Solid-phase Sequence Analysis Research Articles

Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides

Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.

A Selective Precipitation Purification Procedure for Multiple Phosphoseryl-Containing Peptides and Methods for Their Identification

Multiple phosphoseryl-containing sequences of proteins stabilize amorphous calcium phosphate and have been implicated in the regulation of biomineralization, protein structure, and enzyme activity. To facilitate studies on the identification and characterization of multiple phosphoseryl-containing sequences of proteins we have developed a simple and efficient purification procedure involving precipitation of Ca2+/ethanol-induced aggregates of the multiple phosphoseryl-containing peptides from enzymic digests. The multiple phosphoseryl-containing peptides of a tryptic digest of casein were selectively precipitated using Ca2+ (20 mol/mol protein) and 50% (v/v) ethanol at pH 3.5, 4.6, and 8.0. The individual peptides of the precipitates were purified using anion-exchange fast-performance liquid chromatography and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapor-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalyzed β-elimination in the presence of ethanethiol. The modified peptides were sequenced using an Applied Biosystems Inc. automated protein sequencer fitted with a membrane cartridge. Only peptides containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)- were precipitated by Ca2+/ethanol at pH 3.5. The pH 4.6 precipitate contained all the cluster peptides plus two diphosphorylated peptides containing -Ser(P)-Glu-Ser(P)- and -Ser(P)-Thr-Ser(P)-. At pH 8.0, a monophosphorylated peptide containing -Ser(P)-Glu-Glu- was also present in the precipitate with the diphosphorylated and cluster peptides. The recoveries of the peptides in the pH 8.0 selective precipitate ranged from 83 to 95% of that present in the hydrolysate. All of the phosphopeptides in the casein tryptic hydrolysate were selectively precipitated at pH 8.0 by Ca2+/ethanol, except the two monophosphorylated peptides containing -Ser(P)-Ala-Glu- and -Ser(P)-Thr-Glu-, suggesting that, for a monophosphorylated peptide, the minimal motif for Ca2+/ethanol-induced precipitation is -Ser(P)-Glu-Glu-.

Derivatization of Polyvinylidene Difluoride Membranes for the Solid-Phase Sequence Analysis of a Phosphorylated Sea Urchin Embryo Histone H1 Peptide

We describe a simple and effective chemical protocol for attaching polyallylamine to polyvinylidene difluoride (PVDF) membranes. The underivatized PVDF discs were first etched in 0.5 M alcoholic KOH for 1.5 mm and subsequently reacted with polyallylamine of high molecular weight under alkaline conditions. The covalently attached amino groups were reacted with 1,4-phenylene diisothiocyanate (DITC), thus converting the amino-modified PVDF discs to DITC-functionalized discs. The substitution level of the DITC-functionalized discs was found to be lower than that of the equivalent discs available commercially. Lysine-containing polypeptides were immobilized on these DITC-membrane discs and then subjected to either solid-phase manual Edman degradation or automated gas-phase sequencing. The method was found to be well suited for the sequencing of 32P-labeled phosphopeptides by employing the manual solid-phase technique.

Linkage mapping of the human thromboxane A2 receptor (TBXA2R) to chromosome 19p13.3 using transcribed 3' untranslated DNA sequence polymorphisms.

The actions of thromboxane A2 as a prostaglandin mediator are dependent on its recently cloned and sequenced receptor. The identification and characterization of DNA polymorphisms in the thromboxane A2 receptor (TBXA2R) will advance the study of this gene as a candidate in a number of medical disorders. We amplified a 573-nucleotide fragment of the transcribed 3' untranslated region of the TBXA2R gene using the polymerase chain reaction (PCR) and the published cDNA sequence. This region was found to contain two sequence polymorphisms within an Alu. These DNA polymorphisms were demonstrated using an efficient method of direct solid-phase sequence analysis. Three of the four expected alleles were observed in the CEPH families. TBXA2R was localized to chromosome 19 by PCR amplification in a series of monochoromosomal human/rodent somatic cell hybrids. Linkage mapping places TBXA2R closest to the anonymous marker D19S120, with a maximal LOD = 19.55, at a theta = 0.05 in the CEPH panel of DNAs. Multipoint linkage analysis places TBXA2R between the markers D19S120 and PMS207 on the telomeric end of chromosome 19p13.3.

Index to subjects

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gasphase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify α and ϵ amines. The protein is then overlayed with a solution of 1,4-phenylene diisothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.

Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid-phase sequence analysis

A method for the determination of the sites of tyrosine phosphorylation in proteins and peptides at the low picomole level for “cold” phosphopeptides and at the subpicomole level for 32P-labeled phosphopeptides is presented. The procedure is based on solid-phase sequence analysis of phosphopeptides immobilized on carrier discs and the “on-line” detection by reverse-phase high-performance liquid chromatography of the phenylthiohydantoin derivative of phosphotyrosine. The procedure is sensitive and automated and allows the identification of phosphotyrosine derivatives in the same operation as the detection of the derivatives of the other common amino acids. Essentially quantitative extraction of the phosphotyrosine derivatives from the sequencer makes this method ideally suited for the quantitative assessment of protein-tyrosine kinase and protein phosphatase activities and for the determination of their respective recognition sequences.

Three regions of erythrocyte band 3 protein are phosphorylated on tyrosines: characterization of the phosphorylation sites by solid phase sequencing combined with capillary electrophoresis

The major part of band 3 phosphorylation was recently shown to concern the first tryptic peptide of the protein (Yannoukakos et al. (1991) Biochim. Biophys. Acta 1061, 253–266). Tyrosine 8 is the prevalent site of phosphorylation, but other phosphorylated regions were found which could not be analyzed with certainty. Direct characterization of the phosphorylated residues in all these phosphorylated fragments was made possible due to recent advances in protein chemistry techniques, such as solid phase sequence analysis and capillary electrophoresis. The present report establishes that band 3 phosphorylation occurs predominantly on tyrosines: besides tyrosine 8 already known in the N-terminal region, two other tyrosines are demonstrated to be targets for the tyrosine kinase, tyrosine 359 and tyrosine 904. These residues lie in regions of band 3 exposed to the cytoplasm, the junction of the cytoplasmic and the membrane-spanning domains, and the C-terminal end of the protein which is also cytosolic, respectively.

Functionalized membrane supports for covalent protein microsequence analysis

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to > 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine β-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of β-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 μg of bovine α-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (> 10 residues).

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 aminophenyltriethoxysilane 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 α-carboxyl groups and amino acid side-chain carboxyl groups by activation with the water-soluble reagent N-ethyl- N′-(3-dimethylaminopropyl)carbodiimide. Disks containing the covalently attached peptide were directly inserted into the cartridge of an automated sequenator for sequence analysis by the Edman degradation. Peptides prepared in this way could be routinely sequenced through to and including the C-terminal amino acid residue, at extraordinarily low backgrounds. The covalent immobilization of peptide fragments allowed far more flexibility in sequencing conditions, including the use of polar extraction solvents to increase the yield of phenylthiohydantoin (PTH)-His and PTH-Arg and the use of alternative Edman-type sequencing reagents with enhanced detectability, such as the chromophoric compound 4-( N,N′-dimethylamino)azobenzene-4′-isothiocyanate. The potential of this high efficiency immobilization method for contributing to the development of sequencing chemistries with enhanced sensitivity is discussed.

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gasphase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify α and ϵ amines. The protein is then overlayed with a solution of 1,4-phenylene diisothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.

New Methods for the Solid-Phase Sequence Analysis of Nucleic Acid Fragments Using the Sanger Dideoxy Procedure

Abstract 3′-Terminal tailing of a given DNA fragment with 5-bromodeoxyuridine-5 triphosphate or biotinylated deoxyuridine-5′-triphosphate and deoxynucleotidyl terminal transferase allows its immobilization to an anti-bromo-deoxyuridine-antibody column or to a streptavidin column. The immobilized DNA could be subjected to enzymatic sequencing following the usual protocol. The dideoxy-nucleotide-terminated fragments were eluted with buffer containing formamide / dye mixture and directly applied to gel electrophoresis, allowing reading of ca. 600 bases. Several “sequencing cycles” could be performed with the same DNA column. Semi-mechanization of the process is described.

Solid-phase protein sequence analysis

The solutions to many protein analytical problems require flexible chemistries at a high sensitivity. Solid-phase sequence analysis is one way to eliminate limitations inherent in current methods.

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking.

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.

Identity of the heme-not-containing protein in bovine heart cytochrome c1 preparation with the protein mediating c1-c complex formation--a protein with high glutamic acid content.

A heme-not-containing protein was isolated from cytochrome c1 preparation by gel filtration after carboxymethylation and citraconylation. The amino acid sequence of this protein was determined by the analysis of tryptic and chymotryptic peptides as well as by solid-phase sequence analysis. It consisted of 78 amino acid residues and the molecular weight was calculated to be 9,175. This protein contained a high proportion of glutamic acid and glutamine (27% of the total residues) but no methionine, isoleucine, tyrosine, and tryptophan. The most notable feature was an acidic cluster of 8 consecutive glutamic acid residues near the amino(N)-terminus). The secondary structure was predicted to have a high proportion of helical content. The amino acid composition and N-terminal sequence of a protein independently prepared from bovine heart mitochondria, which is essential to the formation of the cytochrome c1-c complex, suggested that this colorless factor and the present heme-not-containing protein are identical. Evidence shows that another protein, called the non-heme protein, isolated from "two-band" cytochrome c1 preparation is also the same protein as that presented in this paper.

Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels. An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda.

An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.