Phosphopeptide Enrichment by Covalent Chromatography After Solid Phase Derivatization of Protein Digests on Reversed Phase Supports.

The isolation of the phosphopeptide constituents from phosphoprotein digests is prerequisite to facilitate the mass spectrometric characterization of phosphorylation events. Here, we describe a chemical proteomics approach which combines solid phase derivatization of phosphoprotein digests with phosphopeptide enrichment by covalent chromatography. The use of the solid phase support for derivatization ensures for speed and completeness of reactions.

N-terminal protein characterization by mass spectrometry using combined microscale liquid and solid-phase derivatization.

A sample-preparation method for N-terminal peptide isolation from protein proteolytic digests has been developed. Protein thiols and primary amines were protected by carboxyamidomethylation and acetylation, respectively, followed by trypsinization. The digest was bound to ZipTip(C18) pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate.

N-terminal protein characterization by mass spectrometry after cyanogen bromide cleavage using combined microscale liquid- and solid-phase derivatization.

A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTipC18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate.

C-terminal protein characterization by mass spectrometry: isolation of C-terminal fragments from cyanogen bromide-cleaved protein.

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2'-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives.

Phosphopeptide enrichment by covalent chromatography after derivatization of protein digests immobilized on reversed-phase supports.

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose.

Optimization of the β-elimination/michael addition chemistry on reversed-phase supports for mass spectrometry analysis of O-linked protein modifications.

We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization.

C-terminal protein characterization by mass spectrometry using combined micro scale liquid and solid-phase derivatization.

A sample preparation method for protein C-terminal peptide isolation has been developed. In this strategy, protein carboxylate glycinamidation was preceded by carboxyamidomethylation and optional α- and ϵ-amine acetylation in a one-pot reaction, followed by tryptic digestion of the modified protein. The digest was adsorbed on ZipTip(C18) pipette tips for sequential peptide α- and ϵ-amine acetylation and 1-ethyl-(3-dimethylaminopropyl) carbodiimide-mediated carboxylate condensation with ethylenediamine.

Phosphopeptide characterization by mass spectrometry using reversed-phase supports for solid-phase β-elimination/Michael addition.

We have adapted the Ba(2+) ion-catalyzed concurrent Michael addition reaction to solid-phase derivatization on ZipTip(C18) pipette tips using 2-aminoethanethiol as a nucleophile. This approach provides several advantages over the classical in-solution-based techniques, including ease of operation, completeness of reaction, improved throughput, efficient use of dilute samples, and amenability to automation. Phosphoseryl and phosphothreonyl peptides, as well as phosphoserine peptides with adjoining prolines, were used to optimize the reaction conditions, which proved highly compatible with the integrity of the samples.

IVICAT for the masses: an improved technique for permethylation of peptides.

To determine the levels of post-translational modifications, we needed a quantitative technique that would allow comparison of the amounts of acetylated versus mono-, di-, and tri-methylated lysines in histones. One method, IVICAT, generates trimethyl-amines and could be used, but is technically challenging. We have modified this technique to be used with standard laboratory equipment so that this chemistry is accessible to most proteomics laboratories.

EGF-induced ERK activation promotes CK2-mediated disassociation of alpha-Catenin from beta-Catenin and transactivation of beta-Catenin.

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of beta-catenin and alpha-catenin, thereby abrogating the inhibitory effect of alpha-catenin on beta-catenin transactivation via CK2alpha-dependent phosphorylation of alpha-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2alpha via the ERK2 docking groove and phosphorylates CK2alpha primarily at T360/S362, subsequently enhancing CK2alpha activity toward alpha-catenin phosphorylation.

Malignant paraganglioma of the urinary bladder in a 44-year-old female: clinicopathological and immunohistochemical study of a rare entity and literature review.

Paraganglioma of the urinary bladder is a rare pathologic entity with no definitive histological, immunohistochemical or molecular features to determine its malignant potency. Malignancy is essentially determined by the presence of deep local invasion, invasion of adjacent structures, and lymph node or distant metastases. So far, up to 180 cases of paraganglioma have been reported, with less than 30 being malignant.

Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity.

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation is poorly understood. We have demonstrated that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates beta-catenin at Ser552 in vitro and in vivo. AKT-mediated phosphorylation of beta-catenin causes its disassociation from cell-cell contacts and accumulation in both the cytosol and the nucleus and enhances its interaction with 14-3-3zeta via a binding motif containing Ser552.

Liquid chromatography-electrospray ionization mass spectrometry of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins.

We report the separation of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by microbore reverse-phase high-performance liquid chromatography and their detection by on-line electrospray ionization mass spectrometry. These compounds are the products of the chemical stepwise degradation of polypeptides using 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate. We describe chromatographic conditions for on-column concentration of the analytes and for baseline separation of the isobaric amino acid derivatives of leucine and isoleucine.

Synthesis of the protein-sequencing reagent 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate and characterization of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins.

We report the synthesis and structural characterization of the novel Edman-type protein-sequencing reagent 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate. A panel of thiohydantoins prepared from this reagent were found stable during liquid chromatography-electrospray mass spectrometry and were detectable at the low femtomole sensitivity level. Furthermore, the signal detected for these compounds in the mass spectrometer was linear from the low femtomole to the low picomole range.

Covalent attachment of peptides for high sensitivity solid-phase sequence analysis.

We have developed a method for the high efficiency covalent immobilization of picomole to nanomole quantities of peptides in a form compatible with high sensitivity gas-liquid or solid-phase sequence analysis. Glass fiber filter paper was derivatized with amino-phenyltriethoxysilane and peptides were applied to circular disks cut to 1-cm diameters. Peptides were covalently immobilized on the aminophenyl-glass fiber paper through their terminal alpha-carboxyl groups and amino acid side-chain carboxyl groups by activation with the water-soluble reagent N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.

Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40.

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells.

Covalent immobilization of proteins for high-sensitivity sequence analysis: electroblotting onto chemically activated glass from sodium dodecyl sulfate-polyacrylamide gels.

We report a new method for the preparation of proteins in a form suitable for high-sensitivity N-terminal amino acid sequence analysis. Proteins separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyanate functional groups. The proteins became covalently coupled to the matrix during the electrotransfer process.

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: separation and identification of contact sequences of neighboring proteins after CNBr fragmentation.

Mammalian ribosomal proteins were cross-linked in situ with the primarily cysteine-selective heterobifunctional reagents N-succinimidyl 2-(4-hydroxy-2-maleimidophenylazo)benzoate (reagent A, maximum range approx. 8 A) and N-succinimidyl 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoate (reagent B, maximum range approx. 12 A).

Reaction detector system for the simultaneous monitoring of primary amino groups and sulfhydryl groups in peptides eluted by high-performance liquid chromatography.

A reaction detector system is described which can be used for simultaneous analysis of primary amino groups and sulfhydryl groups in peptides separated by high-performance liquid chromatography. By use of an automatic split valve system, well-defined portions of the effluent are diverted alternately into one of the two reaction detectors, based on the ninhydrin and Ellman assay, respectively. In the latter case the dithioerythritol included in the elution buffers for thiol preservation is eliminated with arsenite.

Location of the sulfhydryl groups involved in disulfide interaction between the neighboring proteins L6 and L29 in mammalian ribosomes. S-cleavage of the cyanylated proteins in polyacrylamide gels after separation by dodecylsulfate gel electrophoresis.

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60-S ribosomal subparticles and are easily cross-linked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex were subjected to S-cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis.

Isolation of the contact regions of the neighboring mammalian ribosomal proteins L6 and L29. Cyanogen bromide cleavage of the disulfide complex after preparative electrophoresis in non-oxidative polyacrylamide gels.

Proteins L6 and L29 in the 60-S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non-oxidizing conditions had to be maintained throughout the preparation.

Identification and characterization of sulfhydryl-containing proteolytic fragments involved in the Ca2+-induced conformational change of beef brain S-100.

The Ca2+-dependent conformational alteration of the brain-specific S-100 protein was studied by reacting the protein with N-ethyl[2,3-14C]maleimide in the absence and presence of Ca2+ and under denaturing conditions. Peptic hydrolysates of the 14C-labeled protein were analyzed and fractionated by high-performance liquid chromatography. Labeled peptide fractions were characterized by high-voltage electrophoresis and TLC.

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes.

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration.