Method for detecting post-translational modification of peptides
a post-translational modification and peptide technology, applied in the field of methods for detecting post-translational modification of peptides, can solve problems such as inability to obtain complete sequence information
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example 1
Sequencing of [Glu1]Fibrinopeptide B
[0126] [Glu1]Fibrinopeptide B was purchased from Sigma Chemical Co. (St. Louis, Mo.). The reported sequence was: Glu1-Gly-Val-Asn-Asp5-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg14. Matrix assisted laser desorption mass spectrometry gave MW 1570.6 dalton (Calculated: 1570.8 dalton) and showed high purity of the starting peptide. A mixture of PITC plus 5% v / v phenylisocyanate PIC was used in the coupling step. PIC reacts with the NH2— of a polypeptide chain to yield an Nα-phenylcarbamyl-peptide which is stable to the conditions of the Edman degradation. A modification of a standard manual Edman degradation procedure (6) was used. All reactions were carried out in the same 0.5 mL polypropylene microfuge tube under a blanket of dry nitrogen. Peptide (200 pmoles to 10 nmole) was dissolved in 20 ul of pyridine / water (1:1 v / v; pH 10.1); 20 uL of coupling reagent containing PITC:PIC:pyridine:hexafluoroisopropanol (20:1:76:4 v / v) was added to the reaction vial. ...
example 2
[0129] Stepwise solid phase synthesis of the 99 amino acid residue polypeptide chain corresponding to the monomer of the HIV-1 protease (SF2 isolate):
PQITLWQRPLVTIRIGGQLKEALLDTGADDTVLEEMNLPGKWKPKMIGGIGGFIKVRQYDQIPVEI (Aba) GHKAIGTVLVGPTPVNIIGRNLLTQIG (Aba) TLNF99[where Aba = α-amino-n-butyric acid] was undertaken.
[0130] Highly optimized Boc-chemistry instrument-assisted stepwise assembly of the protected peptide chain was carried out on a resin support, according to the method described by S. B. H. Kent (8). Samples (3-8 mg, about lumole each) were taken after each cycle of amino acid addition. The protected peptide-resin samples were mixed in three batches of consecutive samples: (number corresponds to the amino acid after which sample was taken, i.e. residue number in the target sequence.) 99-67; 66-33; 32-1. The first such mixture contained the peptides:
99-Resin98-99-Resin97-98-99-Resin96-97-98-99-Resin. . . (etc.) . . .70 . . . 96-97-98-99-Resin69-70 . . . 96-97-98-99-Resin6...
example 3
Boc / Fmoc Terminations
[0135] Synthesis of the peptide LRRAFGLIGNNPLMAR-amide was performed manually on a 0.2 mmol scale using p-methylbenzhydrylamine resin and 0.8 mmoles amino acid (95 mol % N-α-Boc, 5 mol % N-α-Fmoc) according to the in situ neutralization methods of Schnolzer et al (9). The following side chain protecting groups were used: Boc-Arg, tosyl; Fmoc-Arg, 2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr). Fmoc-Arg(Mtr) was used for its greater stability in trifluoroacetic acid (TFA). After completion of the chain assembly, Fmoc groups were removed using 50% piperidine / DMF, followed by Boc group removal in TFA. The peptide fragments were then cleaved from the resin by treatment with HF-10% p-cresol (0° C., 1 hour). The resulting crude peptide products were precipitated and washed with ether, dissolved in 50% acetic acid, diluted with water and lyophilized. The mass spectra of the reaction mixture thus produced is shown in FIG. 11.
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