A sulfated, phosphorylated 7 kDa secreted peptide characterized by direct analysis of cell culture media (original) (raw)
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1998
A method has been developed for the sample handling and analysis of bio-active peptides. Based on electrospray ionization mass spectrometry (ESI-MS), the method is applicable to the target analysis of known peptides. ESI-MS is used to determine the molecular mass of the peptide and provisional identification is based on matching the molecular mass with that of known peptides in a database. Disulfide bridge reductive alkylation is used to determine the number of cysteines in the peptide as well as the presence of disulfide bridges. The peptide is then enzymatically digested and the digestion products analyzed by ESI-MS. The resulting mass map is compared to the masses predicted from the structure of the peptide. Finally, the peptide and its enzymatic fragments are analyzed by liquid chromatography (LC) ESI-MS using collisional activated disassociation (CAD) conditions which promote the formation of product ions from which it is possible to determine the amino acid sequence of the peptide. The developed method was applied to the analysis and identification of a-conotoxin GI. The monoisotopic molecular mass was determined to be 1436.42 ± 0.13 u (n=6). A search of the in-house database determined that the only match within one mass unit of the calculated molecular mass was a-conotoxin GI with a theoretical molecular mass of 1436.48 u. The error between the calculated and theoretical values was 42 ppm. Reductive alkylation indicated the presence of four cysteines and two intramolecular disulfide bridges which was consistent with the structure of a-conotoxin GI. LC-ESI-MS analysis of the tryptic digestion products indicated the presence of two fragments with masses of 564.18 and 1122.45 u which were in agreement with the predicted products. Under CAD conditions, Y series ions were observed from which the entire sequence of the larger tryptic fragment was determined.
Analytical Biochemistry, 1975
Polyacrylamide gel electrophoresis of Fluorescamine-modified peptides is a rapid (90 mitt), sensitive (< 1 nmole), and reproducible method which may be used to resolve peptides without the limitations with respect to molecular size or water solubility found in other analytical systems. Fluorescamine-modified peptides, ranging in size from 3 to 215 amino acids, have been examined in a variety of buffer systems between pH 2.3 and 9.8 and gel concentrations varying between 4% and 16% acrylamide. Similar peptides were examined by high-voltage paper electrophoresis and detected by Fluorescamine. Peptides of low solubility have been examined in the presence of urea. The method reported here was used to resolve small peptides differing in size by a single amino acid, as well as peptides of the same size differing only by a single charge. It was successfully employed as a criterion for homogeneity of peptides in the course of purification for amino acid sequence analysis.
Journal of Chromatography A, 2009
This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids' properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.
A novel procedure for separating small peptides on polyacrylamide gels
Letters in Peptide Science, 2003
A simple and fast procedure that allows the separation of small(1–3 kDa) peptides on glycine-SDS gels is described. Peptideswere separated by glycine-SDS/PAGE as a result of in situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamidemini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.
Purification of small peptides labeled with Bolton-Hunter reagent
Analytical Biochemistry, 1978
A method utilizing high-voltage electrophoresis on paper is described whereby a pentapeptide (Asp-Ser-Asp-Pro-Arg) labeled with Bolton-Hunter reagent is separated from hydrolyzed reagent and unreacted peptide and is recovered from the electrophoretogram in high yield. The general applicability to other peptides is discussed.
Sample pretreatment techniques for the bioanalysis of peptides
Journal of Controlled Release, 1992
The bioanalysis of peptides requires specific quantitative methods with the sensitivity to measure concentrations in the low fmol/ml range. Although immunoassay techniques offer the required specificity and sensitivity, in practice, endogenous interferences may limit their direct application to the measurement of peptides in biological fluids. For this reason there is an increasing utilisation of combined techniques, particularly radioimmunoassay combined with high performance liquid chromatography and solid-phase extraction. It is the aim of this paper to review the principles and application of such combined techniques for the measurement of peptides. Case studies in the bioanalysis of a number of peptides with relative molecular masses of < 5,000 and composed of < 40 amino acid residues will be used to illustrate the versatility of this approach.
Rapid Peptide Reagent Isolation in a Disposable Microfluidic Cartridge
2010
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Journal of Neurochemistry, 2004
The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d 10-Leu) and used mass spectrometry to measure the biosynthetic rate of c-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d-to H-labeled c-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d-to H-labeled c-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d 10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 lM chloroquine for 3 or 6 h significantly reduced the rate of formation of c-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 lM chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d 10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.