Determining ProGRP and isoforms in lung and thyroid cancer patient samples: comparing an MS method with a routine clinical immunoassay

This paper compares two methods to determine the tumor marker progastrin-releasing peptide (ProGRP): as routine assay, the automated time-resolved immunofluorometric assay (TR-IFMA), which allows total ProGRP determination; and the immunocapture liquid chromatography selected reaction monitoring mass spectrometry (LC–SRM-MS) method, which additionally allows isoform differentiation. The investigation included 60 serum samples from patients suffering from various cancer diseases which may cause elevated ProGRP levels (small cell lung carcinoma; SCLC, non-small cell lung carcinoma; NCLC; and medullary thyroid cancer; MTC, as well as some with unspecific diagnosis). The two methods showed good correlation (R ²?=?0.887). However, the MS method determines the total ProGRP concentration systematically approximately 30 % lower than the TR-IFMA, implying that the absolute values generated by the methods are not interchangeable. The MS method gives additional information about isoform levels in the samples, providing novel insight into isoform expression on the protein level.     

High-Performance Liquid Chromatography of Amphibian Peptides. Selectivity Changes Induced by pH

Abstract

The effect of pH on the retention behavior under reversed- phase liquid chromatography conditions of a series of peptides was examined. Isocratic conditions were used with either methanol or acetonitrile as organic modifiers. The intrinsic hydrophobicity of the peptides was altered by changes in the pH of the eluent mixture. Increased retention at pH 7 relative to pH 4 was correlated with the presence of a histidine residue in a hydrophobic environment. An experimental parameter, α_pH, was defined as the positive quotient of capacity factors at pH 4 and pH 7 for a given eluent. These α_pH values are interpreted as reflecting changes in peptide hydrophobicity introduced by variations in solvent and pH. Identical α_pH values were obtained for homologous peptides, particularly histidine containing peptides. This approach to selectivity effects yielded diagnostic conditions for the analysis of bombesin, a peptide touted as a potential marker for human small-cell lung carcinoma.     

Solid phase synthesis of the signal sequence of E. coli alkaline phosphatase

A synthetic peptide corresponding to the signal sequence ofE. coli alkaline phosphatase has been synthesized by the solid phase method employing the transesterification method of cleavage from the resin. The protected peptide obtained after cleavage was purified to homogeneity by column chromatography on silica gel, followed by partition chromatography on SephadexLH-20 using organic solvents like chloroform and methanol as eluants.     

Anticancer β-hairpin peptides: membrane-induced folding triggers activity

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic β-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.     

Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.     

Global proteome analysis of a human gastric carcinoma

An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.     

Comparison of reversed-phase liquid chromatography and hydrophilic interaction/cation-exchange chromatography for the separation of amphipathic alpha-helical peptides with L- and D-amino acid substitutions in the hydrophilic face

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which has excellent potential for peptide separations. Separations by HILIX/CEX are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlaid on ionic interactions with the cation-exchange matrix. In the present study, HILIC/CEX has been compared to reversed-phase liquid chromatography (RP-HPLC) for separation of mixtures of diastereomeric amphipathic alpha-helical peptide analogues, where L- and D-amino acid substitutions were made in the centre of the hydrophilic face of the amphipathic alpha-helix. Unlike RP-HPLC, temperature had a substantial effect on HILIC/CEX of the peptides, with a rise in temperature from 25 to 65 degrees C increasing the retention times of the peptides as well as improving resolution. Our results again highlight the potential of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP-HPLC.     

Rational and combinatorial design of peptide capping ligands for gold nanoparticles

Based on protein folding considerations, a pentapeptide ligand, CALNN, which converts citrate-stabilized gold nanoparticles into extremely stable, water-soluble gold nanoparticles with some chemical properties analogous to those of proteins, has been designed. These peptide-capped gold nanoparticles can be freeze-dried and stored as powders that can be subsequently redissolved to yield stable aqueous dispersions. Filtration, size-exclusion chromatography, ion-exchange chromatography, electrophoresis, and centrifugation can be applied to these particles. The effect of 58 different peptide sequences on the electrolyte-induced aggregation of the nanoparticles was studied. The stabilities conferred by these peptide ligands depended on their length, hydrophobicity, and charge and in some cases resulted in further improved stability compared with CALNN, yielding detailed design criteria for peptide capping ligands. A simple strategy for the introduction of recognition groups is proposed and demonstrated with biotin and Strep-tag II.     

HPLC Purification of Solid-Phase Generated Synthetic Bombesin

Abstract

A scheme based on ion-exchange and reverse-phase high pressure liquid chromatography has been utilized for the semi-preparative and preparative purification of the solid phase generated model peptide bombesin. The final product showed a purity ≥ 99% in analytical reverse-phase high pressure liquid chromatography and was identical to authentic bombesin as demonstrated by different physico-chemical and biological criteria. The results are discussed and compared to those obtained using countercurrent chromatography.