Sequencing of protein immobilized on polytetrafluoroethylene sup

Details Sequencing of protein immobilized on polytetrafluoroethylene sup Sequencing of protein immobilized on polytetrafluoroethylene sup

1994-03-15

1996-07-30

Redding, David A.

Chemistry: analytical and immunological testing

Peptide, protein or amino acid

Amino acid or sequencing procedure

530334, 530335, 530337, 530344, 530402, 530412, 530417, G01N 3368

Patent

active

055411142

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to the sequencing of peptides immobilized on preferably porous polytetrafluoroethylene supports. More particularly, the invention relates to automated C-terminal and N-terminal sequencing of peptides blotted from a gel onto a porous polytetrafluoroethylene support.


BACKGROUND OF THE INVENTION

Various methods for the N-terminal and C-terminal sequencing of peptides are known. Application of this chemistry to peptides covalently attached to a solid support has facilitated automation. Perceived advantages of covalently immobilizing a peptide or protein to a solid support include: elimination of sample washout thereby resulting in high initial and repetitive yields, the ability to use reagents and solvents optimal for derivatization and washing, and the ability efficiently to wash the sample to remove reaction by-products resulting from thiohydantoin formation, thereby creating a potential for a low chemical background.
The concept of solid phase sequencing for N-terminal Edman chemistry was proposed by Laursen, Eur. J. Biochem. 20:89-102 (1971) and has since been used successfully by a number of groups for the Edman degradation (Laursen, et al., FEBS Lett. 21:67-70 (1972); L'Italien, et al., Anal. Biochem. 127:198:212 (1982); L'Italien, Methods in Protein Microcharacterization (Shively, J. E., Ed.), pp. 279-314, Humana Press, Inc. (1986)).
Several different types of functional supports for the covalent immobilization of polypeptide samples for N-terminal sequencing have been described. These include polystyrene resins, polyacrylamide resins, and glass beads substituted with aminoalkyl or aminophenyl groups. See Laursen, et al., Methods Biochem. Anal. 26:201-284 (1980).
Initial attempts at C-terminal sequencing from covalently attached peptides using thiocyanate chemistry were made by several groups. Williams, et al. FEBS Lett. 54: 353-357 (1975) were able to perform 1-3 cycles on peptides (1 micromol) covalently attached to N-hydroxysuccinimide activated glass beads using 12N HCl for cleavage of the peptidylthiohydantoins. Utilizing this same procedure, Rangarajan, et al., Biochem. J. 157:307-316 (1976) were able to perform six cycles on ribonuclease (1 .mu.mol) covalently coupled to glass beads with a cycle time of 5 to 6 hours. Three successful cycles, with HPLC identification of the released amino acid thiohydantoins, were performed by Meuth, et al., Biochem. 21:3750-3757 (1982) on a 22-amino acid polypeptide (350 nmol) covalently linked to a carbonyldiimidazole activated aminopropyl glass. These authors used thiocyanic acid for derivatization to a peptidylthiohydantoin and acetohydroxamate for cleavage, further reducing the time per cycle to 3 hours. A more recent report by Inglis, et al., Methods in Protein Sequence Analysis (Wittman-Lebold, B., Ed.) pp. 137-144, Springer-Verlag (1989) reports the sequential degradation of nine residues from a synthetic decapeptide (30 nmol) covalently coupled to glass beads with a cycle time of 48 min. However, no experimental details were given. More recent studies have involved the use of carboxylic acid modified PVDF (Bailey, et al., Carboxy terminal sequencing: Automation and application to the solid phase. In Techniques in Protein Chemistry: II (Villafranca, J. J.,Ed.) pp. 115-129 (Academic Press, Inc.) (1991)), carboxylic acid modified polyethylene (Shenoy, et al. Protein Science 1:58-67 (1992), and a disuccinamidoyl carbonate polyamide resin (Hawke, et al., Met. Protein Sequence Analysis (Jornvall/Hoog/Gustavsson Eds.) pp. 35-45, Birkhauser-Verlag, Basel (1991).
Currently PVDF is a preferred support for N-terminal sequencing, and for blotting of purified proteins from gels, such as SDS gels. However, in C-terminal sequencing procedures PVDF turns black and dissolves, frequently limiting some C-terminal sequencing procedures to a single cycle.
In addition to these problems presented by prior art supports, the need for covalent attachment inherently results in sample loss. For that reason, proteins are now blotted o

REFERENCES:
patent: 3843443 (1974-10-01), Fishman
patent: 5061635 (1991-10-01), Shively
Pappin, et al., Analytical Biochemistry 187:10-19 (1990).

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