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At least 55 records · Page 3Linked to original sources
PubMed1994

N-terminal protein sequence analysis of the rabbit erythrocyte lactate transporter suggests identity with the cloned monocarboxylate transport protein MCT1.

An improved purification for the rabbit erythrocyte lactate transporter, using aminoethyl-Sepharose chromatography, is described. The process of purification of the 40-50 kDa transporter, labelled with 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), was followed by Western blotting with anti-DIDS antibodies [Poole, R. C. and Halestrap, A. P. (1992) Biochem. J. 283, 855-862]. Fractions highly-enriched in transporter were further purified by SDS/PAGE and the 40-50 kDa DIDS-labelled polypeptide was subjected to N-terminal protein sequencing. This analysis identified the first 16 amino acids of the protein. With the exception of one conservative substitution, this protein sequence is identical to the N-terminal protein sequence predicted from a cDNA isolated from Chinese hamster ovary cells that encode a monocarboxylate transporter, MCT1 [Kim Garcia, C., Goldstein, J. L., Pathak, R. K., Anderson, R. G. W. and Brown, M. S. (1994) Cell 76, 865-873]. This observation, along with similarities in functional properties, leads us to conclude that lactate transport in rabbit erythrocytes is mediated by the MCT1 monocarboxylate transporter isoform.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Characterization of ribosomal frameshift events by protein sequence analysis.

In cell-free protein synthesis studies with RNA from phage MS2 as template, normal Escherichia coli tRNASer3 promotes two base translocation at GCA alanine codons with a resultant shift of ribosomes to the minus one reading frame. Similarly, normal tRNAThr3 promotes two base translocation at CCG proline codons. These conclusions were reached by amino acid sequencing of tryptic peptides or cyanogen bromide fragments that contained the reading frame shift site. It is proposed that these frameshift events occur by a two-base pair interaction between the anticodons of these exceptional tRNAs and the noncognate codons.

Alanine↗
PubMed1992

Alkylation of cysteine with acrylamide for protein sequence analysis.

Alkylation of cysteine in proteins with acrylamide under mildly alkaline conditions yields a thioether derivative, Cys-S-beta-propionamide (Cys-S-Pam), which is stable during automated Edman degradation. Its phenylthiohydantoin derivative, PTH-Cys-S-Pam, is easily separated from other PTH-amino acids by HPLC and is thus useful for cysteine identification during protein sequencing. PTH-Cys-S-Pam was first noticed during sequencing polypeptides blotted onto polyvinylidene difluoride membranes from polyacrylamide gels, in which cysteine had reacted with residual unpolymerized acrylamide. Cysteine in proteins is easily alkylated by reaction of proteins in aqueous solution with acrylamide. Methods are also presented for alkylation of cysteine in proteins adsorbed on fiberglass disks in the reaction cartridge of a protein sequencer. Finally, PTH-Cys-S-Pam was synthesized chemically. The synthetic compound is unstable in neutral solution, but can be stabilized by acidification. It has the same HPLC retention time as the product formed from cysteine when sequencing proteins alkylated with acrylamide.

Acrylamide↗
PubMed2000

Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis.

The high resolving power of two-dimensional polyacrylamide gel electrophoresis 2D-PAGE and its full analytical and preparative potential have been described with special emphasis on reproducibility and standardization of protein spot patterns, enhanced protein detection sensitivity, and computer analysis database development. New methodologies for peptide mass fingerprinting, peptide, sequence, and fragmentation tagging have been highlighted. Major challenges associated with 2D-PAGE/mass spectrometric protein sequencing were outlined which need to be addressed in the future, including sample enrichment, use of alternative gel matrices, improvements in separation systems interfaced directly to the mass spectrometer, and design of high-sensitivity instruments with very high mass ranges. It is hoped that comparative studies to identify, quantitate, and characterize proteins differentially expressed in normal versus diseased cells would give insight into mechanisms of pathogenesis and allow the development of a way to control both the etiology and the course of diseases.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

The elucidation of protein function by sequence motif analysis.

Protein sequence motifs are acquiring increasing prominence in the area of sequence analysis. This review describes the current methods of their construction and their use in the determination of protein function, and offers guidelines on interpreting data obtained. An appendix is attached which refers to 200 motifs of various kinds.

Amino Acid Sequence↗
PubMed1992

Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes.

A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.

Collodion↗
PubMed2000

PROTEAN. Protein sequence analysis and prediction.

The archaeal, bacterial, andeukaryotic genome projects have overwhelmed our ability to experimentally elucidate the function of each novel gene and gene product. To a certain extent, protein functional assignments can be derived via sequence similarity measures and direct primary sequence analysis using methods to predict hydropathy, secondary structure, amphilicity, and antigenicity. Function can also be inferred on the basis of sequence motifs, such as phosphorylation and lipid binding signatures. These methods, provided in DNASTAR's PROTEAN module, can be used to putatively assign roles for novel proteins from the genome explosion as well as clarify function for better known proteins.

Databases, Factual↗
PubMed1990

A method for the unambiguous identification of tryptophan in automated protein sequence analysis.

Some widely used standard protocols for the separation of phenylthiohydantoin amino acid derivatives by reverse-phase gradient HPLC do not provide separation of the phenylthiohydantoin derivative of tryptophan (PTH-Trp) from diphenylurea (DPU), a by-product generated during Edman degradation of proteins in variable amounts. Furthermore, PTH-Trp is usually recovered in low yield under typical experimental conditions used with automated sequencing equipment. These factors may compromise the unambiguous assignment of tryptophan residues in automated protein sequence analysis, especially when sequencing is performed at high sensitivity. We devised a reverse-phase HPLC method which allows the separation of DPU and PTH-Trp and therefore the correct assignment of PTH-Trp. The method is based on a modification of the HPLC gradient used to elute and separate all PTH amino acids of interest. With Applied Biosystems Model 477A protein sequencers with on-line PTH amino acid identification, the correct assignment of tryptophan was consistent and reproducible even when sequencing at very high sensitivity (5 pmol).

Amino Acid Sequence↗
PubMed1994

SEQSEE: a comprehensive program suite for protein sequence analysis.

SEQSEE (SEQuence SEEker) is a multi-purpose, menu-driven suite of programs designed to provide a fully integrated, state-of-the-art package for the analysis and display of protein sequences and protein databases. It is currently configured to run on most UNIX-based machines including Sun, SGI and NeXT workstations with conversion to other architectures (e.g. Vax or Cray) being a relatively simple task. SEQSEE is capable of performing nearly all of the analytical and comparative tasks found in most comprehensive commercially available software packages. These include sequence/database searching, sequence retrieval, sequence entry and editing, statistical sequence analysis, multiple sequence alignment, flexible pattern matching, and secondary structure prediction. SEQSEE also integrates a number of unique databases which allow it to perform many additional functions such as structure-based sequence alignments and homology-based secondary structure prediction. Additional enhancements to many previously published algorithms have substantially improved the performance of SEQSEE over that found for most other commercial products. The source code, the documentation and all of the required databases for SEQSEE are freely available and may be obtained by anonymous ftp.

Algorithms↗
PubMed1998

Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis.

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.

Amino Acid Sequence↗
PubMed2001

Use of nonspecific cleavage products for protein sequence analysis as shown on calcyclin isolated from human granulocytes.

In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6-calcyclin whose expression in granulocytes has not been described so far.

Amino Acid Sequence↗
PubMed1993

Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis.

Asparaginyl endopeptidase was highly purified from mature seeds of the jack bean (Canavalia ensiformis). The final enzyme preparation showed a single peak in high-performance liquid chromatography on a reversed-phase column, and the material in the peak gave the following NH2-terminal amino acid sequence on Edman degradation for 25 cycles: H-Glu-Val-Gly-Thr-Arg-Trp-Ala-Val-Leu-Val-Ala-Gly-Ser-Asn-Gly-Tyr-Gly-Asn-Tyr- Arg-His-Gln-Ala-Asp-Val-. Behavior of the enzyme toward various protease inhibitors suggested that it belongs to a family of cysteine proteases. Strict substrate specificity of this enzyme was verified by the use of 14 polypeptide substrates including those derived from proteins. Almost all the peptide bonds on the carboxyl side of Asn residues were susceptible to the enzyme. The exceptions were cases where the residue was at the NH2 terminus or the second position from the NH2 terminus of substrates and where it was N-glycosylated Asn. Peptide bonds on the carboxyl side of any other amino acid residues were not cleaved. These properties promise the high utility of this novel endopeptidase in protein sequence analysis. Identity of jack bean asparaginyl endopeptidase with a processing enzyme responsible for maturation of concanavalin A from its precursor is also discussed.

Amino Acid Sequence↗
PubMed1994

Applications of statistical criteria in protein sequence analysis: case study of yeast RNA polymerase II subunits.

We have recently proposed statistical techniques to identify unusual protein sequence features. Extensive mapping of these features to particular groups of proteins may afford new ways of protein classification. Here we present a case study of such analysis by discussing special features of the amino acid sequences of yeast RNA polymerase II, the first eukaryotic RNA polymerase for which all subunits have been sequenced. Specific new suggestions derived from this analysis include: (i) based on unusual charge configurations in some of the sequences, electrostatic forces may play a significant role in subunit interactions; (ii) RPB4, on account of similar charge distribution, may well be grouped together with RNA polymerase II transcription initiation factors.

Amino Acid Sequence↗
PubMed1998

Macaque MDC family of proteins: sequence analysis, tissue distribution and processing in the male reproductive tract.

A large number of sequence-related, cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues from a variety of species. Previous studies in the macaque (Macaca fascicularis) have led to the cDNA cloning and sequence analysis of a number of MDC proteins which are abundantly expressed in the male reproductive tract. We now describe the distribution of seven of these macaque MDC transcripts in a range of different tissues. This description includes a novel macaque testis-derived MDC, tMDC III, whose full-length sequence is reported for the first time. In addition, polyclonal antisera have been used to localize a number of these MDC proteins to spermatogenic cells in testis sections, and to demonstrate their processing on the sperm surface during epididymal transit.

ADAM Proteins↗
PubMed1983

Protein sequence analysis: automated microsequencing.

The automated microsequencing of proteins can now be carried out at the 5- to 10-picomoles (submicrogram) level on polypeptides obtained directly from one- and two-dimensional gel electrophoresis. The techniques are applicable to polypeptides ranging in size from small peptides (less than 10 residues) to large proteins (more than 1000 residues).

Amino Acid Sequence↗
PubMed2001

Chemical C-terminal protein sequence analysis: improved sensitivity, length of degradation, proline passage, and combination with edman degradation.

Use of a C-terminal sequencer with modified solvents, reagent concentrations, chromatographic parameters, temperatures, and reaction cartridge geometry yields four sets of improvements in chemical degradations. They are increased sensitivity, longer runs, passage of Pro residues, and practical combination with N-terminal degradation. Over 200 proteins and protein fragments with sizes between 20 and 600 residues were analyzed. C-terminal sequences could be interpreted for more than 10 residues at high picomole sample levels, while the 10-pmol level gave 4-5 residues. The average initial yield was 15% but up to 30% could be achieved. The improved performance allowed combination of C- and N-terminal degradations from the same sample application. After initial Edman degradation, the sample is moved to the C-terminal instrument for continued sequencing. Proteins available in limited amount are thereby efficiently analyzed. Lys, modified from the N-terminal degradation, may be detected as the alkylated thiohydantoin-phenylthiocarbamyl-Lys derivative in the C-terminal degradation. Notably, C-terminal sequence analysis could be proceeded through Pro residues which unexpectedly were no absolute hindrance. The improved technique provides characterization of truncation patterns and microheterogeneities in proteins down to the 10-pmol level and is a useful approach for analysis of N-terminally blocked polypeptides.

Mass Spectrometry↗