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.     

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.     

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.     

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.     

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.     

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.     

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.     

肽脂质在毛细管胶束电动色谱中的应用

以自主合成的肽脂质(N C5Gly2C16)作为表面活性剂进行毛细管胶束电动色谱(MEKC)研究。测定了不同pH值条件下的迁移时间窗口,并且与十六烷基三甲基溴化铵(CTAB)为准固定相的MEKC模式对比,考察了硫脲的保留行为。结果表明,肽脂质可以在较低的浓度下形成胶束,具有背景较低的特征。在优化条件下,采用此系统成功分离了石芽茶提取物,说明以肽脂质作为表面活性剂的MEKC系统具有特殊的分离选择性。     

Affinity Chromatography of Human Blood Coagulation Factor VIII on Monoliths with Peptides from a Combinatorial Library

FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.     

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.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (aminooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with liquid chromatography and tandem mass spectrometry has been used. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.     

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography‐multiple reaction monitoring mass spectrometry (LC‐MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope‐labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC‐MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients. Copyright © 2012 John Wiley & Sons, Ltd.     

GHGKHKNK octapeptide (P-5m) inhibits metastasis of HCCLM3 cell lines via regulation of MMP-2 expression in in vitro and in vivo studies

P-5m, an octapeptide derived from domain 5 of HKa, was initially found to inhibit the invasion and migration of melanoma cells. The high metastatic potential of melanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in an experimental lung metastasis model, suggesting that P-5m may play an important role in the regulation of tumor metastasis. The aim of this study was to measure the effect of P-5m on tumor metastasis of human hepatocarcinoma cell line (HCCLM3) in vitro and in vivo in a nude mouse model of hepatocellular carcinoma (HCC), and detect the mechanisms involved in P-5m-induced anti-metastasis. By gelatin zymography, matrix metallo-proteinases 2 (MMP-2) activity in HCCLM3 was dramatically diminished by P-5m peptide. In addition, the migration and metastasis of HCCLM3 cells was also inhibited by the peptide in vitro. In an orthotopic model of HCC in nude mice, P-5m treatment effectively reduced the lung metastasis as well as the expression of MMP-2 in the tumor tissues. Overall, these observations indicate an important role for P-5m peptide in HCC invasion and metastasis, at least partially through modulation MMP-2 expression. These data suggests that P-5m may have therapeutic potential in metastatic human hepatocarcinoma.     

亲和色谱中配基的筛选与应用

亲和配基的选择与筛选是发展新的亲和色谱填料或构建一个新的亲和色谱体系所必须解决的首要问题。该文结合作者所在实验室的工作,对配基的选择、筛选与应用方面的一些进展进行了简要评述。作者所在实验室针对特定蛋白质和多肽的多肽亲和配基的筛选,开展了反义肽简并性的研究,发展了基于反义肽的组合化学筛选新方法。与常规的组合合成法相比,该方法简单、快捷、有效,极大地减小了合成和筛选的工作量,降低了筛选后亲和组分结构鉴定的难度。所建立的筛选策略已应用于流感病毒、严重急性呼吸道综合征(SARS)病毒亲和抑制剂的筛选和用于人β-干扰素测定的石英晶体微天平（QCM）生物传感器的构建,均取得了有意义的结果。     

Applications of hydrophilic interaction chromatography to amino acids,peptides, and proteins

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed‐phase liquid chromatography, in a two‐dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.     

Continuous-flow fast atom bombardment mass spectrometry

The continuous-flow fast atom bombardment probe performs equally well with or without a high-performance liquid chromatography column producing clean spectra containing little or no background noise. Its function as a liquid chromatography-mass spectrometry interface for labile and involatile samples has been illustrated with reference to dansylated amino acids. The versatility of the new probe has been exemplified by on-line enzymatic peptide sequencing.     

Multidimensional chromatographic separation of estrogen receptors

Analyses of estrogen and progesterone receptors in biopsies of breast carcinoma play a vital role in the selection of patients likely to respond to hormone manipulation. Sucrose density gradient centrifugation has been the reference method in the determination of estrogen receptors in human breast carcinoma cytosols. To reduce assay time and circumvent prolonged manipulation of labile receptor preparations, high performance liquid chromatography techniques in the size-exclusion and ion-exchange modes were compared as potential alternate methods for the rapid separation of receptor isoforms. Multidimensional analyses were performed by reapplying estrogen receptor isoforms obtained from high performance size-exclusion and ion-exchange chromatography to sucrose density gradients and vice versa. This confirmed that the estrogen-binding components identified by high performance liquid chromatography appear to correspond to estrogen receptor species from sucrose density gradients.