Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Prediction of peptide retention volumes in gradient reversed phase HPLC

A method of computation of retention volumes of linear peptides of known composition that contain no more than 25 amino acids in gradient reversed phase HPLC was developed. Tha method is suitable for various acetonitrile gradient profiles. The calculations were carried out on the basis of a statistical model, the parameters of which were experimental dependences of the retention of individual amino acids on acetonitrile concentration. The method developed was used to predict the chromatographic behavior of 34 peptides in four different acetonitrile gradients. The correlation coefficients between the predicted and experimental retention volumes were more than 0.9, and the average relative error of prediction was less than 15%.     

Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Aligning LC peaks by converting gradient retention times to retention index of peptides in proteomic experiments

MOTIVATION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. RESULTS: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k(0) (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (t(g)) to log k(0). Using the optimized function, t(g) values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale.     

Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome using artificial neural networks

We developed a computational method to predict the retention times of peptides in HPLC using artificial neural networks (ANN). We performed stepwise multiple linear regressions and selected for ANN input amino acids that significantly affected the LC retention time. Unlike conventional linear models, the trained ANN accurately predicted the retention time of peptides containing up to 50 amino acid residues. In 834 peptides, there was a strong correlation (R2 = 0.928) between measured and predicted retention times. We demonstrated the utility of our method by the prediction of the retention time of 121,273 peptides resulting from LysC-digestion of the Escherichia coli proteome. Our approach is useful for the proteome-wide characterization of peptides and the identification of unknown peptide peaks obtained in proteome analysis.     

An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC: its application to protein peptide mapping by off-line HPLC-MALDI MS

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.     

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.     

Solid-phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa-(4-24)] from skin secretion of Xenopus laevis

From the nucleotide sequence of clones isolated from a cDNA library constructed from skin of Xenopus laevis, the existence of PYLa, a peptide comprised of 24 amino acids, was predicted. This peptide was synthesized by solid-phase methods and purified to homogeneity with an overall yield of 61%. The synthetic peptide was used as reference substance to search for its natural counterpart in skin secretion of Xenopus. Two peptides were found which were very similar to PYLa except for the absence of the first three amino acids. These 21-amino-acid peptides, termed PGLa, can be generated from PYLa by cleavage after the single arginine residue present in the latter. The two forms of PGLa differ in their retention time on HPLC but have identical amino acid compositions and terminal sequences. Tryptic hydrolysis of synthetic PYLa after the single arginine yields exclusively PGLa with the shorter retention time on HPLC. The chemical difference between the two forms of PGLa is currently not known. The possible biological role of these newly discovered constituents of frog skin secretion is discussed.     

Discriminating the xanthones in an extract of Swertia franchetiana by retention parameters

The quantitative structure--retention relationship is one of the most actively studied topics in the field of chromatography. In this paper, retention parameters of components were used to discriminate the xanthones in a methanol extract of Swertia franchetiana The extract was analysed by HPLC under two different multistage linear gradient conditions and the retention parameters calculated from these retention data. It was found that the retention parameters of xanthones are in a specific region in the plot of log k(w) vs. S and the xanthones in the extract could be distinguished from other components by this feature. Furthermore, xanthone aglycones and xanthone glucosides could also be discriminated by retention parameters.     

EDMAN SEQUENCING RESEARCH GROUP 2004 STUDY: MODIFIED AMINO ACIDS IN EDMAN SEQUENCING

Edman degradation sequencing relies on comparing high-performance liquid chromatography retention times of the sample phenylthiohydantoin amino acids with phenylthiohydantoin amino acid standards. The elution characteristics of the twenty common amino acids have been well characterized, which aids in making confident assignments. Modified amino acids may present more of a challenge since they are not part of the commonly used standards and because the protein sequencer analyst may not have experience with them. Laboratories requesting a sample were sent a tube containing approximately 775 pmoles of a 20-amino-acid synthetic peptide composed of several modified amino acids that may be found in proteins or are generated during sample preparation. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the Edman Sequencing Research Group website (ESRG). The goal of the study consisted of two parts: assessment of the ability to correctly assign all the amino acids in the peptide, including the modified amino acids; and the collection and compiling of elution time characteristics of modified amino acids for instruments used in the study. The resulting compilation of the modified amino acid elution times and running conditions will be accessible at the Association of Biomolecular Resource Facilities (ABRF) ESRG website for future reference. The ABRF ESRG 2004 sample is the 16th in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.     

Generation of accurate peptide retention data for targeted and data independent quantitative LC‐MS analysis: Chromatographic lessons in proteomics

The emergence of data‐independent quantitative LC‐MS/MS analysis protocols further highlights the importance of high‐quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP‐HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP‐HPLC as a “black box”, while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion‐pairing modifier, stationary phase and column temperature, we describe the “mysterious” effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation—a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S‐values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress.     

Protein modification by diazotized arsanilic acid: synthesis and characterization of the phenylthiohydantoin derivatives of azobenzene arsonate-coupled tyrosine, histidine, and lysine residues and their sequential allotment in labeled peptides

Analytical procedures are elaborated for the sequential allotment of azobenzene arsonate binding sites in proteins and peptides. The reaction of diazotized arsanilic acid with proteins leads to covalent modification of tyrosine, histidine and, in part, lysine residues. Synthetic peptides containing these amino acids were modified with diazotized arsanilic acid and subjected to N-terminal sequence analysis. The amino acid derivatives phenylthiohydantoin(Pth)-azobenzene-arsonate-tyrosine, Pth-azobenzene-arsonate-histidine, and alpha-Pth-epsilon-hydroxycaproic acid are recovered upon Edman degradation of selected peptides. Phenylthiohydantoins of modified and nonmodified amino acids are fully separated by reverse-phase HPLC on a Zorbax-PTH column. For identification purposes, phenylthiohydantoins of azobenzene arsonate-labeled amino acids have been synthetized. They are characterized with respect to spectral absorption characteristics and retention times on reverse-phase supports.     

Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His⁷]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.