Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels

Strickler, J. Rudi; Hunkapiller, Michael W.; Wilson, Kenneth J.

doi:10.1016/0003-2697(84)90207-0

Cited by 41 publications

(9 citation statements)

References 12 publications

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“…It was found that larger molecular forms of cardioditatin of tess than 20 kD circulating in human blood would have been detected if they had constituted more than 1% of the cardiodilatin bioactivity. We therefore confirm earlier suggestions that the small molecular form is the circulating hormone as seen in the rat [19,20] and humans [3,21]. The circulating atrial polypeptide hormone has previously been structurally analysed in the rat [19,20]; these authors, analysing rat plasma, found the 28 amino acid containing homologous polypeptide.…”

Section: Discussionsupporting

confidence: 86%

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Isolation and structural analysis of the circulating human cardiodilatin (alpha ANP)

Forssmann

Hock

Herbst³

et al. 1986

Klin Wochenschr

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A new method was applied to isolate a polypeptide hormone from human blood. The polypeptides from 1,000 1 of hemofiltrate with a molecular weight lower than 20 kDaltons were adsorbed to 2.5 kg alginic acid, then eluted, precipitated, and desalted on a G-25 Sephadex column, thus obtaining a crude lyophilised plasma polypeptide extract. These polypeptides were further submitted to ion-exchange chromatography. Thereafter, two steps of HPLC were carried out to purify a distinct polypeptide which was the circulating form of cardiodilatin (CDD) in this case. The amino acid analysis, C-terminal enzymatic cleavage by carboxypeptidase A, and sequence analysis showed that the only form of circulating cardiodilatin is the 28 amino acid residue containing molecule, cardiodilatin-99-126 cleaved from the C-terminus of cardiodilatin-126 and identical with alpha-ANP (alpha atrial natriuretic polypeptide). Other bioactive molecular forms of the polypeptide hormones of the cardiodilatin family were not detected in the hemofiltrate. The isolation procedure was followed up by a bioassay using in vitro vascular smooth muscle relaxation.

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Section: Discussionsupporting

confidence: 86%

“…Stepwise Edman degradation o f 20 n m o l f r o m the intact peptide is performed on a gas-phase protein sequencer 420 A (from Applied Biosystems, [19]). The PTH-amino acids are identified using HPLC according to Lottspeich [15].…”

Section: Sequence Analysismentioning

confidence: 99%

Isolation and structural analysis of the circulating human cardiodilatin (alpha ANP)

Forssmann

Hock

Herbst³

et al. 1986

Klin Wochenschr

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“…Peptides (50 to 3,000 pmol) isolated from the digests by lysyl endopeptidase and trypsin were analyzed with a gas phase sequencer (model 470A with an on-line model 120A PTH amino acid analyzer; Applied Biosystems), either by a conventional method or by covalently fixing the peptide at the terminal COOH on aminopolystyrene resin (8,16).…”

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confidence: 99%

Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli

et al. 1989

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Chromatographic peptide mapping of lysyl endopeptidase digests of penicillin-binding protein 3 (PBP 3) of Escherichia cofi revealed peptides that differed in retention time between the precursor and mature forms. The peptides were purified from a processing-defective (prc) mutant and a wild-type (prc+) strain. These peptides were identiied as the C-terminal region of the precursor form and mature PBP 3 by amino acid sequencing. Each of the C-terminal peptides was deaved into two fragments by trypsin digestion. By sequencing the resultant carboxyl-side faent derived from the mature form, it was concluded that the C-terminal residue of mature PBP 3 was Val-577, and thus the Val-577-Ble-578 bond is the cleavage site for processing. This conclusion was consistent with the amino acid compositions of the relevant peptides, which suggested that the peptide from the cleavage site to the end of the deduced sequence (Ile-578-Ser-588) was present in the precursor but absent in the mature form. One lysyl peptide bond resisted both lysyl endopeptidase and trypsin and remained undeaved in the peptide analyzed above.It is well known that proteins destined for extracytoplasmic locations are processed by proteolytic cleavage at the N-terminal part during transfer across the cytoplasmic membrane. Penicillin-binding protein 3 (PBP 3) of Escherichia coli, although not an excreted protein, is synthesized as a precursor form and then processed to a mature form (9). PBP 3 participates in septum formation (12,13,17), and a substantial part of the molecule appears to be located in the periplasm, where the murein lies (15). Thus, it was suspected that insertion of PBP 3 into the membrane may involve the cleavage of an N-terminal signal peptide (3, 9). However, the C-terminal part rather than the N-terminal part of PBP 3 appeared to be the site of processing. The evidence for this is described in the accompanying paper (1); substitution of the N-terminal part with the MLP signal peptide lacking its Ala-Cys cleavage site did not affect the processing of PBP 3; the loss of 28 residues at the C-terminus abolished the processing capacity; and cleavage at the C-terminal region was shown by the loss of labeled peptide extended artificially from the normal C-terminus.Finding of a mutant (prc) defective in this novel type of processing permitted us to isolate the precursor PBP 3, and we were able to identify the C-terminal peptide by comparing peptide maps of the precursor and mature forms and to determine the cleavage site, as described in the present report. MATERIALS AND METHODSPRP 3. Bacterial strains used, cultivation of bacteria, and the procedure for isolation of PBP 3 were as described in the accompanying paper (1). PBP 3 was overproduced with pWK7 or pWK9 (7). The precursor and the mature PBP 3 were prepared from JE7860 (prc)(pWK9) and JE7627 (prc') (pWK7), respectively. Both the precursor and the mature * Corresponding author. PBP 3 preparations contained a penicillin-binding protein of about 40 kilodaltons (kDa) molecular mass (40K pro...

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“…However, it is apparent from the designs of the two instruments that the techniques are converging. This is further emphasized by attempts to degrade samples covalently coupled to glass beads in the gas-phase instrument (Strickler et al, 1984).…”

Section: Perspectivesmentioning

confidence: 99%

A rapid solid-phase protein microsequencer

Walker¹,

Fearnley²,

Blows³

1986

Biochemical Journal

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A solid-phase protein microsequencer is described that has been designed to determine protein sequences with subnanomolar quantities of protein. Its utility has been demonstrated by the determination of many sequences in subunits of mitochondrial F1-ATPase, in a protein isolated from mouse gap junctions and in the mitochondrial phosphate-transporter protein. It has a number of advantages over liquid- and gas-phase sequencers. Firstly, the degradation cycle takes 24 min, more than twice as fast as any other sequencer. This helps to reduce exposure of proteins to inimical reagents and increases throughput of samples. Secondly, polar amino acids such as phosphoserine, and polar derivatives formed by active-site photoaffinity labelling with 8-azido-ATP, are recovered quantitatively from the reaction column and can be positively identified. In other types of sequencer these polar derivatives, being somewhat insoluble in butyl chloride, tend to remain in the reaction chamber of the instrument and so are more difficult to identify. The solid-phase protein sequencer is also more suited than the liquid-phase instrument for analysis of proteolipids from membranes. These hydrophobic proteins tend to dissolve in organic solvents during washing steps in the liquid-phase instrument and are lost. Covalent attachment as used in the solid-phase instrument solves this problem.

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Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels

Cited by 41 publications

References 12 publications

Isolation and structural analysis of the circulating human cardiodilatin (alpha ANP)

Isolation and structural analysis of the circulating human cardiodilatin (alpha ANP)

Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli

A rapid solid-phase protein microsequencer

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