Observation of exchangeable protons by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-electrospray ionization mass spectrometry: a useful tool for the hyphenated analysis of natural products

The first high-performance liquid chromatography-nuclear magnetic resonance (HPLC-NMR) investigation of exchangeable protons of low-molecular-mass natural products is reported. Model alkaloids or crude plant extracts were dissolved in 2H2O-1H2O-MeCN (deuterium oxide-water-acetonitrile) or 2H2O-MeCN and, after direct injection or chromatographic separation, examined in a 60-microl NMR flow probe. Exchangeable amino protons initially detected by HPLC-electrospray ionization mass spectrometry were subsequently identified and investigated by stop-flow 1H-NMR, two-dimensional (2D) total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser effect spectroscopy (NOESY). These experiments extend the applicability of HPLC-NMR for the investigation and structure elucidation of natural products.     

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

Detection of the site of phosphorylation in a peptide using Raman spectroscopy and partial least squares discriminant analysis

Normal (non-enhanced) Raman spectroscopy is used to determine the site of phosphorylation on a 13-residue peptide whose sequence derives from the cellular protein pp60(c-src) (protein tyrosine kinase). Raman spectra of serine, threonine and tyrosine amino acids and their phosphorylated derivatives are used to aid in the interpretation of peptide spectra. The purity of the synthetic peptides are confirmed by mass spectroscopy. Peptide Raman measurements are performed using the recently reported drop-coating deposition Raman (DCDR) method, followed by Savistky-Golay second derivative (SGSD) pre-processing and multivariate spectral classification using partial least squares (PLS) discriminant analysis. Leave-one-out training/testing results are displayed using a PLS psuedo-probability score plot and shown to facilitate error-free spectral determination of the site of phosphorylation.     

Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K(m) of 5.9x10(4) M(-1) s(-1). Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.     

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H(2)O(2) induced tyrosine phosphorylation of PLD1, protein kinase C-alpha (PKC-alpha), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-alpha in VSMCs. Stimulation of the cells by H(2)O(2) and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H(2)O(2)-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H(2)O(2)-stimulated PLD activation. The cells stimulated by oxidative stress (H(2)O(2)) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.     

NMR-based mapping of disulfide bridges in cysteine-rich peptides: application to the mu-conotoxin SxIIIA

Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, mu-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. Mu-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with (15)N/(13)C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance ((1)H, (13)C, (15)N) NMR experiments and 2D [(13)C, (1)H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of mu-conotoxins. Mu-SxIIIA was found to be a potent blocker of the sodium channel subtype Na(V)1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides.     

Label-free electrochemical differentiation of phosphorylated and non-phosphorylated peptide by electro-catalyzed tyrosine oxidation

Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)(3)(2+) solution. The limit of detection was about 0.23 microg mL(-1) EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm(-2), respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 microg mL(-1). This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.     

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39.

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.     

Real-time separation of natural products by ultrafast 2D NMR coupled to on-line HPLC

Hyphenated HPLC-NMR is an extremely efficient analytical tool, which makes it possible to perform on-flow experiments where 1D NMR spectra are obtained in real time as the analytes are separated and eluted from the chromatographic column. However, it is incompatible with multidimensional NMR methods that form an indispensible tool for the study of complex mixtures. Recently, Frydman and co-workers have proposed an ultrafast 2D NMR approach, where a complete 2D NMR correlation can be recorded in a single scan, thus providing a solution to the irreversibility of hyphenated techniques. This paper presents the first implementation of on-line ultrafast HPLC-NMR. Ultrafast COSY spectra are acquired every 12 s in the course of a chromatographic run performed on a mixture of natural aromatic compounds. The results, obtained on a commercial HPLC-NMR setup, highlight the generality of the ultrafast HPLC-NMR methodology, thus opening the way to a number of applications in the numerous fields in which HPLC-NMR forms a routine analytical tool.     

Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional activation in an orthogonal quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer. These peptides contain a C-terminal receptor-binding sequence and an N-terminal nonbinding region. When the peptides form a receptor complex, the amide hydrogens of the interacting sequences are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen and oxygen occurs in the gaseous peptide ion prior to its dissociation.     

Implementation of real-time two-dimensional nuclear magnetic resonance spectroscopy for on-flow high-performance liquid chromatography

Directly coupled HPLC-NMR has become a powerful tool for separation and structural elucidation of unknown compounds. However, there are only a few reports on application of on-flow two-dimensional (2D) NMR in HPLC-NMR. Here we present an alternative method for recording real-time 2D-NMR spectrum (total correlation spectroscopy, TOCSY) on a commercial HPLC-NMR system. The method is based on well-established Hadamard matrix for 2D-NMR frequency encoding. In addition, a modified/improved solvent suppression approach is incorporated. This makes it possible to carry out the experiment with both polar and gradient eluents, the widely used chromatographic conditions. The method is example using a synthesized mixture of three amino acids (His, Phe and Try) and a human urine sample. The method demonstrated here may be utilized for high-throughput structural or unknown component identification and fast dynamic study in a variety of applications.     

Primary and secondary structure dependence of peptide flexibility assessed by fluorescence-based measurement of end-to-end collision rates

The intrachain fluorescence quenching of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is measured in short peptide fragments, namely the two strands and the turn of the N-terminal beta-hairpin of ubiquitin. The investigated peptides adopt a random-coil conformation in aqueous solution according to CD and NMR experiments. The combination of quenchers with different quenching efficiencies, namely tryptophan and tyrosine, allows the extrapolation of the rate constants for end-to-end collision rates as well as the dissociation of the end-to-end encounter complex. The measured activation energies for fluorescence quenching demonstrate that the end-to-end collision process in peptides is partially controlled by internal friction within the backbone, while measurements in solvents of different viscosities (H2O, D2O, and 7.0 M guanidinium chloride) suggest that solvent friction is an additional important factor in determining the collision rate. The extrapolated end-to-end collision rates, which are only slightly larger than the experimental rates for the DBO/Trp probe/quencher system, provide a measure of the conformational flexibility of the peptide backbone. The chain flexibility is found to be strongly dependent on the type of secondary structure that the peptides represent. The collision rates for peptides derived from the beta-strand motifs (ca. 1 x 10(7) s(-1)) are ca. 4 times slower than that derived from the beta-turn. The results provide further support for the hypothesis that chain flexibility is an important factor in the preorganization of protein fragments during protein folding. Mutations to the beta-turn peptide show that subtle sequence changes strongly affect the flexibility of peptides as well. The protonation and charge status of the peptides, however, are shown to have no significant effect on the flexibility of the investigated peptides. The meaning and definition of end-to-end collision rates in the context of protein folding are critically discussed.     

Giant macrolactams based on β-sheet peptides

This paper reports the use of natural amino acids, the tripeptide β-strand mimic Hao, and the β-turn mimic δ-linked ornithine to generate water-soluble 54-, 78-, and 102-membered-ring macrolactams. These giant macrocycles were efficiently prepared by synthesis of the corresponding protected linear peptides, followed by solution-phase cyclization and deprotection. The protected linear peptide precursors were synthesized on 2-chlorotrityl chloride resin by conventional Fmoc-based solid-phase peptide synthesis. Macrocyclization was typically performed using HCTU and N,N-diisopropylethylamine in DMF at ca. 0.5 mM concentration. The macrocycles were isolated in 13-45% overall yield after HPLC purification and lyophilization. 1D, 2D TOCSY, and 2D ROESY (1)H NMR studies of the 54- and 78-membered-ring macrolactams establish that these compounds fold to form β-sheet structures in aqueous solutions.     

Identifying the presence of a disulfide linkage in peptides by the selective elimination of hydrogen disulfide from collisionally activated alkali and alkaline earth metal complexes

We report a new method for identifying disulfide linkages in peptides using mass spectrometry. This is accomplished by collisional activation of singly charged cationic alkali and alkaline earth metal complexes, which results in the highly selective elimination of hydrogen disulfide (H2S2). Complexes of peptides possessing disulfide bonds with sodium and alkaline earth metal are generated using electrospray ionization (ESI). Isolation followed by collision induced dissociation (CID) of singly charged peptide complexes results in selective elimination of H2S2 to leave newly formed dehydroalanine residues in the peptide. Further activation of the product yields sequence information in the region previously short circuited by the disulfide bond. For example, singly charged magnesium and calcium ion bound complexes of [Lys8]-vasopressin exhibit selective elimination of H2S2 via low-energy CID. Further isolation of the product followed by CID yields major b- and z-type fragments revealing the peptide sequence in the region between the newly formed dehydroalanine residues. Numerous model peptides provide mechanistic details for the selective elimination of H2S2. The process is initiated starting with a metal stabilized enolate anion at Cys, followed by cleavage of the S-C bond. An examination of the peptic digest of insulin provides an example of the application of the selective elimination of H2S2 for the identification of peptides with disulfide linkages. The energetics and mechanisms of H2S2 elimination from model compounds are investigated using density functional theory (DFT) calculations.     

Caveolin-1 is involved in reactive oxygen species-induced SHP-2 activation in astrocytes

Recent evidence supports a neuroprotective role of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) against ischemic brain injury. However, the molecular mechanisms of SHP-2 activation and those governing how SHP-2 exerts its function under oxidative stress conditions are not well understood. Recently we have reported that reactive oxygen species (ROS)-mediated oxidative stress promotes the phosphorylation of endogenous SHP-2 through lipid rafts, and that this phosphorylation strongly occurs in astrocytes, but not in microglia. To investigate the molecules involved in events leading to phosphorylation of SHP-2, raft proteins were analyzed using astrocytes and microglia. Interestingly, caveolin-1 and -2 were detected only in astrocytes but not in microglia, whereas flotillin-1 was expressed in both cell types. To examine whether the H2O2-dependent phosphorylation of SHP-2 is mediated by caveolin-1, we used specific small interfering RNA (siRNA) to downregulate caveolin- 1 expression. In the presence of caveolin-1 siRNA, the level of SHP-2 phosphorylation induced by H2O2 was significantly decreased, compared with in the presence of control siRNA. Overexpression of caveolin- 1 effectively increased H2O2-induced SHP-2 phosphorylation in microglia. Lastly, H2O2 induced extracellular signal-regulated kinase (ERK) activation in astrocytes through caveolin-1. Our results suggest that caveolin-1 is involved in astrocyte-specific intracellular responses linked to the SHP-2-mediated signaling cascade following ROS-induced oxidative stress.     

Prepatellamide A, a new cyclic peptide from the ascidian Lissoclinum patella

A new cyclic peptide, prepatellamide A (1), along with three known cyclic peptides (2)-(4), was isolated from the ascidian Lissodinum patella. The structure of prepatellamide A was determined from one- and two-dimensional H and 13C NMR spectra. The known cyclic peptides were identified as patellamides A (2), B (3) and C (4).     

Reverse phase high pressure liquid chromatography for the separation of peptide hormone diastereoisomers

Diastereoisomers of specifically labeled oxytocin derivatives were resolved using reverse phase high pressure liquid chromatography. The peptides [1-hemi-DL-[alpha-2H]cystine]oxytocin, [6-hemi-DL-[alpha-2H]cystine]oxytocin, [2-DL-[alpha-2H]tyrosine]oxytocin and[8-DL-[2-13C]leucine]oxytocin were readily separated using the conditions described. The diastereoisomers of the oxytocin analog [3-DL-[2-13C]leucine]oxytocin also demonstrated baseline resolution under the same conditions. The procedure offers the investigator a rapid method for screening synthetic oxytocin peptides for undesirable diastereoisomeric by-products.     

Cluster-phase reactions: gas-phase phosphorylation of peptides and model compounds with triphosphate anions

Molecular clusters provide a unique environment in which chemical reactions between cluster components can occur. In the present study, electrospray ionization is used to examine the behavior of anionic clusters of triphosphate with choline, acetylcholine, and betaine, and the behaviors of cationic clusters of triphosphate with the peptides bradykinin (RPPGFSPFR) and ARRPEGRTWAQPGY. Phosphorylation of a hydroxyl group, when one is present, is shown to be a facile process when the cluster is subjected to collisional activation. Of particular interest is the selective phosphorylation of the hydroxyl substituent in serine and threonine residues of peptides. Less conclusive results are obtained with three peptides containing tyrosine, but the data obtained are consistent with phosphorylation on tyrosine residues. In the absence of residues with hydroxyl substituents, the C-terminus of a peptide is observed to be phosphorylated. The unique chemical reactions reported in this study represent the first examples of gas-phase phosphorylation of alcohols and are also interesting in that they occur at a site remote from charged functional groups in the same molecule. This facile process may have interesting implications for the synthesis of key molecules at the threshold of life.     

Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined

The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal "lid" (residues 1-24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1-109)MDM2 and its Ser17-phosphorylated analogue (1-109)MDM2 pS17 as well as (1-109)MDM2 S17D and (25-109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1-109)MDM2 by the (15-29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation.     

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.