[36] High-sensitivity sequencing with a gas-phase sequenator

Hunkapiller, Michael W.; Hewick, Rodney M.; Dreyer, William J.; Hood, Leroy

doi:10.1016/s0076-6879(83)91038-8

Cited by 656 publications

(217 citation statements)

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“…Disulfide-linked peptides thus identified were finally analyzed in a pulsed-liquid-phase sequenator 477A from Applied Biosystems combined with a model 120A PTH Analyzer using only DTT-free reagents [28]. Di-phenylthiohydantoin-cystine was identified after its release in the corresponding cycle as a double peak [29,30].…”

Section: Methodsmentioning

confidence: 99%

Identification of disulfide bonds in the ninth component (C9) of human complement

1996

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Section: Methodsmentioning

confidence: 99%

Identification of disulfide bonds in the ninth component (C9) of human complement

1996

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“…Table 1 represents five disulfide containing peptides isolated from the skin secretions of R. Arvalis and R. ridibunda. Their structures were elucidated earlier by mass spectrometry and confirmed by Edman degradation [12,13,22,23].…”

Section: Resultsmentioning

confidence: 99%

Oxidation versus carboxamidomethylation of s-s bond in ranid frog peptides: Pro and contra for de novo MALDI-MS sequencing

Samgina

Artemenko

Gorshkov

et al. 2008

J. Am. Soc. Mass Spectrom.

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Five natural peptides isolated from ranid skin secretions of European frog species of Rana ridibunda and Rana arvalis (molecular masses 3516, 26743516, , 26363516, , 18743516, , and 1810 were studied by MALDI-TOF/TOF to compare two procedures of disulfide bond cleavage: (1) performic oxidation and (2) reduction/carboxamidomethylation. The processes are relevant for the elucidation of the amino acid sequence inside the seven-member cystine ring at the C-terminus. The results clearly demonstrated that oxidation of the disulfide bond led to notably higher abundances of b-and y-ions, corresponding to the C-terminal peptide bonds, than reduction/carboxamidomethylation. This conclusion is true for all five peptides studied. Besides that, the oxidation procedure is simpler than carboxamidomethylation, as it is a one-step process with no purification required. The oxidation is more reproducible. The results were similar each time the peptide was subjected to the process. It was successfully applied to all five peptides while reduction/carboxamidomethylation failed in the case of brevinin-1Ra, despite all variations of reaction conditions. (J Am Soc Mass Spectrom 2008, 19, 479 -487) © 2008 American Society for Mass Spectrometry A skin secretion produced by granular skin glands of anurans in response to a variety of stimuli contains biologically active peptides [1][2][3][4]. Our interest was focused on the Rana genus containing a notable array of common frogs spread around the territory of Eurasia and America. A major part of ranid peptides has a disulfide cycle at the C-terminus [1,5]. This peculiarity, called "Rana box" [6], complicates sequencing of these peptides. The amino acid sequence inside the cycle is practically unattainable by means of mass spectrometry without preliminary chemical derivatization. There is only one communication on direct sequence identification inside the C-terminus disulfide cycle using ESI MS/MS in the negative ion mode. It deals with some bioactive peptides isolated from genus Crinia (Australia) and with brevinin-1E R. ridibunda, (Russia) [7]. In this case, a combination of the positive and negative mass spectra has produced complete sequences for all the investigated peptides. Unfortunately not all peptides give reasonable negative ion spectra.Mass spectrometric sequencing of disulfide containing peptides requires preliminary modification of the S-S bond. There are two general methods to cleave this bond: (1) reduction followed by the alkylation of cysteines [8 -10]; (2) oxidation with performic acid [11]. The first procedure is traditionally used to break S-S bonds in proteins with associated polypeptide chains before their enzymatic digestion. We applied it for the skin peptides of the Rana genus with an internal disulfide cycle [12,13]. Reduction of the S-S bond followed by carboxamidomethylation allows reliable sequencing at the C-terminus. LTQ FTICR mass spectrometric results completely match those obtained by Edman degradation.Performic oxidation is a well-known method for the cl...

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“…Two additional aliquots of the same amount of TPCK-trypsin were added at the end of 1 and 2 h, respectively, and digestion was continued for another 2 h. The peptides were dried, redissolved in 19'0 TFA and then purified by chromatography on a Vydac 214 TP column (RPC C4) using a Hewlett-Packard HP-1090 HPLC system. Well-resolved peptides with good recovery were sequenced using the Applied Biosystem model 470A gas-phase amino acid sequencer [17] with an on-line PTH amino acid analyzer (Applied Biosystem model 120A).…”

Section: Methodsmentioning

confidence: 99%

Proteolytic signal sequences (PEST) in the mammalian aminoacyl‐tRNA synthetase complex

et al. 1988

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Eight aminoacyl-tRNA synthetases together with three unidentified proteins are associated as a multi-enzyme complex in mammalian cells. Partial peptide sequences for lysyl-and aspartyl-tRNA synthetases are determined and no highly hydrophobic peptides are found. The partial amino acid sequences for two of the unidentified proteins in the complex are shown to have substantial homology and each has a number of unique sequences. The results suggest that the two unidentified proteins are fragments of synthetases. The partial sequences revealed the presence of PEST sequences in at least three proteins. Inasmuch as PEST sequences are signals for intracellular degradation, the mammalian synthetase complex may have evolved to protect these synthetases against intracellular proteolysis.Aminoacyl-tRNA synthetase; Multi-enzyme complex; Amino acid sequence

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[36] High-sensitivity sequencing with a gas-phase sequenator

Cited by 656 publications

References 9 publications

Identification of disulfide bonds in the ninth component (C9) of human complement

Identification of disulfide bonds in the ninth component (C9) of human complement

Oxidation versus carboxamidomethylation of s-s bond in ranid frog peptides: Pro and contra for de novo MALDI-MS sequencing

Proteolytic signal sequences (PEST) in the mammalian aminoacyl‐tRNA synthetase complex

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