Partial acetylation of lysine residues improves intraprotein cross-linking

Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.     

Parallel fabrication of RNA microarrays by mechanical transfer from a DNA master

A new method for fabrication of RNA microarrays is described. The approach involves cohybridization of a short, biotinylated DNA oligonucleotide and an RNA probe sequence to DNA templates spotted onto a master array. Next, the short DNA sequence and the RNA probe are linked using a T4 DNA ligase. Finally, a poly(dimethylsiloxane) (PDMS) monolith modified on the surface with streptavidin is brought into conformal contact with the master array. This results in binding of the biotinylated DNA/RNA oligonucleotides to the PDMS surface. When the two substrates are mechanically separated, the DNA/RNA oligonucleotides transfer to the PDMS replica, and the DNA oligonucleotides remaining on the master array are ready to template another RNA replica array. This sequence can be repeated for at least 18 cycles using a single master array. RNA arrays consisting of up to three different oligonucleotide sequences and consisting of up to 2500 individual approximately 70 microm spots have been prepared.     

Reduction in database search space by utilization of amino acid composition information from electron transfer dissociation and higher-energy collisional dissociation mass spectra

With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.     

Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR

Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.     

Mass spectrometry evidence for cisplatin as a protein cross-linking reagent

Cisplatin is a potent anticancer drug, which functions by cross-linking adjacent DNA guanine residues. However within 1 day of injection, 65-98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification but result in more comprehensive MS/MS fragmentation and can assist in the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.     

Analysis of phosphorylated peptides by ion mobility-mass spectrometry

An ion mobility-mass spectrometry technique for rapid screening of phosphopeptides in protein digests is described. A data set of 43 sequences (ranging in mass from 400 to 3000 m/z) of model and tryptic peptides, including serine, threonine, and tyrosine phosphorylation, was investigated, and the data support our previously reported observation (Ruotolo, B. T.; Verbeck, G. F., IV; Thomson, L. M.; Woods, A. S.; Gillig, K. J.; Russell, D. H. J. Proteome Res. 2002, 1, 303.) that the drift time-m/z relationship for singly charged phosphorylated peptide ions is different from that for nonphosphorylated peptides. The data further illustrate that a combined data-dependent IM-MS/MS approach for phosphopeptide screening would have enhanced throughput over conventional MS/MS-based methodologies.     

Improvement of the MS/MS fragment ion coverage of acidic residue-containing peptides by amidation with 15N-substituted amine

Tandem mass spectrometry (MS/MS) is a powerful tool for peptide sequencing and characterization. However, the selective cleavage at acidic residues, aspartic acid, and glutamic acid prevents the generation of enough product ions to elucidate the entire sequence. We attempted to solve the problem by converting the residues into the corresponding amides, asparagine and glutamine. The amidation suppressed the cleavage at the converted residues, and the product ions derived from dissociation at other sites became abundant. Incorporation of nitrogen isotope (15)N in the amine constituent for amidation minimized the mass change from -0.984 016 to +0.013 019, allowing easy discrimination of acidic and amide residues in the original sequences by MS/MS database search. In addition, the amidated and unchanged peptides had the same nominal mass, even when the transformation was incomplete, which was approximately 70% in the current condition. The unmodified acidic residues remaining were rather useful to give marker fragments by the dominant dissociation. These results demonstrate that (15)N-amidation is effective in improving the performance of MS/MS to elucidate amino acid sequences of peptides.     

Investigation of neutral loss during collision-induced dissociation of peptide ions

MS/MS fragmentation of peptides is dominated by overlapping b and y ion series. However, alternative fragmentation possibilities exist, including neutral loss. A database was generated containing 8400 MS/MS spectra of tryptic peptides assigned with high probability to an amino acid sequence (true positives) and a set of certified false (true negative) assignments for analysis of the amino terminus. A similar database was created for analysis of neutral loss at the carboxy termini using a data set of chymotryptic peptides. The analysis demonstrated that the presence of an internal basic residue, limiting proton mobility, has a profound effect on neutral loss. Peptides with fully mobile protons demonstrated minimal neutral loss, with the exception of amide bonds with proline on the carboxy terminal side, which created an intense neutral loss peak. In contrast, peptides with partial proton mobility contained many amino acids on either side of the amide bond associated with a strong neutral loss peak. Most notable among these was proline on the carboxy terminal side of an amide bond and aspartic acid on the amino terminal side of a bond. All results were found to be consistent for doubly and triply charged peptides and after adjustment for pairings across the amide bonds with particularly labile residues. The carboxy terminal of chymotryptic peptides also demonstrated significant neutral loss events associated with numerous amino acid residues. Clarification of the rules that govern neutral loss, when incorporated into analysis software, will improve our ability to correctly assign spectra to peptide sequences.     

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).     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of alpha-amino acids.

Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of alpha-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides containing beta-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (omega) of alpha-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying number of backbone atoms. The beta-turn, a ubiquitous structural feature formed by two residue (alphaalpha) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramolecular 4-->1 hydrogen bond. Hybrid turns may be classified by comparison with their alphaalpha counterparts. The available crystallographic information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual omega-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of alpha-peptides. Examples of both peptide helices and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all alpha-residue sequences. The use of stereochemically constrained omega-residues promises to expand the range of peptide design strategies to include omega-amino acids. This approach is exemplified by well-folded structures like the C12 (alphagamma) and C14 (gammagamma) helices formed in short peptides containing multiply substituted gamma-residues. The achiral gamma-residue gabapentin is a readily accessible building block in the design of peptides containing gamma-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.     

Unique tryptic peptides specific for bovine and human hemoglobin in the detection and confirmation of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygen-carrying capacity of circulating blood, they are excellent candidates for abuse in human and equine athletes. To deter athletes from blood doping with HBOCs such as Hemopure and Oxyglobin (OXY), a method for detection, confirmation, quantification, and distinguishing of HBOCs from native hemoglobin in test samples is needed. The purpose of this study was to identify unique peptides specific for bovine Hb and human Hb that are useful in the detection and confirmation of HBOCs in test samples. The LC-MS chromatographic peak profiles of tryptic digests from OXY, bovine Hb, human Hb, and equine Hb were compared, and unique tryptic peptides specific for bovine Hb, human Hb, and equine Hb were identified. The peptides specific for bovine Hb and OXY are related to bovine Hb alpha chain residues 69-90 and beta chain residues 40-58. The peptides specific for human Hb are related to human Hb alpha chain residues 63-91 and beta chain residues 42-60 and 68-83. The amino acid sequences of these unique tryptic peptides were confirmed by their characteristic MS/MS spectra. MS/MS spectra, b-ion series and y-ion series, and LC retention time of the tryptic peptides are essential pieces of information for the unequivocal identification, detection, and confirmation of HBOCs. The results of this study provide useful and defensible data on identification, detection, and confirmation of HBOCs of bovine Hb or human Hb origin. In addition, in-ESI-source fragmentation of tryptic peptides was observed in this study. The fragmentation was undesired since it decreased intensities of the trypic peptide ions, but it was helpful to elucidating sequences of the tryptic peptides thanks to the fragment peptide ions produced from the fragmentation.     

华根霉(Rhizopus chinensis)前导肽脂肪酶基因的克隆及其在Pichia pastoris中的表达

通过3次PCR程序克隆得到了华根霉(Rhizopus chinensis CCTCCM201021)脂肪酶全基因序列,该基因的开放阅读框长1170bp,不含内含子,编码一个389个氨基酸残基的蛋白质,包括26个氨基酸的信号肽,94个氨基酸的前导序列和269个氨基酸的成熟肽,其推断的氨基酸序列与一些已报道的根霉脂肪酶序列同源性为86%.在巴斯德毕赤酵母(Pichia pastoris)GS115中分泌表达前导肽序列.SDS-PAGE分析表明表达蛋白的分子量约37kD.N-端氨基酸序列分析表明该重组蛋白分泌过程中前导序列N-端的67个氨基酸被切割掉,表达的重组脂肪酶由前导序列C-端的27个氨基酸和成熟肽的269个氨基酸组成.发酵132h后上清中重组脂肪酶的表达量最高,蛋白含量约5.4mg/mL,橄榄油乳化法测水解酶活为161U/mL,其比活比野生型华根霉脂肪酶高约43倍.     

Protein quantification by isotope dilution mass spectrometry of proteolytic fragments: cleavage rate and accuracy

The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U = 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.     

Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.     

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.     

小麦印度腥黑穗病菌的分子检测

根据GenBank中登录Tilletia属的20个种的核糖体转录间隔区(ITS)序列差异设计1对引物T1／T2，可以特异地从T.indica和T.wallceri菌株中扩增到一条450bp左右的条带，而从其他供试菌株中不能扩增出条带，表明该对引物可以将这二个种与Tilletia其他种区分开。进一步根据T．indica和T．walked的线粒体DNA序列的差异设计一对引物M1／M2，只能特异地从T．indica DNA中扩增到一条250bp左右的条带，表明该对引物可以将T．indica与T．walked区分开。利用上述2对引物分别与ITS区通用引物ITS1/ITS4．和线粒体引物Ti-1／Ti4组合建立套式PCR检测体系，能够直接将T．indica与其他相似或相关种区分开，且灵敏度可达1个冬孢子。此外，还观察到在反应体系中加入适量的(NH4)2SO4对PCR反应具有增效作用，可以提高检测灵敏度。上述结果提示建立的套式PCR反应体系可望能应用于口岸对T．indica的检疫。     

Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC/MS/MS peptide identification for proteomics

Tryptic digests of human serum albumin and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MS/MS with strong cation exchange (SCX) and reversed-phase chromatography and continuous gradient elution. The peptide elution conditions combined simultaneous pH gradient with ammonium acetate salt gradient elution modes. A novel empirical SCX peptide elution score was developed, which accounts for both the number of basic and acidic residues and, in part, their location within a sequence of a peptide. Average scores calculated for the fractionated peptide sequences correlated well with the pH of SCX elution fractions. Multiple peptides with identical amino acid sequences, but differing in cysteine tags possessing different positive charge and different SCX elution properties, were obtained by subjecting the samples to reduction and alkylation with different cysteine alkylating reagents: iodoacetamide, 4-vinylpyridine, and (3-acrylamidopropyl) trimethylammonium chloride. The structurally similar peptides were used as elution standards.     

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.