The amino-acid sequence in the glycyl chain of insulin. 1. The identification of lower peptides from partial hydrolysates

Sanger, F.; Thompson, Eop

doi:10.1042/bj0530353

Cited by 549 publications

(98 citation statements)

References 12 publications

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“…(c) The number of defects can be quantified via the ultraviolet (UV) absorbance or fluorescence of a label specifically binding to these defects. In this study, the label used is the Sanger reagent 51,52 . In turn, these last two features enable the theoretical analysis of the labelling data of the homologous series of DP in order to quantify the number of defects for different g. As we shall discuss, the number of defects increases significantly for 1,000 PG6 thus suggesting the onset of SIS in the vicinity of g max .…”

Section: Resultsmentioning

confidence: 99%

“…There is no universal label and the choice of the reagent should be customized to the synthetic chemistry used. When NH 2 groups are involved one may use the Sanger reagent 51,52 or Dansyl [54][55][56] . The Sanger reagent is less prone to aggregation and its smaller size is advantageous at high g. (iv) The labelling method can quantify all free ends for 1rgtg max , that is,…”

Section: Discussionmentioning

confidence: 99%

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Synthetic regimes due to packing constraints in dendritic molecules confirmed by labelling experiments

Zhang

Schlüter

et al. 2013

Nat Commun

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Classical theory predicts that branching defects are unavoidable in large dendritic molecules when steric congestion is important. Here we report first experimental evidence of this effect via labelling measurements of an extended homologous series of generations g ¼ 1y6 of dendronized polymers. This system exhibits a single type of defect interrogated specifically by the Sanger reagent thus permitting to identify the predicted upturn in the number of branching defects when g approaches g max and the polymer density approaches close packing. The average number of junctions and defects for each member of the series is recursively obtained from the measured molar concentrations of bound labels and the mass concentrations of the dendritic molecules. The number of defects increases at g ¼ 5 and becomes significant at g ¼ 6 for dendronized polymers where the g max was estimated to occur at 6.1 rg max r 7.1. The combination of labelling measurements with the novel theoretical analysis affords a method for characterizing high g dendritic systems.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Synthetic regimes due to packing constraints in dendritic molecules confirmed by labelling experiments

Zhang

Schlüter

et al. 2013

Nat Commun

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“…Sequential degradation with phenylisothiocyanate was performed according to Gray [ll] and dinitrophenylation was carried out by the procedure of Sanger and Thompson [12]. The terminal amino acid was determined substractively by amino acid analysis of an acid hydrolysate of residual peptide or Dnppeptide.…”

Section: Sequential Degradation Of Peptidesmentioning

confidence: 99%

Carboxyl‐Terminal Region of Human and Bovine Erythrocyte Carbonic Anhydrases

Andersson¹,

Po²,

Nilsson³

et al. 1968

European Journal of Biochemistry

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A methionine-containing tryptic peptide from acetamidinated human carbonic anhydrase B has been isolated. This peptide has been shown to bridge a previously known carboxyl-terminal fragment prepared by CNBr degradation with the rest of the enzyme molecule. Sequence studies on the methionine-containing peptide have extended the previous knowledge of the carboxylterminal sequence of the enzyme.Corresponding bridge peptides from human carbonic anhydrase C and acetamidinated bovine carbonic anhydrase B have also been isolated and their amino acid compositions have been determined.The preceding paper [l] gives the carboxyl-terminal sequences of human carbonic anhydrase forms B and C and bovine carbonic anhydrase formB, (for nomenclature see [2]). The results were obtained by characterization of carboxyl-terminal peptides derived from the proteins by splitting with CNBr. From the specificity of this reagent [3] one would expect a methionine residue to be located next to the sequences determined.The present investigation is a study of methioninecontaining tryptic peptides bridging the previously determined carboxyl-terminal sequence with t,he rest of the enzyme molecule. MATERIALS AND METHODS Preparation of Carbonic AnhydrasesHuman carbonic anhydrases B and C, and bovine carbonic anhydrase B were purified as described previously [l]. Acetamidination of Human Carbonic Anhydrase B and Bovine Carbonic Anhydrase BAcetamidination was performed as described elsewhere [4]. Proteolytic Digestions with EndopeptidasesTryptic digestions were performed a t room temperature in a pH-stat (Radiometer, Copenhagen : Titrator, type TTTlc, in combination with Titri- Unusual Abbreviations. Di-isopropyl phosphorofluoridate, DFP; 2,4-dinitrophenyl, Dnp-; sulfoethyl, SE-; glntamine or glutamic acid, Glx. graph, typeSBR2c) a t pH 9.0 with trypsin (3 x crysf., salt free, TRL 61 13, Worthington Biochemical Corporation, Freehold, New Jersey 07728) treated with diphenyl-carbamyl chloride [5]. A 1 solution of human carbonic anhydrase C in water was adjusted to pH 9.0 with 0.05 M NaOH. The protein was denatured by keeping the solution in a boiling water bath for 15 min. Trypsin, dissolved in 2.5 mM HC1, was added. After 6 h another amount of trypsin solution was added. After about 10 h from start, the consumption of NaOH had almost ceased. The heat denaturation was then repeated and an additional aliquot of trypsin was added. The total amount of trypsin added then corresponded to a molar ratio of substrate/trypsin of 100. When no more NaOH was consumed (usually after 13-14 h) pH was decreased to about 3 with glacial acetic acid.Tryptic digestions of acetamidinated human enzyme B and bovine enzyme B were performed as described for human enzyme C but the final molar ratio of substrate/trypsin was 50 and the initial concentration of substrate 1.5-2 Oi0. Under these conditions the hydrolyses took 4.5-6 h.A digestion of a peptide with subtilisin was carried out by dissolving the peptide (9.4 pmoles) in 2.5 ml of 0.1 M Tris-C1 buffer, pH 9.0, and adding...

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“…The analytical application of acid hydrolysis for generating peptides can be dated back to the classic protein sequencing work of Sanger and Thompson [13]. Prior to the use of MS as a major tool for protein identification, limited acid hydrolysis had been used as a means of generating peptides from proteins separated by SDS-PAGE for peptide mapping and Edman microsequencing [14,15].…”

mentioning

confidence: 99%

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Zhong

Marcus

2005

J. Am. Soc. Mass Spectrom.

150

142

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Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 g of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/ l or 37 fmol/ l. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein. (J Am Soc Mass Spectrom 2005, 16, 471-481)

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The amino-acid sequence in the glycyl chain of insulin. 1. The identification of lower peptides from partial hydrolysates

Cited by 549 publications

References 12 publications

Synthetic regimes due to packing constraints in dendritic molecules confirmed by labelling experiments

Synthetic regimes due to packing constraints in dendritic molecules confirmed by labelling experiments

Carboxyl‐Terminal Region of Human and Bovine Erythrocyte Carbonic Anhydrases

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

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