorescence spectroscopy, circular dichroism, ellipsometry, z potential, and photon correlation spectroscopy measurements. The effect of the peptides regarding leakage induction of both zwitterionic and anionic liposomes increased with increasing peptide length, as did the peptide-induced killing of Enterococcus faecalis and Bacillus subtilis bacteria. The peptides, characterized by a random coil conformation both in buffer and when attached to the liposomes (helix content less than 20%), displayed an increased adsorption with increasing peptide length, and plateau adsorption for the longest peptides corresponded to 1 peptide per 65 and 17 lipid molecules for zwitterionic and anionic membranes, respectively. Control experiments with uncharged peptide analogues as well as experiments at high excess electrolyte concentration showed that peptide charges are important both for peptide adsorption and leakage induction. These observations, together with observations of the liposome z potential at different peptide additions as well as a comparison between the results for zwitterionic and anionic liposomes, suggest that electrostatically affected local packing effects are crucial for the action of these peptides, although pore formation such as that observed for many AMPs cannot be excluded at present.     

Influence of a ring substituent on the tendency to form H(2)O adducts to Ag(+) complexes with phenylalanine analogues in an ion trap mass spectrometer

In a previous report we showed that certain binary Ag(+)-amino acid complexes formed adduct ions by the attachment of a single water and methanol molecule when stored in an ion trap mass spectrometer: complexes with aliphatic amino acids and with 4-fluorophenylalanine formed the adduct ions whereas complexes with phenylalanine and tryptophan did not. In this study we compared the tendency of the Ag(+) complexes derived from phenylalanine, 4-fluorophenylalanine, 4-hydroxyphenylalanine (tyrosine), 4-bromophenylalanine, 4-nitrophenylalanine and aminocyclohexanepropionic acid to form water adducts when stored, without further activation, in the ion trap for times ranging from 1 to 500 ms. Because the donation of pi electron density to the Ag(+) ion is a likely determining factor in complex reactivity, our aim in the present study was to determine qualitatively the influence of para-position substituents on the aromatic ring on the formation of the water adducts. Our results show that the reactivity of the complexes is influenced significantly by the presence of the various substituents. Decreases in [M + Ag](+) ion abundance, and increases in adduct ion abundance, both measured as a function of storage time, follow the trend -NO(2) > -Br > -F > -OH > -H. The complex of Ag(+) with 4-nitrophenylalanine was nearly as reactive towards water as the Ag(+) complex with aminocyclohexanepropionic acid, the last being an amino acid devoid of pi character in the ring system. Collision induced dissociation of the [M + Ag](+) species derived from the amino acids produces, among other products, Ag(+) complexes with a para-substituted phenylacetaldehyde: complexes that also form adduct species when stored in the ion trap. The trends in adduct ion formation exhibited by the aldehyde-Ag(+) complex ions were similar to those observed for the precursor complexes of Ag(+) and the amino acids, confirming the influence of the ring substituent.     

Origin and removal of adducts (molecular mass = 98 u) attached to peptide and protein ions in electrospray ionization mass spectra

Electrospray ionization of peptides and proteins often produces intense adduct ions resulting from the attachment of a moeity with mass 98 u. The formation of these adduct ions results in a substantial reduction in the mass spectrometric sensitivity and an undesirable increase in the complexity of the mass spectra. In the present study it was shown that the removal of the attached adducts from peptide and protein ions can be affected by collisional activation and that the adducts arise from the attachment of sulfuric acid or phosphoric acid to peptide and protein ions. When sulfate and phosphate ions are removed from the samples by chemical means, adduct free ions are obtained from proteins yielding spectra with improved quality and sensitivity.     

Self-assembly of peptide porphyrin complexes: toward the development of smart biomaterials

The anionic porphyrin, meso-tetrakis(4-sulfonatophenyl)porphine, is found to tightly bind to an engineered 14-residue peptide, resulting in induced alpha-helix formation when mixed in aqueous solutions. The small porphyrin-peptide dissociation constant (2 muM) observed is related to the energetics of peptide helix formation coupled with electrostatic interactions between the anionic porphyrin and cationic residues in the coiled peptide. Analytical ultracentrifugation measurements indicate the porphyrin-peptide complexes dimerize, probably into a coiled coil, and weakly associate to form even higher order structures.     

Intrinsic Ca2+ affinities of peptides: Application of the kinetic method to analogs of calcium-binding site III of rabbit skeletal troponin C

We extended the kinetic method to determine the intrinsic affinities of nonvolatile organic molecules with divalent metal ions and then applied the amended method to determine the calcium affinities of peptide analogs of the calcium-binding site III of rabbit skeletal troponin C. Metal-bis(peptide) complexes of the composition ([H2Pi + H2Pii] - H + Ca)+, where H2P is a neutral peptide, were introduced into the gas phase by fast atom bombardment. The extended kinetic method recognizes that the dissociation characteristics of a singly charged, bis(peptide) complexes of divalent metal ions are determined by not only the metal-ion affinity but also the proton affinities of the neutral and deprotonated peptides. The modified method requires one to measure the relative abundances of [H2P - H + Ca]+, [H2P + H]+, and [H2P - H]- ions that form upon collisional activation of mixed peptide/metal complexes, proton-bound peptide dimers, and deprotonated peptide dimers, respectively. We found, by using the modified method, that the set of peptides has a different affinity order than that in solution. Peptides with one aspartic acid have a higher intrinsic Ca2+ affinity than those with two aspartates. The location of the aspartic acid (Asp) residues at various positions also affects the Ca2+ affinity. Those peptides with one Asp in the middle of the chain have higher Ca2+ affinities than those with Asp on the end because the former peptides offer greater polarizability to stabilize the charge. Peptides with two Asp's located in close proximity have higher intrinsic calcium affinities than those with aspartates positioned further apart.