Gas-phase Protein Sequencer Research Articles

Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by western blotting.

A technique has been developed for efficient deblocking and subsequent microsequencing of N-terminally blocked proteins immobilized on a polyvinylidene difluoride (PVDF) membrane at the picomole levels. In this technique, proteins were first separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane by Western blotting. The electroblotted proteins with N-terminal acetylserine or acetylthreonine could be deblocked on-membrane by treatment with trifluoroacetic acid vapor and sequenced by a gas-phase protein sequencer. Similarly, N-formylated proteins could be deblocked on-membrane in HC1 solution and then directly sequenced from the N-terminal amino acid. Proteins with N-terminal pyroglutamic acid were enzymatically deblocked by in situ pyroglutamyl peptidase digestion, and N-acetylated proteins were also enzymatically deblocked with acylamino acid-releasing enzyme (AARE) after on-membrane digestion with trypsin to generate the N-terminal peptide fragment. This tryptic digestion was required since AARE can remove the acetylamino acid only from a short peptide. Based on these four deblocking methods, we present a strategy for sequential deblocking and subsequent N-terminal sequence analysis of N-blocked protein immobilized on PVDF membrane.

Endothelin 1 hydrolysis by rat kidney membranes

Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC, Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser 5Leu 6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu 17Asp 18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by μs [(1992) Eur. J. Biochem. 204, 547–552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of K m =71.5 μM and k cal=1.49 s −1, giving a ratio of k cal K m =2.08 × 10 4 s −1·M −1. The K m value was much higher and the k cal/K m value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.

Sensitization of gas-phase protein sequencer using fluorescein isothiocyanate FITC

Sensitization of gas-phase protein sequencer using fluorescein isothiocyanate FITC

Correlation between carbohydrate-binding specificity and amino acid sequence of carbohydrate-binding regions of Cytisus-type anti-H(O) lectins

A carbohydrate-binding peptide of the di- N-acetylchitoblose-binding Cytisus sessilifolius anti-H(O) lectin I (CSA-I) was isolated from the endoproteinase Asp-N digest of CSA-I by affinity chromatography on a column of N-acetyl- d-glucosamine oligomer-Sepharose (GIcNAc oligomer-Sepharose). The amino acid sequence of the carbohydrate-binding peptide of CSA-I was determined to be DTYFGKTYNPW using a gas-phase protein sequencer. This sequence corresponds to the sequence from Asp-129 to Trp-139 based on the primary structure of CSA-I, and shows a high degree of homology to those of the putative carbohydrate-binding peptide of the Laburnum alpinum lectin I (LAA-I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA-II) (DSYFGKTYNPW). The binding of these three anti-H(O) lectins is known to be inhibited by di- N-acetylchitobiose but not by l-fucose. These results strongly suggest that there is a good correlation between the carbohydrate-binding specificity and the amino acid sequence of the carbohydrate-binding regions of di- N-acetylchitobiose-binding lectins.

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.

Sequence Analysis of Cysteine and Cystine Containing Peptides and Proteins by a Gas-Phase Sequencer with Isocratic Separation of PTH-Amino Acids

Abstract A facile method for routine sequence analysis of cysteine and cystine containing peptides or proteins is described. An automated gas-phase protein sequencer, Shimadzu PSQ-1 System, consisting of an Edman-reaction unit and an isocratic separation mode for the phenylthiohydantoin amino acid analysis with equivalent sensitivity and resolution to that of the current gradient mode, allows easy determination of the intact second half cystine, of which phenylthiohydantoin derivatives appear as a dithiothreitol adduct of dehydroalanine, cysteine and S- pyridylethylated cysteine. The optimal sequencing protocol for in situ pyridylethylation was established. This protocol gives an N-terminal amino acid sequence with half cystines, which are automatically converted to the S-pyridylcthyl derivative in the sequencer. Sequence analyses both intact and S-pyridyiethylated cysteine can identify a disulfide form if only one disulfide linkage exists in the sample. Sequence analysis of synthetic peptides containing ...

A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides.

A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.

Structural homology between semidwarfism-related proteins and glutelin seed protein in rice (Oryza sativa L.)

Two semidwarfism-related proteins, SRP-1 and SRP-2, were detected as major spots in a long-culm rice cultivar, Norin 29 and its semidwarf near-isogenic line, SC-TN1, respectively, by two-dimensional gel electrophoresis (2D-PAGE). The testcross showed that SRP-1 and SRP-2 are controlled by codominant alleles, Srp-1 and Srp-2, respectively, at a single locus Srp. This locus was considered to be closely linked with the semidwarfing gene locus sd-1. SRP-1 and SRP-2 were separated by 2D-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and sequenced by a gas-phase protein sequencer. The N-terminal amino acid sequences, however, could not be determined due to the blockage of the N-terminals of these proteins. After removal of the N-terminal residue with pyroglutamyl peptidase given to the membrane, the amino acid sequence in the N-terminal region was determined. The N-terminal and internal amino acid sequences of SRP-1 and SRP-2 were highly homologous with those of the glutelin α-subunits of seed endosperm storage protein, which were deduced by the cDNA sequences. In the seed endosperms of Norin 29 and SC-TN1, a total of eight glutelin α-subunits was identified by 2D-PAGE. The amino acid sequences in the N-terminal and internal regions of these proteins were determined. This experiment confirmed that SRP-1 and SRP-2 are almost identical in structure with the glutelin α5a- and α5b-subunits, respectively, which were identified in several organs such as endosperms, embryos, and leaves, unlike the other glutelin α-subunits.

Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites.

The present studies examine the effects of in vivo and in situ progesterone treatment in the regulation of site-specific phosphorylation of the chicken oviduct progesterone receptor (PR). By gas-phase protein sequencing we have identified three hormonally regulated phosphorylation sites: Ser-211, Ser-260, and Ser-530. We determined phosphorylation stoichiometries by analyzing the amounts of phosphorylated and dephosphorylated serine at each site. Stoichiometries of sites 211 and 260 were about 20% under basal conditions and increased 1.5-2-fold by in situ progesterone treatment. Site 530 was virtually absent under basal conditions and induced to greater than 33% by in situ progesterone treatment. We tested several protein kinases for phosphorylation of the PR in vitro on these sites or peptides containing these sites. We found that the catalytic subunit of cAMP-dependent protein kinase mimicked the in vivo, hormone-induced altered mobility of PRs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the in vivo and in vitro alterations were reversed by alkaline phosphatase. Finally, we showed that cAMP-dependent protein kinase phosphorylated Ser-528.

Amino Acid Sequence Analysis by Gas-Phase Protein Sequencer

A gas-phase protein sequencer which has been developed for last several years allows the determination of N-terminal amino acid sequence of 2030 residues from picomole amounts of proteins. Recently, using the gas-phase sequencer, a new technique for protein microsequence analysis has been established. In this method, picomole amounts of proteins are first separated by gel electrophoresis, then electroblotted from the gel onto sequencer stable membrane supports, and finally sequenced directly with the gas-phase sequencer. This method frequently permits sequence determination of proteins that could not be easily purified by conventional column chromatographies. In this review, the new instrument and technique for protein microsequence analysis are described.

Gas-phase protein sequencing: gas flow control by positive displacement

A modification of an automatic gas-phase protein/peptide sequencing apparatus is described; this eliminates the effect of the sample on cell gas flow rates. Consistent sequencing chemistry is achieved, yielding data from material that is intractable using standard equipment.

Indirect fluorescence detection of native amino acids in capillary zone electrophoresis.

Amino acids are but one of several important classes of small chemical compounds in biological chemistry that have an inherent lack of analytically useful physical properties. Amino acids, peptides, fatty acids, sugars, many mono-, di-, and tricarboxylic acids, and phosphorylated intermediates in glycolysis and metabolism show little, if any, UV or visible absorption, fluorescence, or electrochemical activity. As the emphasis of biochemical research shifts to smaller samples where, for example, picomolar quantities of amino acids are analyzed in gas phase protein sequencing or in microliter samples of the extracellular fluid of the mammalian brain, the analytical problem becomes even more challenging due to the small volume of sample available for analysis. In this work, laser-induced fluorescence spectroscopy is performed on-column to detect the bands separated with capillary zone electrophoresis (CZE). CZE is an instrumental form of zone electrophoresis where chemical species are separated purely on the basis of their electrophoretic mobility, since no supporting gel is utilized. Both anions and cations can be separated in the same run because of the large electroosmotic flow generated in small diameter capillaries. This technique has already been used successfully in the rapid, efficient separation of dansyl-amino acids.

Identification of a polypeptide secreted by human breast cancer cells (MCF-7) as the human estrogen-responsive gene (pS2) product

Human epidermal growth factor-like immunoreactive factor (designated as EGF-LI) synthesized and secreted by human breast cancer cells, strain MCF-7, was isolated in pure form. Thirty-seven micrograms of EGF-LI was purified by anion-exchange, gel permeation, and reverse-phase high-performance liquid chromatography from 2 liters of serum-free medium conditioned by the cells. The sequence of the first 36 amino acids from the N-terminus was determined with a gas-phase protein sequencer. Computer-assisted screening revealed, quite unexpectedly, this sequence to be completely identical to that of the translational product encoded by pS2, the human estrogen-responsive gene, over the region extending from residue 25 to 60 (Jakowlew, S. B. et al. (1984) Nucleic Acids Res., 12, 2861-2878).

Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product.

The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method. To confirm the deduced amino acid sequence of the tail sheath protein (gp18) that is encoded by gene 18, gp18 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography. Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified. Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N- and C-terminal sequences. Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined.

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain Comparison of predicted amino acid sequences from G-protein α-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human G i2α gene and rat G i2α cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat G i1α and G oα cDNAs, respectively.

Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

Primary structure of striped dolphin renal metallothionein II.

The complete amino acid sequence of metallothionein II isolated from kidneys of the striped dolphin, Stenella coeruleoalba, is reported. The sequence was established by a gas phase protein sequencer using the fragments obtained by cyanogen bromide cleavage and protease digestions of the S-carboxamidomethylated protein. The N-terminal was identified as acetylmethionine by 1H-NMR and MS using the N-terminal dipeptide obtained by formic acid hydrolysis. The C-terminal amino acid was assigned as alanine using both carboxypeptidase and trypsin digests. Remarkable sequence homology was observed between dolphin and mammalian metallothionein II.

Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.

Purification and Partial Amino Acid Sequence of Human Platelet Membrane Glycoproteins lib and IIIa

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes by immunoaffinity chromatography using a monoclonal antibody specific for GP IIb-IIIa. GP IIb and IIIa were further separated in the presence of sodium dodecyl sulfate (SDS) by gel filtration high-performance liquid chromatography (HPLC). Two cycles of this procedure yielded almost complete separation of homogeneous preparations of GP IIb and IIIa. Each protein was then digested with lysyl endopeptidase (Achromobacter protease I), which cleaves at the carboxyl side of lysine residues, and the resulting oligopeptides from GP IIb and IIIa were fractionated with HPLC using a C18 reverse-phase column. Comparison of the elution profiles showed no obvious homology between the two proteins. Amino acid sequences of selected oligopeptides from each glycoprotein were determined using a gas-phase protein sequencer. Sixty amino acid residues (26 residues for IIb and 34 residues for IIIa) were identified.