Sequential degradation of proteins and peptides from the N-termi

Details Sequential degradation of proteins and peptides from the N-termi Sequential degradation of proteins and peptides from the N-termi

1994-07-13

1995-07-11

Schain, Howard E.

Chemistry: analytical and immunological testing

Peptide, protein or amino acid

Amino acid or sequencing procedure

436 90, 436 92, 530345, 530402, 530408, G01N 3368, C07K 1107

Patent

active

054320933

DESCRIPTION:

BRIEF SUMMARY
FIELD OF INVENTION

This invention relates to the sequential degradation of proteins and peptides from the N-terminus. In particular, the invention permits N-terminal degradation of protein and peptide samples in the picomole to subpicomole range. The amino acid derivatives are rapidly detectable at high sensitivity by electrospray mass spectrometry.


BACKGROUND OF THE INVENTION

Proteins have a central role in all biological processes including the development and treatment of human disease. Recent advances make it possible to isolate proteins of biological interest which are present in tissues in subpicomole quantities. Study of the structure function aspects of these proteins requires sequence information prerequisite to cloning and expression thereof. There is a consequential need for improved methods to sequence proteins and protein fragments at sample levels several orders of magnitude smaller than is now possible.
Currently, protein sequence analysis is primarily accomplished with the use of an automated sequencer using chemistry developed by Edman over 40 years ago (Edman, 1950) (FIG. 1). Since that time improvement in the instrumentation has resulted in the ability to sequence smaller and smaller sample quantities (mmole to pmol), although the original chemistry has remained essentially unchanged. Current automated instrumentation permits 10-20 cycles of sequence determination of 10-50 pmol of sample (Simpson et al., 1989).
The major limitation of Edman chemistry is the approximately one picomole practical detection limit of the PTH amino acids. The current method involves separation of the phenylthiohydantoin (PTH) amino acids by high-performance liquid chromatography (HPLC) followed by UV detection.
Various proposals to increase the sensitivity of the PTH's by use of radiolabeled, chromophoric, or fluorescent isothiocyanate reagents have been described. 4-(N,N'-Dimethylamino)azobenzene-4'-isothiocyanate (DABITC), a highly chromophoric reagent, was introduced by Chang (Chang et al., 1976). Fluorescent reagents, such as fluorescein isothiocyanate (Maeda and Kawauchi, 1968; Muramoto et al. 1984), and dansyl-containing isothiocyanates (Hirano and Wittmann-Liebold, 1986; Hirano and Wittmann-Liebold, 1987; Jin et al., 1986; Jin et al., 1989; Salnikow et al. 1987) have also been evaluated as sensitivity enhancing reagents. Synthetic amino acid analogues prepared using these reagents have shown subpicomole sensitivity by HPLC analysis. However, the use of these reagents in automated sequencing has not surpassed the sensitivity of the standard Edman methodology. It is postulated that the large chromophore of these reagents interferes with the derivatization and cleavage reactions of the Edman degradation. Radiolabeled reagents undergo autoradiodegradation which results in decreasing product yields and increasing amounts of labeled by-products.
An alternative method involves treatment of the anilinothiazolinone (ATZ) derivative normally formed in Edman chemistry with a fluorescent amine (Tsugita et al., 1989). The advantage is that the derivatization and cleavage reactions of the Edman chemistry remain unchanged. Theoretically this chemistry should permit sequencing on femtomole levels of sample. However, investigation has revealed a number of problems rendering this chemistry of little value toward the goal of more sensitive sequencing. Foremost is the instability of the ATZ-amino acids which are required for reaction with the fluorescent amine. The ATZ-amino acids, in particular the hydrophilic amino acids such as histidine, glutamate, and aspartate, rearrange to the PTH derivative so rapidly that reaction with the fluorescent amine was not possible. A possible solution to this problem is to convert the PTH amino acid back to the ATZ amino acid so that reaction with the fluorescent amine will be possible.
The aminolysis of PTH amino acids is discussed in detail by Pavlik et al. (1992).
Replacement of the fluorescent amine with a reagent such as N,N-dimethylethylenediamine (DMED) has been found by appli

REFERENCES:
patent: 5008205 (1991-04-01), Horn
Aebersold et al, Protein Science 1: 494-503 (1992).
Pavlik et al, Anal. Biochem 201: 9-16 (1992).
Simpson et al, Anal Biochem. 177: 221-236 (1989).
Tsugita et al, J. Biochem. 106: 60.65 (1989).

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