Positive identification of phospho-proteins using motif-specific, context-independent antibodies coupled with database searching

A method for the positive identification of cellular phosphoproteins using motif-specific, context-independent antibodies coupled with database searching, said method comprising the steps of:

• (a) fractionating a cellular preparation comprising a plurality of phosphoproteins on the basis of mass and/or PI;
• (b) contacting the fractionated preparation of step (a) with a plurality of motif-specific, context-independent antibodies that each bind a different phosphorylated motif of known sequence, thereby to detect at least one phosphoprotein comprising two or more of the different motifs;
• and
• (c) utilizing a search program to search one or more protein database(s) for reported proteins having a sequence comprising the two or more different motifs detected in step (b) and having a mass and/or PI substantially matching that determined in step (a), thereby to positively identify at least one cellular phosphoprotein in said preparation as a reported protein of known sequence and mass and/or PI.

A method for the positive identification of cellular phosphoproteins using motif-specific, context-independent antibodies coupled with database searching, said method comprising the steps of:

• (a) fractionating a cellular preparation comprising a plurality of phosphoproteins on the basis of mass and/or isoelectric point (PI);
• (b) contacting the fractionated preparation of step (a) with a plurality of motif-specific, context-independent antibodies that each bind a different phosphorylated motif of known sequence, wherein the precise target sequences preferably bound by said antibodies has first been determined, thereby to detect at least one phosphoprotein comprising two or more of said precise target sequences;
• and
• (c) utilizing a search program to search one or more protein database(s) for reported proteins having a sequence comprising the two or more precise target sequences detected in step (b) and having a mass and/or PI substantially matching that determined in step (a), thereby to positively identify at least one cellular phosphoprotein in said preparation as a reported protein of known sequence and mass and/or PI.