Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-07-18
2004-09-07
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S068100, C435S176000, C435S177000, C435S180000, C435S810000
Reexamination Certificate
active
06787330
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the identification or characterisation of one or more polypeptides which have been isolated on a gel, typically from polyacrylamide gel electrophoresis (PAGE) and to a kit for use in the method. It is especially useful in proteomics (the large scale identification and characterisation of proteins).
2. Description of the Related Art
In proteomics, massively parallel protein identification and characterisation techniques are required. The identification of proteins or other polypeptides merely by PAGE, even using two-dimensional gels (2D-PAGE), is laborious and often uncertain. Many different methods have been developed to identify and partially characterise proteins from complex biological samples. Some of them use Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MMI-TOF MS) techniques to analyse peptide “fingerprints” produced by fragmenting the proteins with enzymes. Several software programs have been developed to compare mass spectra of the peptides obtained from MALDI-TOF MS experiments with theoretical spectra from proteins. The subject has been reviewed by M. Kussmann and P. Roepstorff, Spectroscopy 1998, 14, 1-27. These authors noted three ways in which proteins separated by gel electrophoresis could be digested with enzymes to yield fragment peptides:
1. The digestion can be carried out in a plug of excised gel and the peptides recovered by elution. This is the authors' own preference.
2. The protein can be first electroleluted from an excised gel plug and then digested in solution.
3. The protein can be electroblotted onto a membrane and subsequently digested on the membrane.
These types of processes are not practical for the sequencing of polypeptides which have been run on the same gel, since the cutting out of the polypeptide bands from the gel has to be done sequentially and the plugs thus obtained placed in tubes for further analysis. Also, losses occur when the polypeptides adhere to the walls of the tube.
Two of the present inventors have experimented with a different method, which they have termed one-step digestion transfer (OSDT). See U.S. patent application Ser. No. 09/107 991 filed Jun. 30, 1998 and corresponding Canadian Patent Application No. 2 244 947 filed Sep. 24, 1998 entitled “Methods of identifying polypeptides”, the disclosure of which is herein incorporated by reference. They have found that the proteins or other polypeptides separated on a gel can be cleaved into fragments, for example by digestion with an enzyme, and that these fragments are presented very satisfactorily for analysis, especially by MALDI-TOF NS, if the cleaving reagent is immobilised on a hydrophilic membrane and interposed as the “filling” in a blotting “sandwich” between the separation gel as one “slice” of the sandwich and a hydrophobic collection member, exemplified as a conventional polyvinylidene fluoride (PVDF) membrane, as the other “slice” of the sandwich. In this way, the fragments are collected on the hydrophobic member and can then be formulated in an appropriate way for the MALDI-TOF MS. It is only necessary that the transblotting is carried out so that the proteins have a long enough residence period in the proximity of the immobilised cleaving reagent to ensure that a reasonable amount of the fragments is produced, but, of course, not so long as to allow undesired diffusion. With electroblotting, i.e. blotting assisted by an electric field, this is easily achievable by varying appropriately the current used in the electroblotting, e.g. by pulsing the current or using a unsymmetrical alternating current. Further, when an enzyme is used as the cleaving agent and when the enzyme is immobilised securely on a hydrophilic membrane, especially by covalent bonding to the solid phase, autodigestion (cleavage of the enzyme by itself) is inhibited.
The OSDT method gives good results for many proteins, but very strongly basic proteins such as lysozyme are not easily transferred under the conditions which are optimal for use of the preferred enzyme, trypsin. Trypsin gives best digestion in a buffer of pH about 8.4. Also, the OSDT method does not give good digestion of very high molecular weight proteins.
SUMMARY OF THE INVENTION
The present invention Is an improvement to the OSDT method and is based in part on the discovery of another technology which the inventors have termed “in full gel digestion” (IFG). This procedure involves dehydrating the gel and then rehydrating it, adding to gel a polypeptide-cleaving reagent such as an enzyme, e.g. in the rehydration buffer. After the IFG, the digested proteins are then electroblotted in a conventional way. One drawback of this technique is the loss of low molecular weight proteins (those of m.w. less than 40 kDa) by diffusion during the in-gel digestions.
It has now been found that by combining the IFG procedure, optionally modified, with OSDT, satisfactory digestion of the proteins (or other polypeptides), accompanied by improved identification, can be achieved for polypeptides having a wide range of molecular weights. Moreover, high molecular weight proteins can be satisfactorily immunoblotted to yield fragments which can be identified as epitopes.
In a preferred “combined procedure”, the gel is dehydrated and at least partially, preferably only partially, rehydrated with a buffer containing the polypeptide-cleaving reagent, IFG is performed and this is then followed by OSDT.
In one aspect the invention provides a method of identifying or characterising polypeptides which have been isolated on a gel by electrophoresis, comprising:
a) providing a gel on which at least one polypeptide has been isolated;
b) incorporating a first polypeptide-cleaving reagent in the gel (preferably by dehydrating the gel and at least partially rehydrating it with a buffer containing the polypeptide-cleaving reagent);
c) providing adjacent to the gel at least one hydrophilic membrane on which is immobilised at least one second polypeptide-cleaving reagent;
d) providing a hydrophobic collection member (preferably a membrane) suitable for receiving thereon fragments of polypeptide transferred thereto from the gel by transblotting, preferably by electroblotting, said hydrophobic member being positioned beyond the hydrophilic membrane in a direction of movement of the fragments of polypeptide;
e) transblotting the polypoptide or polypeptides from the full gel, on which the polypeptide or polypeptides were isolated, through the hydrophilic membrane or membranes, under conditions effective to cause it or them to be cleaved into fragments by the second polypeptide-cleaving reagent, to the hydrophobic member; and
f) identifying or characterising the fragments collected on the hydrophobic collection layer.
Preferably the method further comprises
g) identifying or characterising the polypeptide from which the fragments were derived.
The invention also includes a kit for use in the method of the invention, said kit comprising:
a) a first polypeptide-cleaving reagent suitable for incorporating in an electrophoretic gel;
b) at least one hydrophilic membrane suitable for use in transblotting of polypeptides separated on an electrophoretic gel, the membrane having at least one second polypeptide-cleaving reagent imobilised thereon; and
c) a hydrophobic collection member suitable for receiving thereon fragments of the separated polypeptides transferred thereto by transblotting.
Elements b) and c) may be provided as separate components, e.g. in separate containers, or as a pre-formed assembly.
The term “cleaving a polypeptide”, as used herein, refers to any step in which a group, residue or any chain of groups or residues is split off from the remainder of the molecule. It includes cleavage in the main chain of amino acids or in a side-chain or of any terminal or side-chain group or residue, e.g. removal of a C-terminal amino acid by carboxypeptidase, N-terminal amino group by an aminopeptidase or a glycosyl side-chain by a glycosidase is included.
The me
Bienvenut Willy Vincent
Hochstrasser Denis Francois
Sanchez Jean-Charles
Naff David M.
University of Geneva
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