Sharply resolving labeled protein molecular weight standards

Abstract

Pre-labeled protein standards useful in electrophoresis that have sharp, consistent separation characteristics that are substantially the same as those of their unlabeled counterparts are provided. The invention provides pre-labeled protein standard sets that include a plurality of labeled proteins that are labeled on a first amino acid, in which side reactions of the label with amino acids not targeted for labeling are reduced.

1 . A pre-labeled protein standard set comprising a plurality of labeled proteins, wherein one or more of the plurality of labeled proteins is a selectively labeled protein comprise a labeling compound on a first amino acid, wherein the selectively labeled protein has fewer than one residue of a second amino acid that reacts with the labeling compound per ten kilodalton (kDa) molecular weight of the selectively labeled protein. 
     
     
         2 . The pre-labeled protein standard set of  claim 1 , wherein the selectively labeled protein lacks residues of a second amino acid that reacts with the labeling compound. 
     
     
         3 . The pre-labeled protein standard set of  claim 1 , wherein the first amino acid is lysine and the second amino acid is one or more of cysteine, histidine, or tryptophan; the first amino acid is cysteine and the second amino acid is one or more of lysine, histidine, or tryptophan; the first amino acid is glutamate and the second amino acid is apartate; the first amino acid is aspartate and the second amino acid is glutamate; the first amino acid is histidine and the second amino acid is one or more of cysteine, lysine, or tryptophan; the first amino acid is tryptophan and the second amino acid is one or more of cysteine, lysine, histidine, or asparagine; the first amino acid is asparagine and the second amino acid is one or more or cysteine, lysine, histidine, or tryptophan; or the first amino acid is tyrosine and the second amino acid is one or more of cysteine, lysine, histidine, or tryptophan; or the first amino acid is methionine and the second amino acid is one or more of cysteine, lysine, histidine, tyrosine, or tryptophan. 
     
     
         4 . The pre-labeled protein standard set of  claim 3 , wherein the first amino acid is lysine and the second amino acid is cysteine. 
     
     
         5 . The pre-labeled protein standard set of  claim 3 , wherein the first amino acid is cysteine and the second amino acid is lysine. 
     
     
         6 . The pre-labeled protein standard set of  claim 1 , wherein two or more of the plurality of labeled proteins are selectively labeled proteins that comprise a labeling compound on a first amino acid and have fewer than one residue of a second amino acid that reacts with the labeling compound per ten kilodalton (kDa) molecular weight of the selectively labeled protein. 
     
     
         7 . The pre-labeled protein standard set of  claim 4 , comprising three or more selectively labeled proteins that comprise a labeling compound on a first amino acid and have fewer than one residue of a second amino acid that reacts with the labeling compound per ten kilodalton (kDa) molecular weight of the selectively labeled protein. 
     
     
         8 . The pre-labeled protein standard set of  claim 4 , comprising four or more selectively labeled protein that comprise a labeling compound on a first amino acid and have fewer than one residue of a second amino acid that reacts with the labeling compound per ten kilodalton (kDa) molecular weight of the selectively labeled protein. 
     
     
         9 . The pre-labeled protein standard set of  claim 4 , comprising five or more selectively labeled proteins that comprise a labeling compound on a first amino acid and have fewer than one residue of a second amino acid that reacts with the labeling compound per ten kilodalton (kDa) molecular weight of the selectively labeled protein. 
     
     
         10 . The pre-labeled protein standard set of  claim 1 , wherein at least one selectively labeled protein comprises one or more copies of a sequence of at least 40 contiguous amino acids that has at least 80% identity with an amino acid sequence of a naturally-occurring protein. 
     
     
         11 . The pre-labeled protein standard set of  claim 10 , wherein at least one selectively labeled protein comprises one or more copies of a sequence of at least 50 contiguous amino acids that has at least 80% identity with an amino acid sequence of a naturally-occurring protein. 
     
     
         12 . The pre-labeled protein standard set of  claim 6 , wherein at least two selectively labeled proteins comprise one or more copies of a sequence of at least 40 contiguous amino acids that has at least 80% identity with an amino acid sequence of a naturally-occurring protein. 
     
     
         13 . The pre-labeled protein standard set of  claim 12 , wherein at least two selectively labeled proteins comprise one or more copies of the same sequence of at least 40 contiguous amino acids that has at least 80% identity with an amino acid sequence of a naturally-occurring protein. 
     
     
         14 . The pre-labeled protein standard set of  claim 10 , wherein first amino acid is lysine and the second amino acid is cysteine. 
     
     
         15 . The pre-labeled protein standard set of  claim 14 , wherein the naturally-occurring protein is an ADP-ribosylation factor or a  Bacillus megaterium  spore protein. 
     
     
         16 . The pre-labeled protein standard set of  claim 10 , wherein the first amino acid is cysteine and the second amino acid is lysine. 
     
     
         17 . The pre-labeled protein standard set of  claim 16 , wherein the naturally-occurring protein is a seed storage protein, the L domain of HIV or Ebola viruses, or a WNK protein. 
     
     
         18 . The pre-labeled protein standard set of  claim 10 , wherein the amino acid sequence has a reduced number of residues of the second amino acid compared to the amino acid sequence of a naturally-occurring protein. 
     
     
         19 . The pre-labeled protein standard set of  claim 18 , wherein the amino acid sequence lacks residues of the second amino acid. 
     
     
         20 . The pre-labeled protein standard set of  claim 1 , wherein at least one of the one or more proteins that is selectively labeled on a first amino acid is labeled with a chromophore. 
     
     
         21 . The pre-labeled protein standard set of  claim 1 , wherein at least one of the one or more proteins that is selectively labeled on a first amino acid is labeled with a textile dye. 
     
     
         22 . The pre-labeled protein standard set of  claim 21 , wherein the textile dye is remazol brilliant blue R, uniblue A, malachite green isothiocyanite, or orange 16. 
     
     
         23 . The pre-labeled protein standard set of  claim 1 , wherein at least one of the one or more proteins that is selectively labeled on a first amino acid is labeled with a fluorophore. 
     
     
         24 . The pre-labeled protein standard set of  claim 23 , wherein the fluorophore is an Alexa fluor dye, a BODIPY dye, fluoroscein or a derivative thereof, eosin or a derivative thereof, tetramethylrhodamine, rhodamine or a derivative thereof, Texas red or a derivative thereof, pyridyloxazole or a derivative thereof, NBD chloride, NBD fluoride, ABD-F, lucifer yellow or a derivative thereof, 8-anilino-1-naphthalenesulfonic acid (8-ANS) or a derivative thereof, or Oregon green or a derivative thereof. 
     
     
         25 . A pre-labeled protein standard set comprising a plurality of labeled proteins, wherein one or more of the plurality of labeled proteins is a selectively labeled protein that comprises a labeling compound on a first amino acid, wherein the selectively labeled protein comprises an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein, wherein the amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein comprises at least 40 contiguous amino acids that have at least 80% identity with the amino acid sequence of a naturally-occurring protein, wherein the amino acid sequence has a reduced number of residues of a second amino acid that reacts with the labeling compound compared to the amino acid sequence of the naturally-occurring protein. 
     
     
         26 . The pre-labeled protein standard set of  claim 25 , wherein the first amino acid is lysine and the second amino acid is one or more of cysteine, histidine, or tryptophan; the first amino acid is cysteine and the second amino acid is one or more of lysine, histidine, or tryptophan; the first amino acid is glutamate and the second amino acid is apartate; the first amino acid is aspartate and the second amino acid is glutamate; the first amino acid is histidine and the second amino acid is one or more of cysteine, lysine, or tryptophan; the first amino acid is tryptophan and the second amino acid is one or more of cysteine, lysine, histidine, or asparagine; the first amino acid is asparagine and the second amino acid is one or more or cysteine, lysine, histidine, or tryptophan; or the first amino acid is tyrosine and the second amino acid is one or more of cysteine, lysine, histidine, or tryptophan; or the first amino acid is methionine and the second amino acid is one or more of cysteine, lysine, histidine, tyrosine, or tryptophan. 
     
     
         27 . The pre-labeled protein standard set of  claim 26 , wherein the first amino acid is lysine and the second amino acid is cysteine. 
     
     
         28 . The pre-labeled protein standard set of  claim 26 , wherein the first amino acid is cysteine and the second amino acid is lysine. 
     
     
         29 . The pre-labeled protein standard set of  claim 25 , wherein two or more of the plurality of labeled proteins is a selectively labeled protein that comprise a labeling compound on a first amino acid, and wherein the two or more selectively labeled proteins each comprise an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein, wherein the amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein comprises at least 40 contiguous amino acids that have at least 80% identity with the amino acid sequence of a naturally-occurring protein, wherein the amino acid sequence has a reduced number of residues of a second amino acid that reacts with the labeling compound compared to the amino acid sequence of the naturally-occurring protein. 
     
     
         30 . The pre-labeled protein standard set of  claim 29 , wherein at least two selectively labeled proteins comprise one or more copies of the same amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein. 
     
     
         31 . The pre-labeled protein standard set of  claim 30 , wherein the amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein has less than 1 residue of the second amino acid per 10 kDa. 
     
     
         32 . The pre-labeled protein standard set of  claim 31 , wherein the amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein lacks residues of the second amino acid. 
     
     
         33 . The pre-labeled protein standard set of  claim 29 , wherein different selectively labeled proteins of the pre-labeled protein standard set comprise different numbers of copies of the amino acid sequence derived from the sequence homologous to an amino acid sequence of a naturally-occurring protein. 
     
     
         34 . The pre-labeled protein standard set of  claim 25 , wherein the naturally-occurring protein is a nucleotide-disulfide oxidoreductase. 
     
     
         35 . The pre-labeled protein standard set of  claim 34 , wherein the nucleotide-disulfide oxidoreductase is a lipoamide dehydrogenase, glutathione reductase, or thioredoxin. 
     
     
         36 . The pre-labeled protein standard set of  claim 34 , wherein the nucleotide-disulfide oxidoreductase is a thioredoxin. 
     
     
         37 . The pre-labeled protein standard set of  claim 36 , wherein the nucleotide-disulfide oxidoreductase is a bacterial thioredoxin. 
     
     
         38 . The pre-labeled protein standard set of  claim 37 , wherein the nucleotide-disulfide oxidoreductase is  E. coli  thioredoxin. 
     
     
         39 . The pre-labeled protein standard set of  claim 36 , wherein the amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein is homologous to a truncated thioredoxin sequence. 
     
     
         40 . The pre-labeled protein standard set of  claim 25 , wherein at least one of the one or more proteins further comprises one or more additional amino acid sequences. 
     
     
         41 . The pre-labeled protein standard set of  claim 40 , wherein at least one of the one or more additional amino acid sequences is homologous to an amino acid sequence of a second naturally-occurring protein. 
     
     
         42 . The pre-labeled protein standard set of  claim 40 , wherein at least one of the one or more additional amino acid sequences is a peptide or protein tag sequence. 
     
     
         43 . The pre-labeled protein standard set of  claim 42 , wherein the peptide or protein tag sequence is a multiple histidine (his) sequence, a HAT sequence, a T7 gene 10 sequence, a hemagglutinin sequence, a FLAG tag sequence, a myc tag sequence, a glutathione-S-transferase sequence, a maltose binding protein sequence, a calmodulin binding peptide sequence, or a chitin-binding sequence. 
     
     
         44 - 72 . (canceled) 
     
     
         73 . A kit comprising a pre-labeled protein standard set comprising a plurality of labeled proteins, wherein two or more proteins of the plurality of labeled proteins comprise a label on residues of a first amino acid, and lack residues of a second amino acid that reacts with the label; and one or more of: one or more gel cassettes, a gel loading buffer, a gel loading dye, a reducing agent, a blotting membrane, a lysis buffer, a purification matrix, or an unlabeled protein standard set. 
     
     
         74 - 97 . (canceled) 
     
     
         98 . The pre-labeled protein standard set of  claim 1 , comprising at least 5 labeled proteins. 
     
     
         99 . The pre-labeled protein standard set of  claim 1 , comprising at least 12 labeled proteins. 
     
     
         100 . The pre-labeled protein standard set of  claim 25 , comprising at least 5 labeled proteins. 
     
     
         101 . The pre-labeled protein standard set of  claim 25 , comprising at least 12 labeled proteins.