High-performance Liquid Chromatography Of Peptides Research Articles

Paradigm Shift: Major Role of Ion-Pairing-Dependent Size Exclusion Effects in Bottom-Up Proteomics Reversed-Phase Peptide Separations.

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Å fully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Å pore size materials─was more pronounced for smaller peptides. These findings contradict the central dogmas that underlie the development of all peptide RP-HPLC applications: (i) a larger surface area leads to higher retention and (ii) increasing the pore size should benefit the retention of larger analytes. Based on our intriguing findings, we compared reversed-phase high-performance liquid chromatography peptide retention for a total of 20 columns with pore sizes between 60 and 300 Å using FA- and TFA-based eluents. Our results unequivocally attest that the larger size of ion pairs in FA- vs TFA-based eluents leads to the observed impact on selectivity and peptide retention. For FA, peptide retention peaks at 200 Å pore size, compared to between 120 and 200 Å for TFA. However, the decrease in retention for narrow-pore particles is more profound in FA. Our findings suggest that common assumptions about analyte size and accessible surface area should be revisited for ion-pair RP separation of small peptides, typical for proteomic applications that are predominantly applying FA eluents. Hybrid silica-based materials with pore sizes of 130-200 Å should be specifically targeted for bottom-up proteomic applications to obtain both superior peak shape and peptide retentivity. This challenging task of attaining the best RPLC column for proteomics calls for closer collaboration between LC column manufacturers and proteomic LC specialists.

Synthesis and Purification of Homogeneous Lipid-Based Peptide Nanocarriers by Overcoming Phospholipid Ester Hydrolysis.

Despite the therapeutic promise of phospholipid-based nanocarriers, a major obstacle to their widespread clinical translation is a susceptibility to fatty acid ester hydrolysis, leading to lack of quality control and inconsistencies in self-assembly formulations. Using electrospray ionization mass spectrometry fragmentation in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have demonstrated a method to detect hydrolysis of one or both of the fatty acid esters in a PEGylated phospholipid, DSPE-PEG, in conditions commonly applied during nanocarrier production. Because such carriers are increasingly being used to deliver peptide-based therapeutics, we further investigated the hydrolysis of phospholipid esters in conditions used for solid-phase peptide synthesis and high-performance liquid chromatography of peptides. We ultimately detail a synthetic strategy to reliably produce pure phospholipid–peptide bioconjugates (peptide amphiphiles), while avoiding unintended or unnoticed hydrolyzed byproducts that could lead to polymorphic nanotherapeutics with dampened therapeutic efficacy. We believe that such an approach could help standardize phospholipid–peptide-based therapeutic development, testing, and clinical translation.

Peptide Retention Standards and Hydrophobicity Indexes in Reversed-Phase High-Performance Liquid Chromatography of Peptides

The growing utility of peptide retention prediction in proteomics would benefit from the development of a universal peptide retention standard for better alignment of chromatographic data obtained using various liquid chromatography (LC) platforms. We describe a six-peptide mixture designed for this purpose; its members cover a wide range of hydrophobicity for the most popular modes of reversed-phase peptide high-performance liquid chromatography (HPLC): C18 sorbents with trifluoroacetic/formic acid as the ion-pairing modifier and separations at pH 10. We propose a hydrophobicity index (HI) describing the concentration of organic solvent (typically acetonitrile) that yields a retention factor of 10 under isocratic elution conditions for any peptide. This measure is a fundamental characteristic of peptide-sorbent interaction, depending only on the type of sorbent and the ion-pairing modifier used. Spiking a sample with a standard peptide mixture provides a measurement of the HI values of all detected species during gradient separation. In addition to alignment of data and calibration of chromatographic runs when peptide retention prediction protocols are used, these values obtained from proteomics experiments can be utilized directly in method development for large-scale preparative separations.

Immunomodulating effects of water-soluble extracts of traditional French Alps cheeses on a human T-lymphocyte cell line

The immunomodulation potential of four pressed-curd cheeses was studied in vitro on human T lymphocytes. T lymphocyte proliferation and metabolic activity were evaluated with optimized 5-bromo-2′-deoxyuridine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays, respectively. The up to 4.6-fold stimulation of T-lymphocyte proliferation and metabolic activity by Abondance and Tomme de Savoie water-soluble extracts (WSEs) at the lag phase suggested the presence of immunomodulating compounds in these cheeses. Since peptides released from milk proteins during cheesemaking are potential immunomodulatory compounds, the high-performance liquid chromatography peptide profiles of the cheese WSEs were determined. While no correlation between peptide composition and in vitro immunomodulation of T-lymphocyte cells could be established, peptide quantity, size and hydrophobicity were related to cheesemaking technological parameters such as cheese age and cooking temperature.

Alternative high-performance liquid chromatographic peptide separation and purification concept using a new mixed-mode reversed-phase/weak anion-exchange type stationary phase

This article describes a new complementary peptide separation and purification concept that makes use of a novel mixed-mode reversed-phase/weak anion-exchange (RP/WAX) type stationary phase. The RP/WAX is based on N-(10-undecenoyl)-3-aminoquinuclidine selector, which is covalently immobilized on thiol-modified silica particles (5 μm, 100 Å pore diameter) by radical addition reaction. Remaining thiol groups are capped by radical addition with 1-hexene. This newly developed separation material contains two distinct binding domains in a single chromatographic interactive ligand: a lipophilic alkyl chain for hydrophobic interactions with lipophilic moieties of the solute, such as in the reversed-phase chromatography, and a cationic site for anion-exchange chromatography with oppositely charged solutes, which also enables repulsive ionic interactions with positively charged functional groups, leading to ion-exclusion phenomena. The beneficial effect that may result from the combination of the two chromatographic modes is exemplified by the application of this new separation material for the chromatographic separation of the N- and C-terminally protected tetrapeptide N-acetyl-Ile-Glu-Gly-Arg- p-nitroanilide from its side products. Mobile phase variables have been thoroughly investigated to optimize the separation and to get a deeper insight into the retention and separation mechanism, which turned out to be more complex than any of the individual chromatography modes alone. A significant anion-exchange retention contribution at optimal pH of 4.5 was found only for acetate but not for formate as counter-ion. In loadability studies using acetate, peptide masses up to 200 mg could be injected onto an analytical 250 mm × 4 mm i.d. RP/WAX column (5 μm) still without touching bands of major impurity and target peptide peaks. The corresponding loadability tests with formate allowed the injection of only 25% of this amount. The analysis of the purified peptide by capillary high-performance liquid chromatography (HPLC)-UV and HPLC–ESI-MS employing RP-18 columns revealed that the known major impurities have all been removed by a single chromatographic step employing the RP/WAX stationary phase. The better selectivity and enhanced sample loading capacity in comparison to RP-HPLC resulted in an improved productivity of the new purification protocol. For example, the yield of pure peptide per chromatographic run on RP/WAX phase was by a factor of about 15 higher compared to the standard gradient elution RP-purification protocol.

Quantitative Prediction of logk of Peptides in High-Performance Liquid Chromatography Based on Molecular Descriptors by Using the Heuristic Method and Support Vector Machine

A new method support vector machine (SVM) and the heuristic method (HM) were used to develop the nonlinear and linear models between the capacity factor (logk) and seven molecular descriptors of 75 peptides for the first time. The molecular descriptors representing the structural features of the compounds only included the constitutional and topological descriptors, which can be obtained easily without optimizing the structure of the molecule. The seven molecular descriptors selected by the heuristic method in CODESSA were used as inputs for SVM. The results obtained by SVM were compared with those obtained by the heuristic method. The prediction result of the SVM model is better than that of heuristic method. For the test set, a predictive correlation coefficient R = 0.9801 and root-mean-square error of 0.1523 were obtained. The prediction results are in very good agreement with the experimental values. But the linear model of the heuristic method is easier to understand and ready to use for a chemist. This paper provided a new and effective method for predicting the chromatography retention of peptides and some insight into the structural features which are related to the capacity factor of peptides.

Rapid screening of urinary proline–hydroxyproline dipeptide in bone turnover studies

In a recent report [J. Chromatogr. B 678 (1996) 165] a urinary hydroxyproline-containing peptide has been preliminarily suggested as a possible alternative to hydroxyproline (HP) determination in bone resorption studies. For this purpose a simple and practical procedure was developed for a rapid high-performance liquid chromatographic (HPLC) assay of the peptide in non-hydrolyzed urine samples. Hundreds of randomly selected urine samples were assayed for both the peptide and HP, the latter in hydrolyzed urine, and a high correlation between them was found. The promising results prompted us to search for the postulated biomarker of bone resorption in urine samples of postmenopausal women examined as osteoporosis suspects. As an alternative to the HPLC determination, an equally rapid procedure has been developed for the peptide assay using capillary gas chromatography (GC) with flame ionization detection (FID). By means of a solid-phase and a liquid–liquid phase extraction, involving ethyl chloroformate (ECF) as the derivatizing agent, two dipeptides and some urinary amino acids could be analyzed within 5 min. A high correlation between both HPLC and GC peptide assay was confirmed ( r=0.944) and the compound was identified as proline–hydroxyproline (PHP) dipeptide.

Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized α-chymotrypsin

Reversed-phase high-performance liquid chromatography (RP-HPLC) separation was used for the comparison of peptide maps of pepsin after its digestions by different forms of immobilized α-chymotrypsin. Porcine pepsin was hydrolysed with soluble α-chymotrypsin, with α-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with α-chymotrypsin attached to poly(acrylamide-allyl glycoside) copolymer or to glycosylated hydroxyalkyl methacrylate copolymer Separon or to agarose gel Sepharose 4B. Efficiency of enzymatic protein cleavage with regard to peptide mapping of porcine pepsin has been examined by the use of α-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with α-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. α-Chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified α-chymotrypsin was not suitable for efficient pepsin cleavage.

Ruggedness Study of HPLC Peptide Mapping for the Identity of a Drug Compound: A Chemometrics Approach

A statistically more reliable approach than the traditional visual inspection of peptide maps to identify a drug compound is to generate a set of reference standards from a designed experiment that incorporates many possible factors that affect variation of peptide mapping. In fact, the experiment can be done for a ruggedness study as part of a high-performance liquid chromatography (HPLC) method validation. Once the ruggedness is proved with the study, those articles in the experiment may form a set of reference standards, and future articles can be compared to the set later to prove identity. A quantitative analysis of the ruggedness study can be done using a chemometrics approach, principal component analysis (PCA). The analysis is used to reduce the many channels of peptide maps to a few manageable dimensions. The scores projected onto the reduced dimensions are used to test factor effects of the ruggedness study. As a by-product, the analysis provides visual inspection of the set of articles in the experiment for any outliers and anomalies.

Size-exclusion high-performance liquid chromatography of peptides: a review

Gel filtration, now referred to as size exclusion chromatography (SEC), on soft gels has been employed for over 40 years for the separation, desalting and molecular weight estimation of peptides and proteins. Developments in rigid particle technology during the 1970s led to the commercial availability of pre-packed columns giving rapid run times and increased resolution, referred to as size-exclusion high-performance chromatography (SE-HPLC). Initially these packings were more suitable for the separation of proteins than of peptides. Today, supports that operate in the fractionation range <10000 Daltons (Da) are available. In this review, the theoretical basis of SEC is discussed and examples are given of its application to the study of peptides, especially regarding purification, estimation of molecular weight and study of molecular associations.

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

High-performance liquid chromatography of peptides on a microspherical carbon column

The properties of a microspherical carbon column for the separation of peptides and proteins are described. One hundred and thirty-three peptides, with from 1 to 148 amino acid residues, were applied on a carbon column and eluted with a linear gradient of acetonitrile [10–70% (v/v), 30 min] in 0.1% (v/v) trifluoroacetic acid solution. The elution behaviour of the peptides on the carbon column and on an octadecylsilica (ODS) column were compared under the same elution conditions. Relationships between the logarithm of the hydrophobicity of peptides and their capacity factors were approximately linear on both columns, but some exceptions were observed with peptides with aromatic residues, indicating stronger adsorption on the carbon than on the ODS column. These results suggest that the major factor for the separation of peptides on both the carbon and ODS columns is hydrophobic interactions. The stronger adsorption of aromatic side-chains in peptides on the carbon column could be understood by an interaction based on the aromatic of graphitic nature of the surface of the microspherical carbon packings.

Electrochemical activity of 6-aminoquinolyl urea derivatives of amino acids and peptides. Application to high-performance liquid chromatography with electrochemical detection

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (6-AQC) is a reagent used to increase the detection-sensitivity of amino acids and peptides in high-performance liquid chromatography with fluorescence detection. In this paper, the electrochemical characteristics of the derivatives of 6-AQC are described. Electrochemical detection of 6-AQC amino acids and peptide derivatives following reverse-phase HPLC are also reported. The response linearity of the derivatives on an amperometric detector was studied in the range of 5 pmol (0.5 μ M) to 2500 pmol (250 μ M). Approximately 2.5 pmol of the amino acid derivatives could be detected. The quantitative results of amino acids in plasma and a bovine serum albumin hydrolysate agreed well with the values reported in the literature.

1] High-resolution reversed-phase high-performance liquid chromatography of peptides and proteins

Publisher Summary Reversed-phase high-performance liquid chromatography (RP-HPLC) has become a commonly used method for the analysis and purification of peptides and proteins. The extraordinary popularity of RP-HPLC can be attributed to a number of factors, including the excellent resolution that can be achieved for closely related as well as structurally disparate substances under a large variety of chromatographic conditions; the experimental ease, with which chromatographic selectivity can be manipulated through changes in mobile phase composition; the generally high recoveries, even at ultramicroanalytical levels; the excellent reproducibility of repetitive separations carried out over long periods of time, due in part to the stability of the various sorbents under many mobile phase conditions; the high productivity in terms of cost parameters; and the potential that is only now being addressed for the evaluation of different physicochemical aspects of solute-eluent or solute-hydrophobic sorbent interactions and assessment of their structural consequences from chromatographic data. The RP-HPLC experimental system usually comprises an n-alkylsilica-based sorbent, from which peptides or proteins are eluted, with gradients of increasing concentration of an organic solvent, such as acetonitrile, containing an ionic modifier, e.g., trifluoroacetic acid (TFA). With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated and low picomolar amounts of resolved components can be collected. Separations can be easily manipulated, by changing the gradient slope, temperature, ionic modifier, or the organic solvent composition. The technique is equally applicable to the analysis of enzymatically derived mixtures of peptides and also for the analysis of synthetically derived peptides.

High-speed high-performance liquid chromatography of peptides and proteins.

Over the last thirty years the name HPLC has been synonymous with high-speed liquid chromatography and during the last ten years we have experienced a dramatic increase in the speed of analysis particularly as far as the separation of biological macromolecules, such as proteins, is concerned. With a solid grounding in the chromatographic theories, column technology has been mainly responsible for the advances in this field. Recent development shows that columns packed with micropellicular or gigaporous stationary phases of the bidisperse or the bimodal type facilitate rapid mass transfer between the mobile and stationary phases and thus can deliver high resolution separations in a very short time. This suggest that HPLC has the potential to be the prime analytical technique for on-line monitoring of biotechnological processes in real time. Further enhancement of the speed of separation comes from the use of elevated temperatures. The role of temperature in HPLC has largely been ignored and most commercial instruments are not equipped with appropriate temperature control. Results presented here strongly suggest, however, that elevated column temperature may find increasing use in the HPLC of large molecules. In such analytical applications temperature programming may also play a major role provided columns with low heat capacity, such as packed fused-silica capillaries, gain wider employment in HPLC.

Selectivity optimization of reversed-phase high-performance liquid chromatographic peptide and protein separations by varying bonded-phase functionality

Several chemical bonded-phase modified silicas were prepared using sterically protected monofunctional silane reagents which varied widely in structure and polarity. Since some of these bonded-phase packing materials are highly polar (hydrophilic), resistance to acid-catalyzed bonded-phase loss by hydrolysis was examined, and observed to remain high even for the highly polar Diol bonded-phase functionality. Modification of the surface of 300 Å pore size, fully hydroxylated and base-deactivated silica microspheres with these sterically protected silanes yielded HPLC column packing materials for examination of separation selectivities in reversed-phase separations of peptide and protein mixtures. Distinct separation selectivities were apparent for each bonded-phase functionality. Selectivity differences ranged from limited band spacing changes for steric-protected C 18 and C 8 bonded-phases, to reversal of elution order for the more polar C 3 and CN bonded phases. The use of column-based selectivity differences between sequential reversed-phase separation steps is used for the two-step HPLC isolation of a recombinant human amyloid precursor polypeptide fragment from a crude bacterial extract.

Cowpea aphid borne mosaic virus-Morocco and South African Passiflora virus are strains of the same potyvirus.

High performance liquid chromatography (HPLC) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the Moroccan isolate of cowpea aphid-borne mosaic virus (CABMV). Some previous reports have suggested CABMV to be very closely related to blackeye cowpea mosaic virus (B1CMV) while other reports have concluded that this relationship is distant. In this report a tryptic digest of the coat protein of CABMV-Morocco was compared with those of the coat proteins of B1CMV-Type, B1CMV-W, the mild mottle strain of peanut stripe virus (PStV-MM) and the NY15 strain of bean common mosaic virus (BCMV-NY15), all of which are now recognised as strains of BCMV. The comparisons also included the NL-3 strain of bean necrosis mosaic virus (BNMV-NL3), which had previously been classified as a strain of BCMV. The HPLC peptide profiles indicated that CABMV-Morocco was distinct from BCMV and BNMV. Amino acid sequence analysis of peptides accounting for more than half of the coat protein confirmed that CABMV-Morocco was not a strain of BNMV or BCMV but was a distinct member of the BCMV subset of viruses that previously has been shown to include BCMV, BNMV, soybean mosaic virus, zucchini yellow mosaic virus, passionfruit woodiness virus and South African Passiflora virus (SAPV). Comparison of the partial sequence data with these and other published sequences revealed that the coat protein of CABMV-Morocco is very similar to that of SAPV suggesting that they are strains of the same virus. Since CABMV was described over 25 years earlier than SAPV, the name CABMV should take precedence and SAPV should be renamed CABMV-SAP, the South African Passiflora strain of CABMV.

Solid-phase derivatization of amino acids and peptides in high-performance liquid chromatography

This paper presents solid-phase derivatization of amino acids and peptides on a hydrophobic polymeric reagent. Using cationic surfactants as a basic pH

Molecular changes during the photooxidation of alpha-crystallin in the presence of uroporphyrin.

Singlet oxygen reacts preferentially with three amino acids in proteins, His, Trp and Met. In order to study the specific molecular events that result from such oxidations, calf alpha-crystallin was photooxidized in the presence of uroporphyrin and the reactions were investigated by high performance liquid chromatography peptide mapping using a photodiode array detector followed by fast atom bombardment mass spectrometry (FAB-MS). From these studies, the following conclusions can be inferred: (1) Upon photooxidation residue Met-68 of the B chain is oxidized to Met sulfoxide, whereas residue Trp-60 remains intact. (2) Two of the 16 His residues in alpha-crystallin are photooxidized with an apparent pKa of ca 7.0 (3) FAB-MS analysis suggests that residue Lys-166 close to the C-terminal end of the A chain forms a cross-link with the His-7 residue close to the N-terminal end of the A chain. This may be either an inter- or intramolecular cross-link.

Strains of Peanut Stripe Potyvirus Rapidly Identified by Profiling Peptides of the Virion Proteins

AbstractVirion proteins of five potyvirus isolates from groundnut in Thailand which induce a range of symptoms were compared with those from the type strains of peanut stripe potyvirus (PStV) and peanut mottle potyvirus (PeMoV). Profiles obtained by high performance liquid chromatography (HPLC) of tryptic peptide digests of the virion proteins of all five Thai isolates and PStV were very similar, whereas that of PeMoV was quite different. The results indicate that the Thai isolates are strains of PStV, and reaffirm the value of HPLC peptide profiling for rapidly identifying potyviruses.