共查询到20条相似文献,搜索用时 15 毫秒
1.
Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides 总被引:1,自引:0,他引:1
Huang RH Wang DC 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,803(1):167-172
The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2. 相似文献
2.
The primary structure of rat liver ribosomal protein L39 总被引:4,自引:0,他引:4
The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1. 相似文献
3.
The primary structure of spinach acyl carrier protein 总被引:7,自引:0,他引:7
Acyl carrier protein (ACP) from spinach leaves has been purified to homogeneity by high-performance liquid chromatography with an anion-exchange column. The amino acid sequence of one major ACP in spinach leaves, ACP-I, has been determined by automated Edman degradation. It consists of the following 82 amino acids: (sequence in text). Sequencing of the intact polypeptide provided data for the first 57 residues. Cleavage of the succinylated ACP with CNBr at Met-46, followed by sequencing of the fragment mixture, provided data for the final 36 residues. The C-terminal alanine was confirmed by carboxypeptidase Y digestion. The spinach ACP has 40, 70, and 25% homology with Escherichia coli, barley, and rabbit ACPs, respectively. The results not only provide the first complete sequence of a plant ACP, but also provide insight into the structural and evolutionary relationships among plant, animal, and bacterial ACPs. 相似文献
4.
Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects. 相似文献
5.
Mating factor is a peptide excreted into the culture fluid by alpha-mating type cells of Saccharomyces cerevisiae X-2180 1B. The purification of the mating factor was carried out by ion exchange chromatography on phosphocellulose and Amberlite IRC 50 columns, followed by gel filtration on a Sephadex LH 20 column. The factor thus prepared was a peptide composed of Lys1, His1, Trp2, Gln2, Pro2, Gly1, Met1, Leu2 and Tyr1, and was able to induce morphological changes on alpha-mating type cells at a concentration of 5 pg/ml. The amino acid sequence of the mating factor was determined by the manual Edman degradation method using intact mating factor and its thermolytic peptides. The C-terminal amino acid residue was determined by digesting the factor with carboxypeptidase A. The complete amino acid sequence of the mating factor was established to be as follows: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. 相似文献
6.
The complete amino acid sequence of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The protein consists of a single chain of 119 amino acids cross-linked by seven disulfide bridges and has a formula weight of 13,578. The main fragmentation of the peptide chain was accomplished with a staphylococcal protease specific for glutamoyl bonds. A cyanogen bromide fragment and tryptic peptides were used to align the five major staphylococcal protease peptides. The sequence was determined by Edman degradation using the direct phenylthiohydantoin method and with carboxypeptidase A. Notexin is shown to be homologous to both porcine pancreatic phospholipase A and a phospholipase A from the venom of Naja melanoleuca. 相似文献
7.
G Hortin A C Chan K F Fok A W Strauss J P Atkinson 《The Journal of biological chemistry》1986,261(19):9065-9069
The predominant form of the fourth component of complement in human and murine plasma (C4p) has an Mr approximately 5,000 less than C4 secreted by hepatocytes or macrophages (C4s). Here we demonstrate that the difference in Mr results from excision of a peptide from the COOH terminus of the alpha-chain of C4s. The site of truncation of the alpha-chain of C4 in plasma was established by sequence analysis of the COOH-terminal cyanogen bromide fragment isolated by high performance liquid chromatography. Sequential Edman degradation, digestion with carboxypeptidase Y, analysis of amino acid composition, and partial sequence analysis of an overlapping tryptic peptide established the sequence of this peptide to be Glu-Ala-Asn-Glu-Asp-Tyr-Glu-Asp-Tyr-Glu-Tyr-Asp-Glu-Leu-Pro-Ala. Comparison of our results with reported sequences establishes the COOH-terminal alanine to be residue 748 in the alpha-chain. This identifies the site at which C4s is cleaved by an extracellular protease and suggests that the protease has an elastase-like activity. 相似文献
8.
The 11 subunits of beef heart bc1 complex can be separated either by a new SDS-PAGE system or by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence of the smallest subunit was determined by complete solid-phase Edman degradation and was confirmed by sequencing the N-terminal part and the C-terminal tryptic fragment by liquid-phase Edman degradation. The protein consists of 56 amino acid residues; the Mr was calculated to be 6363. The protein ('ISP binding factor') might be entangled in the reassembly of the iron-sulfur protein with the bc1 subcomplex. 相似文献
9.
Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. 总被引:28,自引:0,他引:28
B Agerberth J Y Lee T Bergman M Carlquist H G Boman V Mutt H J?rnvall 《European journal of biochemistry》1991,202(3):849-854
We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin-P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria. 相似文献
10.
Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli 总被引:42,自引:0,他引:42
S Aimoto T Takao Y Shimonishi S Hara T Takeda Y Takeda T Miwatani 《European journal of biochemistry》1982,129(2):257-263
A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue. 相似文献
11.
A rabbit antiserum was raised against a synthetic peptide corresponding to residues 403 to 417 of human chromogranin B. This peptide was chosen to match the potential C-terminal end of a putative proteolytic fragment of the protein located between dibasic doublets in positions 366-367 and in positions 418-419 of the precursor. A radioimmunoassay based on this antiserum was developed and used to detect the protein or a fragment thereof in a pheochromocytoma tumor extract. One fragment was purified to homogeneity by successive reverse-phase HPLC chromatographies. The N-terminal sequence established by automated Edman degradation, was N-Y-P-S-L-E-L-D-K-M-A-H-G-Y-G-E-E-S-E-E-E-R corresponding to the 368-389 sequence of human chromogranin B. Taking into account the specificity of the antiserum used for peptide identification and alignment with the precursor sequence, we deduced that the purified peptide was chromogranin B (368-417) and represented a new peptide generated by limited proteolysis of chromogranin B. Combining electrospray mass-spectrometry and enzymatic dephosphorylation, we demonstrated that this peptide was phosphorylated. 相似文献
12.
I S Lee S Wakabayashi K Miyata K Tomoda M Yoneda K Kangawa N Minamino H Matsuo H Matsubara 《Journal of biochemistry》1984,96(5):1409-1418
Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment. 相似文献
13.
C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests 总被引:1,自引:0,他引:1
A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests. 相似文献
14.
Sequence analysis of rat hypothalamic corticotropin-releasing factor with the o-phthalaldehyde strategy 总被引:6,自引:0,他引:6
Sequence analysis was performed on a 41-residue polypeptide that has been identified as the predominant form of high intrinsic corticotropin-releasing activity of rat hypothalamus. The sequence of residues 1-39 of this corticotropin-releasing factor (CRF) was determined by Edman degradation of a partially purified peptide in a highly sensitive spinning cup sequencer after selective blocking of CRF or its main contaminant with o-phthalaldehyde. This approach was validated by peptide mapping of CRF of a highly purified preparation. Peptide mapping was accomplished with reverse-phase high-pressure liquid chromatography of CRF fragments obtained by digestion with clostripain. The identities of the fragments cleaved from CRF were established by chromatographic comparison with synthetic peptides, amino acid analysis, and Edman degradation. On the basis of these experiments, the primary structure of rat hypothalamic CRF was established to be H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn - Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. It is expected that the o-phthalaldehyde strategy will facilitate the sequence analysis of partially purified peptides containing proline residues. 相似文献
15.
A D Bacharach F S Markland A Pellino B H Weber 《Biochemical and biophysical research communications》1977,74(1):165-171
Previous studies have shown that yeast 3-phosphoglycerate kinase is inhibited by nitration of a single tyrosine residue. Chymotryptic fragmentation of the nitrated protein followed by peptide mapping revealed approximately fifty peptides, one of which was shown to contain a nitrotyrosine residue. Isolation of this unique peptide was accomplished by gel filtration and high voltage paper electrophoresis. The sequence as established by Edman degradation and carboxypeptidase hydrolysis is: Lys-NO2Tyr-Phe-Phe-Lys. Independent observations on the X-ray crystallographic model of yeast phosphoglycerate kinase provides supportive evindence of this sequence. Additionally, a peptide has been isolated containing an active-site carboxyl residue following modification of the enzyme with [14C]methoxyamine. 相似文献
16.
E J Van Damme H Kaku F Perini I J Goldstein B Peeters F Yagi B Decock W J Peumans 《European journal of biochemistry》1991,202(1):23-30
Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments. 相似文献
17.
A modified Edman degradation method where fluorescent derivatives of amino acids were generated from the postcleavage products of a peptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manual sequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus amino acid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1′-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl-CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ-CIA-amino acids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for amino acids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio = 3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manual sequencing of insulin chain B composed of 30 amino acids. 相似文献
18.
Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin 总被引:31,自引:0,他引:31
V M Dixit G A Grant S A Santoro W A Frazier 《The Journal of biological chemistry》1984,259(16):10100-10105
Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain. 相似文献
19.
T A Zargarova G K Kovaleva O O Favorova N B Levina I N Telezhinskaia 《Bioorganicheskaia khimiia》1989,15(10):1307-1312
Method of isolation of the bovine pancreas tryptophanyl-tRNA synthetase is improved and a protein with greater than or equal to 99% purity, according to PAGE-SDS, is obtained. The pure enzyme is digested with clostripain and the hydrolysate is separated by FPLC anion-exchange chromatography followed by reversed phase HPLC. Amino acid sequences of 6 individual peptides, including C-terminal one, were determined by the automated Edman degradation. A peptide is also revealed which is encoded with the low degeneracy. 相似文献
20.
Fresh frozen bovine parathyroid glands were defatted in acetone, when extracted with phenol. Following trichloroacetic acid precipitation, the resultant peptides were chromatographed on Sephadex G-100. Parathyroid hormone (BPTH) characteristically elutes in the fourth peak. However, we also observed significant hormonal activity, both biological and immunological, in the fifth elution peak. The peak V material had potent hypercalcemic activity in the rat and chick, and stimulated adenylate cyclase activity in the rat renal cortex bioassay. This material was further purified by ion exchange chromatography on carboxymethylcellulose in 8 M urea. The biological activity of the purified peptide (3700 MRC units/mg) was equivalent to that of the native hormone on a molar basis. Amino acid analysis, carboxypeptidase digestion, and partial Edman sequence analysis identified this material as BPTH 1-65, a hormonal fragment lacking the C-terminal 19 residues of the 84 residue hormone molecule. Several immunoassays using different anti-PTH antisera had variable reactivity toward the BPTH 1-65 fragment, showing that it may be useful for further characterizing antibody recognition sites. The presence of a lysine residue at position 65 suggests a tryptic-like cleavage may be responsible for the genesis of this hormonal fragment. Further investigation will be necessary to determine if this peptide has physiological significance. 相似文献