Reagents and a method for saturation labelling of proteins
A matched set of fluorescent dyes is provided, wherein each dye of the set is capable of covalent attachment to a protein and wherein each of the dyes has a molecular structure and a charge that is matched one with the other, such that relative electrophoretic mobility of a protein labeled with one dye of the set is the same as the electrophoretic mobility of the protein labeled with a different dye of the set. The matched set comprises at least two different fluorescent dyes of formula: wherein n is 1, 2, or 3; Z 1 and Z 2 independently represent the carbon atoms necessary to complete a phenyl or naphthyl ring system; one of groups R 1 and R 2 is a target bonding group; remaining group R 1 or R 2 is selected from —(CH 2 ) 4 —W or —(CH 2 ) r —H; group R 3 is hydrogen, except when either R 1 or R 2 is —(CH 2 ) r —H, in which case R 3 is W; and W is selected from sulphonic acid and sulphonate. The invention also provides a method for saturation labeling of a protein with a fluorescent dye so as to label all available target amino acid, suitably cysteine, residues in the protein, thereby giving a single population of labeled protein molecules.
1. A matched set of fluorescent dyes comprising two or more different fluorescent dyes of formula (I):
wherein n is different for each said dye and is 1, 2, or 3;
Z 1 and Z 2 independently represent the carbon atoms necessary to complete a phenyl or naphthyl ring system;
one of groups R 1 and R 2 is the group:
where Y is a target bonding group and in each dye of the set of dyes is the same and is selected from the group consisting of maleimido groups and iodoacetamido groups;
remaining group R 1 or R 2 is selected from —(CH 2 ) 4 —W or —(CH 2 ) r —H;
group R 3 is hydrogen, except when either R 1 or R 2 is —(CH 2 ) r —H, in which case R 3 is W;
W is selected from sulphonic acid and sulphonate;
p is an integer from 3 to 6;
q is 2 or 3; and
r is an integer from 1 to 5;
and salts thereof
and further wherein when n of two of said dyes differs by +1, one of p, q and r of said two dyes differs by −1.
2. A matched set of fluorescent dyes comprising at least two different fluorescent dyes of formula (II):
wherein n is different for each said dye and is 1, 2, or 3;
one of groups R 1 and R 2 is the group:
where Y is a target bonding group and in each dye of the set of dyes is the same and is selected from the group consisting of maleimido groups and iodoacetamido groups;
remaining group R 1 or R 2 is selected from —(CH 2 ) 4 —W or —(CH 2 ) r —H;
group R 3 is hydrogen, except when either R 1 or R 2 is —(CH 2 ) r —H, in which case R 3 is W;
W is selected from sulphonic acid and sulphonate;
p is an integer from 3 to 6;
q is 2 or 3; and
r is an integer from 1 to 5;
and salts thereof;
and further wherein when n of two of said dyes differs by +1, one of p, q and r of said two dyes differs by −1.
3. The matched set of dyes of claim 1 or claim 2 comprising at least two different fluorescent dyes wherein:
n is 1 or 2;
p is 4 or 5;
q is 2 or 3; and
r is 1, 2 or 3.
4. The matched set of dyes of claim 1 or claim 2 , wherein in each said dye Y is a maleimido group.
5. The matched set of dyes of claim 1 or claim 2 wherein said salts are selected from salts K + , Na + , NH 4 + , or containing (R) 3 NH + and (R) 4 N + wherein R is C 1 to C 4 alkyl.
6. A matched set of dyes selected from the group consisting of:
Set 1
1-(6-{[2-(2,5-dioxo-2,5 -dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-2-[(1E,3E)-3-(1-ethyl-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium (Compound I); and
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-3,3-dimethyl-2-[(1E,3E,5E)-5-(1,3,3-trimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium (Compound II);
Set 2
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-2-[(1E,3E)-3-(1-propyl-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium (Compound III); and
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-3,3-dimethyl-2-[(1E,3E,5E)-5-(1-ethyl-3,3-trimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium (Compound IV);
Set 3
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-2-[(1E,3E)-3-(1-ethyl-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium (Compound I); and
1-(5-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxopentyl)-3,3-dimethyl-2-[(1E,3E,5E)-5-(1-ethyl-3,3-trimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium (Compound V);
Set 4
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-2-[(1E,3E)-3-(3,3-dimethyl(1-sulpho-butyl)-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium (Compound VI); and
1-(5-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxopentyl)-3,3-dimethyl-2-[(1E,3E,5E)-5-(3,3-dimethyl-(1-sulpho-butyl)-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium (Compound VII);
Set 5
1-(6-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl]amino}-6-oxohexyl)-2-[(1E,3E)-3-(1-ethyl-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium (Compound VIII); and
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-3,3-dimethyl-2-[( 1E,3E,5E)-5-(1-ethyl-3,3-trimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium (Compound IV); and
Set 6
1-(6-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl]amino}-6-oxohexyl)-2-[(1E,3E)-3-(3,3-dimethyl(1-sulpho-butyl)-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium (Compound IX); and
1-(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-3,3-dimethyl-2-[(1E,3E,5E)-5-(3,3-dimethyl-(1-sulpho-butyl)-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium (Compound X).
7. A kit comprising a matched set of fluorescent dyes comprising at least two different fluorescent dyes having the formula (I):
wherein n is different for each said dye and is 1, 2, or 3;
Z 1 and Z 2 independently represent the carbon atoms necessary to complete a phenyl or naphthyl ring system;
one of groups R 1 and R 2 is the group:
where Y is a target bonding group and in each dye of the set of dyes is the same and is selected from the group consisting of maleimido groups and iodoacetamido groups;
remaining group R 1 or R 2 is selected from —(CH 2 )4—W or —(CH 2 ) r —H;
group R 3 is hydrogen, except when either R 1 or R 2 is —(CH 2 ) r —H, in which case R 3 is W;
W is selected from sulphonic acid and sulphonate;
p is an integer from 3 to 6;
q is 2 or 3; and
r is an integer from 1 to 5;
and salts thereof;
and further wherein when n of two of said dyes differs by +1, one of p, q and r of said two dyes differs by −1.