Production and separation of peptides from proteins stained with Coomassie brilliant blue R-250 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Proteins stained with Coomassie brilliant blue on polyacrylamide gels were digested with lysylendopeptidase in the presence of sodium dodecyl sulfate. Peptide production was similar to that under ordinary conditions of digestion. Peptides were recovered easily and efficiently from the gel pieces and separated by HPLC. The present method for preparation of peptides from proteins separated by sodium dodecyl sulfate gel electrophoresis is quite simple and can be used for sequence analysis of proteins in general at the subnanomolar level.     

Fixation and immunoperoxidase staining of oligopeptides after isoelectric focusing in thin polyacrylamide slab gels

In this paper a new method for immunological detection of small peptides after isoelectric focusing in thin (200 micron) polyacrylamide slab gels is presented. The peptides are immobilized immediately after focusing by pressing a sheet of glutaraldehyde-impregnated filter paper onto the gel. By this procedure, the gel adheres to the paper and this press-blot can be stained using immunoperoxidase staining procedures. Using the two-step peroxidase conjugate and the three-step peroxidase-antiperoxidase method, several oligopeptides could be visualized after focusing. The detection limit of this method appears to be in the nanogram range.     

Quantification of proteins in the subnanogram and nanogram range: comparison of the AuroDye, FerriDye, and India ink staining methods

The usefulness of three sensitive dyes, AuroDye, FerriDye, and India ink, for the quantification of proteins and peptides bound to nitrocellulose paper has been assessed. In general, the staining intensity varies linearly with the logarithm of protein concentrations. The detection limit of small peptides (Mr less than 5000) is higher than that of large peptides and proteins, but the sensitivity is independent of the molecular weight. Oligopeptides of four or less amino acids either stain with very high detection limits or do not stain at all. The detection limit of proteins stained by AuroDye is approximately 1 ng, and in a number of cases even lower. The useful range for quantification of proteins extends to around 100 ng. The FerriDye and India ink staining methods are less sensitive and can be used to quantify proteins over a wide nanogram range. Among the methods tested, the India ink staining method has the highest protein to protein variation in sensitivity.     

Adrenocorticotropin and beta-endorphin are colocalized in the nervous system of rat duodenum

Adrenocorticotropin and beta-endorphin-like immunoreactivities were visualized by an immunohistochemical method on adjacent serial sections of the nervous system of the rat duodenum. Perikarya lying in the myenteric plexus and stained alternately for ACTH or beta-endorphin, showed on two adjacent serial sections a co-existence of these two peptides within one and the same perikarya. A co-localization of beta-endorphin and ACTH is not demonstrated with certainty in the submucous plexus. These results may be evidence for a common occurrence of the two peptides within perikarya of the rat duodenum.     

Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression

This protocol details a method to identify CD4+ T cells that respond to antigens. The method relies on detection of CD154, a costimulatory cell surface protein that is expressed by CD4+ T cells upon activation, and can be used to purify live CD4+ T cells of diverse function. To detect CD154, fluorescently labeled antibodies are cultured with cell samples, peptides (or whole antigens) and monensin during a 6- to 24-h stimulation period. (Note that the assay is not compatible with brefeldin A.) After stimulation, cells are stained with any other antibodies of interest and then are analyzed by flow cytometry or purified by cell sorting. Unlike other assays, this method allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability. This protocol can be completed in 9 h.     

Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes

Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.     

Biochemical studies on the isolation and characterization of human spleen haemosiderin.

Haemosiderin was isolated from thalassaemic human spleens by centrifugation through concentrated KI solutions. A method for solubilizing haemosiderin was developed which leaves the iron oxyhydroxide cores and constituent polypeptides intact, facilitating further purification and analysis. Purified haemosiderin contained no detectable haem, trace amounts of carbohydrate, and iron and phosphorus in a molar ration of 6:1; much of the phosphate may be present as core-adsorbed. Several lipids were present, but it is not certain whether these are contaminants or components of the haemosiderin granules. In all preparations examined, a characteristic group of six to seven peptides of apparent Mr 12 900-17 800 were found, with a major band at Mr 14 500 and, in addition, a minor component of Mr 42 000; these peptides co-chromatographed with the cores. Negatively stained electron micrographs suggest that these peptides form an incomplete shell about the cores, consistent with the view that haemosiderin is a proteolytic product of ferritin.     

An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins

Thiol-rich peptides such as phytochelatins (PCs) and metallothioneins (MTs) are important cellular chelating agents which function in metal detoxification and/or homeostasis. The variations in molecular sizes and lack of chromophores of these peptides make their analysis difficult. This paper reports an electrophoresis-based method for a broad screen of thiol-rich peptides and proteins. The method uses the thiol-selective fluorescent tag, monobromobimane, coupled with Tricine--sodium dodecyl sulphate--urea polyacrylamide gel electrophoresis for a sensitive determination of both PCs and MTs. Results for PCs were confirmed by two-dimensional NMR and HPLC-tandem MS analyses. Sample throughput is substantially improved over chromatography-based methods through parallel sample analysis in 1 h of electrophoretic separation. The method is versatile in that peptides ranging from glutathione to large proteins can be analysed by simple modification(s) of the extraction and electrophoretic conditions, and the nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.     

蛋白质及多肽的C端分析

本文采用（异）硫氰酸化学法,以乙酸酐为活化试剂,四丁铵硫氰酸为偶联试剂,溴甲基萘为烷化试剂,将蛋白质或多肽的Ｃ端依次转化并裂解为ＡＴＨ－氨基酸,在２５４ｎｍ进行检测。分析了若干种天然的、基因工程表达的蛋白质或多肽,测定了不同长度的Ｃ端序列,并确证了Ｃ端的修饰及突变情况。为蛋白质和多肽的完整性、均一性,基因工程产品及合成多肽的质控提供了重要的Ｃ端信息。目前,可在１￣２ｎｍｏｌ水平上测定了３￣５个Ｃ端     

Antibody characterization using novel ERLIC-MS/MS-based peptide mapping

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.     

Nile blue A for staining Escherichia coli in flow cytometer experiments

Nile blue A is used as a stain for polyhydroxyalkanoic acid-accumulating microorganisms or to detect polyhydroxyalkanoic acids in microorganisms. Here we show that Escherichia coli cells that do not accumulate detectable polyhydroxyalkanoic acids can be stained with Nile blue A and that this staining is sufficient for identifying these cells in fluorescence-activated cell sorting (FACS) experiments. Nile blue A staining did not affect either surface display of peptides or specific labeling of these peptides by a second fluorescence. Staining E. coli for flow cytometry using Nile blue A is an easy-to-handle and low-cost alternative to other fluorescent dyes or the intracellular expression of, for example, green fluorescent protein.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.     

Multiple immunolabeling in histology: a new method using thermo-inactivation of immunoglobulins

We describe a new method for successive immunofluorescence detection of multiple peptides in single paraffin tissue sections. Immunoglobulins were inactivated at 130 degrees C after each labeling step while tissues were protected with a mixture of phosphate-buffered saline and glycerin. The feasibility of the method is demonstrated by double-staining of rat pituitary corticotrophs with anti-ACTH[1-24] and anti-ACTH[17-39] antiserum. The method is applicable to brain tissue, since single neurons of ovine paraventricular hypothalamus could be triple stained with anti-vasopressin, anti-neurophysins I and II, and anti-CRF antiserum. The usual absorption controls and crossreactivity tests, as well as peptide staining, can be performed on the same tissue section. This new labeling procedure should prove useful in endocrinology, clinical pathology, and the neurosciences.     

A group additivity model for analyzing absorption spectra of organic compounds: applications to partial structural analysis and molecular weight determinations of polymers, nucleotides, and peptides.

A new method for the analysis of organic structural groups from absorption spectra is described. The method is based on the integrated intensity of absorption. It requires only micrograms of analyzed compound, which may be fully recovered after analysis from its solution. The method can be used for different kinds of structural and/or kinetic studies. For example, the reaction kinetics and tautomeric equilibria can be easily studied by this method. The method can also be used for the determination of molecular weight and quantitative composition of polymers, nucleotides, and/or peptides. Hydrolysis or derivatization of the studied compounds is not necessary. On the basis of this method, automatic molecular weight, nucleotide, and peptide analyzers can be constructed.     

Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomics

A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3- and d0-acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N-acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R+H1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae.     

A simple method of prediction of visibility of peptides in mass spectrometry with electrospray ionization

A new method for selection of essential peptides applicable for protein detection and quantification analysis in the targeted positive electrospray mass spectrometry has been proposed. It is based on the prediction of the normalized abundance of the mass spectrometric peaks by using a linear regression model. This method has the following a priori restrictions: first selection of peptides must be arranged so that at pH 2.5 the tested peptides must be presented mainly as the 2⁺ and 3⁺ ions. Only peptides containing C-terminal lysine or arginine residues should be considered. The amino acid composition of the peptide, the peptide concentration, the ratio of the polar surface of peptide to common surface and ratio of the polar volume to the common volume are used as independent variables. Among several considered combinations of variables the best linear regression model had a determination coefficient in leave-one-out cross-validation procedure of 0.54. This model confidently discriminated peptides with high response ability and peptides with low response ability, and therefore it is applicable for selection of the most favorable peptides among peptides selected by means of simple criteria. This simple and fast screening method can be successfully applied to reduce the list of observed peptides.     

Distribution of retinol-binding protein in the human digestive tract.

By employing polyclonal antibodies for retinol-binding protein (RBP), its distribution in the human pancreas and digestive tract mucosa was compared with those of transthyretin (TTR) and various peptide hormones. The materials used included surgically removed pancreas, esophagus, stomach, small and large intestines. Paraffin sections were stained by the indirect immunoenzyme method. The results indicate that RBP-containing cells are found in the pancreas and the gastrointestinal mucosa, but most frequently in the gastric antrum and duodenum. In the pancreas, RBP-containing cells are found in the islets and among acinar and ductal epithelial cells, and consistently stain for chromogranin A. RBP-containing cells in the gastrointestinal mucosa showed typical features of endocrine cells and also stained for chromogranin A. The distribution of TTR in these tissue sites resembled that of RBP, but the immunoreactive intensities of both peptides altered independently. Comparison of the distribution of RBP, TTR, and various gastrointestinal peptide hormones revealed that the distribution of RBP coincided with none of the other peptides, although some of the RBP-containing cells stained for most of the peptides examined and vice versa. These results suggest that RBP may be a consistent component of gastrointestinal endocrine cells.     

Sulfo‐NHS‐SS‐biotin derivatization: A versatile tool for MALDI mass analysis of PTMs in lysine‐rich proteins

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo‐NHS‐SS‐biotin derivatization of lysine side chain can help to detect PTMs in lysine‐rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5‐lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.     

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.