Radiolytic modification of acidic amino acid residues in peptides: probes for examining protein-protein interactions

Hydroxyl radical-mediated footprinting coupled with mass spectroscopic analysis is a new technique for mapping protein surfaces, identifying structural changes modulated by protein-ligand binding, and mapping protein-ligand interfaces in solution. In this study, we examine the radiolytic oxidation of aspartic and glutamic acid residues to probe their potential use as structural probes in footprinting experiments. Model peptides containing Asp or Glu were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The radiolysis products were characterized by electrospray mass spectrometry including tandem mass spectrometry. Both Asp and Glu are susceptible to radiolytic oxidization by gamma-rays or synchrotron X-rays. Radiolysis results primarily in the oxidative decarboxylation of the side chain carboxyl group and formation of an aldehyde group at the carbon next to the original carboxyl group, giving rise to a characteristic product with a -30 Da mass change. A similar oxidative decarboxylation also takes place for amino acids with C-terminal carboxyl groups. The methylene groups in the Asp and Glu side chains also undergo oxygen addition forming ketone or alcohol groups with mass changes of +14 and +16 Da, respectively. Characterizing the oxidation reactions of these two acidic residues extends the number of useful side chain probes for hydroxyl radical-mediated protein footprinting from 10 (Cys, Met, Trp, Tyr, Phe, Arg, Leu, Pro, His, Lys) to 12 amino acid residues, thus enhancing our ability to map protein surface structure and in combination with previously identified basic amino acid probes can be used to examine molecular details of protein-protein interactions that are driven by electrostatics.     

Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry.

Radiolysis of peptide and protein solutions with high-energy X-ray beams induces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radiolysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniques were used to characterize radiolytic modification to amino acids cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity order of Cys, Met > Phe, Tyr, > Trp > Pro > His, Leu was determined under aerobic reaction conditions from MS/MS analysis of the radiolyzed peptide products. Radiolysis of peptides in 18O-labeled water under aerobic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine and methionine side chains reacted with hydroxyl radicals generated from radiolysis of water as well as molecular oxygen. Phenylalanine and tyrosine residues were modified predominantly by hydroxyl radicals, and the source of modification of proline was exclusively through molecular oxygen.     

Performance of combinatorial peptide libraries in capturing the low-abundance proteome of red blood cells. 2. Behavior of resins containing individual amino acids

Sixteen different amino acids (Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val) have been separately linked to chromatographic beads and used for studying the mechanism of binding of such baits to proteins, as represented by the cytoplasmic proteome of the human red blood cell (RBC). The 16 different amino acid columns were confronted with equal amounts of RBC lysate, washed to remove unbound material, and eluted with denaturing agents. All eluates were analyzed by nanoLC-MS/MS. The results: there appears to be a dichotomy between a class of "Grand Catchers" (Arg, His, Ile, Lys, Phe, Trp, Tyr, Val), all able to bind from 330 up to 441 unique gene products, and the "Petite Catchers" (Asn, Asp, Gln, Glu, Gly, Pro, Ser, Thr), that bind in general half as much, with the notable exception of Glu that under the described conditions seems to bind only traces of proteins. By comparing homogeneous classes of amino acids (e.g., the basic, the hydrophobic aromatic, the neutral hydrophilic, etc.), it is found that, in general, more than half as many proteins are held in common among the members of each family. In a 16-way comparison, 72 proteins (less than 10% of the total amount, which amounts to 800 unique, nonredundant, identified proteins) appear to be the common catch of all 16 amino acids, suggesting that such proteins might have either multiple binding sites or general motifs recognized by any generic bait. By far, it would appear that the strongest interactions and thus the strongest catches occur with the three aromatic moieties of Phe, Trp, and Tyr, all able to capture a practically identical number of proteins. Ionic interactions, which in principle should be the strongest ones, appear to behave in a peculiar way: they are quite strong with the three basic amino acids (Arg, His, Lys) but almost inexistent with their acidic counterparts. This suggests a peculiar mechanism of interaction: upon formation of the ion pair, the linkage between the protein and the bait is stabilized by the hydrophobicity of the underlying chain (e.g., a butyl in the case of Lys).     

Radiolytic modification of sulfur-containing amino acid residues in model peptides: fundamental studies for protein footprinting

Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using gamma-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the gamma-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine approximately tryptophan > cystine is observed.     

基因工程类丝弹性蛋白聚合物(SELP)水凝胶在药物控制释放方面的研究进展

类丝弹性蛋白聚合物(SELP)是一种通过基因工程方法合成的蛋白质嵌段共聚物,其结构由类丝蛋白(GAGAGS)和类弹性蛋白(GVGVP)的肽段单元串连重复组成。由于具有良好的生物识别性,生物相容性和生物降解性,且能在生理条件下发生原位不可逆溶胶-凝胶转变,SELP在生物医学工程方面展示了广泛的应用前景,尤其作为用于药物控制释放的新型生物医学材料。本文就SELP的生物合成、性质及其水凝胶作为新型的生物材料基质在药物控制释放方面的应用做一综述。     

Resolving isomeric peptide mixtures: a combined HPLC/ion mobility-TOFMS analysis of a 4000-component combinatorial library.

A reversed-phase high-performance liquid chromatography (HPLC) separation approach has been combined with ion mobility/time-of-flight (TOF) mass spectrometry in order to characterize a combinatorial peptide library designed to contain 4000 peptides of the general form NH2-Xxx-Xxx-XXX-CO2H, NH2-Ala-Xxx-Xxx-Xxx-CO2H, NH2-Ser-Ala-Xxx-Xxx-Xxx-CO2H and NH2-Leu-Ser-Ala-Xxx-Xxx-Xxx-CO2H (where Xxx represents a randomization over 10 different amino acids: Ala, Arg, Asp, Glu, Gly, Leu, Lys, Phe, Ser, and Val). Addition of the gas-phase mobility separation between the HPLC separation and TOF measurement dimensions makes it possible to resolve many peptide isomers that have identical retention times (and masses).     

Cleavage N-terminal to proline: analysis of a database of peptide tandem mass spectra

Fragmentation at the Xxx-Pro bond was analyzed for a group of peptide mass spectra that were acquired in a Finnigan ion trap mass spectrometer and were generated from proteins digested by enzymes and identified by the Sequest algorithm. Cleavage with formation of a + b + y ions occurred more readily at the Xxx-Pro bond than at other locations in these peptides, and the importance of this cleavage varied by the identity of Xxx. The most abundant Xxx-Pro relative bond cleavage ratios were observed when Xxx was Val, His, Asp, Ile, and Leu, whereas the least abundant cleavage ratios occurred when Xxx was Gly or Pro. Rationalization for these cleavage ratios at Xxx-Pro may include contribution of the Asp or His side chain to enhanced cleavage or the conformation of Pro, Gly, and the aliphatic residues Val, Ile, and Leu at the Xxx location in the Xxx-Pro bond. Although unusual fragmentation behavior has been noted for Pro-containing peptides, this analysis suggests that fragmentation at the Xxx-Pro bond is predictable and that this information may be used to improve the identification of proteins if it is incorporated into peptide sequencing algorithms.     

Near-infrared spectrometric determination of di- and tripeptides synthesized by a combinatorial solid-phase method

A new method based on near-infrared (NIR) spectrometry and partial least-squares analysis has been developed for the noninvasive and nondestructive determination of the identity and sequences of amino acid residues in di- and tripeptides. The di- and tripeptides were synthesized from six amino acids with similar structures (Gly, Ala, Leu, Met, Phe, Val) on two different polymer beads (bead with and without a linker) using the solid-phase peptide synthetic method. The developed NIR method is capable of determining the identity of sequences of these di- and tripeptides (with and without the Fmoc protecting group) directly on the polymer beads. It can distinguish not only dipeptides from tripeptides but also peptides with very similar structures (e.g., bead-Gly-Ala-Ala, bead-Gly-Ala-Phe, bead-Gly-Ala-Leu, bead-Gly-Ala-Val, and bead-Gly-Ala-Met). More importantly, the method is capable of distinguishing di- and tripeptides with the same amino acid residues but different sequences (e.g., bead-Gly-Leu-Val from bead-Gly-Val-Leu).     

Side-chain losses in electron capture dissociation to improve peptide identification

Analysis of a database of some 20 000 conventional electron-capture dissociation (ECD) mass spectra of doubly charged ions belonging to tryptic peptides revealed widespread appearance of w ions and related u ions that are due to partial side chain losses from radical z* ions. Half of all z* ions that begin with Leu or Ile produce w ions in conventional one-scan ECD mass spectra, which differentiates these isomeric residues with >97% reliability. Other residues exhibiting equally frequent side chain losses are Gln, Glu, Asp, and Met (cysteine was not included in this work). Unexpectedly, Asp lost not a radical group like other amino acids but a molecule CO2, thus giving rise to a radical w* ion with the possibility of a radical cascade. Losses from amino acids as distant as seven residues away from the cleavage site were detected. The mechanism of such losses seems to be related to radical migration from the original site at the alphaCn atom in a zn* ion to other alphaC and betaC atoms. The side chain losses confirm sequence assignment, improve the database matching score, and can be useful in de novo sequencing.     

Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.     

Radiolytic modification of basic amino acid residues in peptides: probes for examining protein-protein interactions

Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examining protein-protein interactions in solution. Hydroxyl radicals generated by radiolysis or chemical methods efficiently react with many amino acid residue side chains, including the aromatic and sulfur-containing residues along with proline and leucine, generating stable oxidation products that are valuable probes for examining protein structure. In this study, we examine the radiolytic oxidation chemistry of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidation products. Model peptides containing arginine, histidine, and lysine were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The rates of oxidation and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidation, giving rise to a characteristic product with a 43 Da mass reduction as a result of the loss of guanidino group and conversion to gamma-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidation studies. Histidine was oxidized to generate a mixture of products with characteristic mass changes primarily involving rupture of and addition to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.     

Part III: Surface-enhanced Raman scattering of amino acids and their homodipeptide monolayers deposited onto colloidal gold surface

Surface-enhanced Raman scattering (SERS) spectra were measured for monolayers of various amino acids: L-methionine (Met), L-cysteine (Cys), L-glycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, Leu-Leu, Phe-Phe, and Pro-Pro) deposited onto a colloidal gold surface. Orientation of amino acids and their homodipeptides, as well as specific-competitive interactions of their functional groups with the gold surface, were predicted by detailed spectral analysis of the obtained SERS spectra. The analysis performed allowed us to propose a particular surface geometry for each amino acid and homodipeptide on the gold surface. In addition, we compared the structures of these molecules adsorbed on colloidal gold and silver surfaces.     

Adsorption of amino acids by fullerenes and fullerene nanowhiskers

Abstract

We have investigated the adsorption of some amino acids and an oligopeptide by fullerene (C₆₀) and fullerene nanowhiskers (FNWs). C₆₀ and FNWs hardly adsorbed amino acids. Most of the amino acids used have a hydrophobic side chain. Ala and Val, with an alkyl chain, were not adsorbed by the C₆₀ or FNWs. Trp, Phe and Pro, with a cyclic structure, were not adsorbed by them either. The aromatic group of C₆₀ did not interact with the side chain. The carboxyl or amino group, with the frame structure of an amino acid, has a positive or negative charge in solution. It is likely that the C₆₀ and FNWs would not prefer the charged carboxyl or amino group. Tri-Ala was adsorbed slightly by the C₆₀ and FNWs. The carboxyl or amino group is not close to the center of the methyl group of Tri-Ala. One of the methyl groups in Tri-Ala would interact with the aromatic structure of the C₆₀ and FNWs. We compared our results with the theoretical interaction of 20 bio-amino acids with C₆₀. The theoretical simulations showed the bonding distance between C₆₀ and an amino acid and the dissociation energy. The dissociation energy was shown to increase in the order, Val < Phe < Pro < Asp < Ala < Trp < Tyr < Arg < Leu. However, the simulation was not consistent with our experimental results. The adsorption of albumin (a protein) by C₆₀ showed the effect on the side chains of Try and Trp. The structure of albumin was changed a little by C₆₀. In our study Try and Tyr were hardly adsorbed by C₆₀ and FNWs. These amino acids did not show a different adsorption behavior compared with other amino acids. The adsorptive behavior of mono-amino acids might be different from that of polypeptides.     

Ab initio study of the binding of collagen amino acids to graphene and A-doped (A = H, Ca) graphene

We present a theoretical study of the binding of collagen amino acids (AA, namely glycine, Gly; proline, Pro; and hydroxyproline, Hyp) to graphene (Gr), Ca-doped graphene and graphane (Gra) using density functional theory calculations and ab initio molecular dynamics (AIMD) simulations. It is found that binding of Gly, Pro and Hyp to Gr and Gra is thermodynamically favorable yet dependent on the amino acid orientation and always very weak (adsorption energies E_ads range from −90 to −20 meV). AIMD simulations reveal that room-temperature thermal excitations are enough to induce detachment of Gly and Pro from Gr and of all three amino acids from Gra. Interestingly, we show that collagen AA binding to Gr is enhanced dramatically by doping the carbon surface with calcium atoms (corresponding E_ads values decrease by practically two orders of magnitude with respect to the non-doped case). This effect is result of electronic charge transfers from the Ca impurity (donor) to Gr (acceptor) and the carboxyl group (COOH) of the amino acid (acceptor). The possibility of using Gr and Gra as nanoframes for sensing of collagen amino acids has also been investigated by performing electronic density of states analysis. It is found that, whether Gr is hardly sensitive, the electronic band gap of Gra can be modulated by attaching different number and species of AAs onto it. The results presented in this work provide fundamental insights on the quantum interactions of collagen protein components with carbon-based nanostructures and can be useful for developments in bio and nanotechnology fields.     

Secondary reactions and strategies to improve quantitative protein footprinting

Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH2 (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.     

Part I: surface-enhanced Raman spectroscopy investigation of amino acids and their homodipeptides adsorbed on colloidal silver

Surface-enhanced Raman scattering spectra (SERS) were measured for various amino acids: L-methionine (Met), L-cysteine (Cys), Lglycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, LeuLeu, Phe-Phe, and Pro-Pro) in silver colloidal solutions. The geometry and orientation of the amino acids or dipeptides on the silver surface, and their specific interaction with the surface, were deducted by detailed spectral analysis of the SERS spectra. This analysis has allowed us to propose the particular surface geometry of amino acids or dipeptides and also implied that C-C bonds were almost parallel to the surface, as evidenced by the absence of marker bands in the skeletal C-C stretching region of the spectra. Additionally, using "time-dependent" SERS measurements we solved an existing controversy regarding the binding specificity of Gly-Gly on the silver surface.     

Analysis of protein phosphorylation in the regions of consecutive serine/threonine residues by negative ion electrospray collision-induced dissociation. Approach to pinpointing of phosphorylation sites

Pinpointing of phosphorylation sites by positive ion collision-induced dissociation (CID) in phosphopeptides containing consecutive Ser/Thr residues (Ser/Thr clusters) is frequently hampered by the lack of backbone cleavage between adjacent Ser/Thr or pSer/pThr sites. In this study, we demonstrate that in negative ion collision-induced dissociation phosphorylated and unmodified residues of Ser/Thr clusters exhibit a very selective behavior toward cleavage of their N-Calpha bonds. Ser/Thr clusters were defined as two and more consecutive serine or threonine residues in phosphopeptide sequences. Dissociation reactions at pSer are significantly more abundant than those of unmodified sites. Thr residues exhibit the same effect, but the cleavages occurring at pThr are generally less prominent than those at pSer. The correlation observed between the facility of the amine backbone bond dissociation of phosphopeptides and the presence of the phosphate group on the side chain residues of Ser and Thr is attributed to the different magnitudes of electron density on the Calpha atoms of the amino acid in phosphorylated and unmodified forms. The results of this study indicate that the intensity ratio of the fragments generated by N-Calpha bond cleavage within the phosphopeptide Ser/Thr clusters represents a reliable and general marker for pinpointing of phosphorylation sites. The presented data illustrate that negative ion electrospray CID is superior over the standard positive ion mode approach for the localization of protein phosphorylation inside Ser/Thr clusters.     

Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry

Protein surfaces are important in most biological processes, including protein-protein interactions, enzymatic catalysis, and protein-ligand binding. We report a method in which hydroxyl radicals generated by a rapid-UV irradiation of a 15% hydrogen peroxide solution were utilized to oxidize specific amino acid side chains of two model proteins (lysozyme, beta-lactoglobulin A), according to the residues' chemical reactivities and the solvent accessibility of the reactive carbons and sulfurs in the residue. Oxidized peptides generated by tryptic digestion were identified by electrospray-Fourier transform mass spectrometry. The specific sites of the stable modification were then identified by reverse-phase liquid chromatography coupled to quadropole ion trap tandem mass spectrometry. The solvent accessibility of the residue was shown to directly affect the rate of oxidation by this method (with the exception of methionine), supporting its use as a rapid measure of the solvent accessibility of specific residues, and in some cases, individual atoms.     

Determination of deamidation artifacts introduced by sample preparation using (18)o-labeling and tandem mass spectrometry analysis

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in (18)O water. When using H(2)(18)O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the (18)O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two (18)O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of (18)O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using (18)O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.     

Application of Fourier transform Raman spectroscopy for prediction of bitterness of peptides

The potential application of Fourier transform (FT) Raman spectroscopy to predict the bitterness of peptides was investigated. FT-Raman spectra were measured for the amino acid Phe and 9 synthetic di-, tri-, and tetra peptides composed of Phe, Gly, and Pro. Partial least squares regression (PLS)-1 analysis was applied to correlate the FT-Raman spectra with bitterness intensity values (R(caf) and log 1/T) reported in the literature. Using full cross-validation, Model 1 based on the single spectral data set for the nine peptides yielded a high correlation coefficient for calibration (R = 0.99), but a low correlation coefficient for prediction (R = 0.56). Two models were constructed using the data sets including replicate spectra for the calibrations and were validated using full cross-validation. Using leave-one-sample-set-out calibrations, Model 2, which was developed with the data for the peptides as well as Phe, yielded a low correlation coefficient (R = 0.533) for the prediction of the bitterness, while Model 3 developed with only the peptide data provided better correlation coefficients (R = 0.807 and 0.724 for R(caf) and log 1/T values, respectively). The correlation coefficients for prediction were 0.975 (R(caf) values) and 0.874 (log 1/T values) for Model 4, which was developed using subtracted spectral data (spectra of peptides with higher R(caf) values minus spectra of peptides with lower R(caf) values). Examination of the PLS regression coefficients at wavenumbers most highly correlated with bitterness revealed the importance of hydrophobicity and peptide length on bitterness. This study indicates the potential of FT-Raman spectroscopy as a useful tool for predicting bitterness of peptides and amino acids.