Barcoded XTEN polypeptides and compositions thereof, and methods for making and using the same

A method of detecting truncation of a fusion polypeptide in a sample,

• wherein the fusion polypeptide comprises at least one extended recombinant (XTEN) polypeptide fused to the N-terminus or the C-terminus of a biologically active polypeptide comprising a reference fragment,
• wherein the XTEN polypeptide is from 150 to 1,000 amino acids in length, comprises a plurality of non-overlapping sequence motifs each between 9 and 14 amino acids in length, and at least 90% of the amino acid residues of the XTEN polypeptide are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P),
• wherein the XTEN comprises a barcode fragment, which is from 4 to 20 amino acids in length, occurs only once in the fusion polypeptide, and is located within 100 amino acids from the N-terminus of the XTEN polypeptide if the XTEN polypeptide is N-terminal to the biologically active polypeptide or from the C-terminus of the XTEN polypeptide if the XTEN polypeptide is C-terminal to the biologically active polypeptide,
• wherein the sample comprises a first set of polypeptides and a second set of polypeptides, wherein each polypeptide of the first set of polypeptides (a) has the same sequence as, or is truncated from, the fusion polypeptide and (b) retains the barcode fragment, and each polypeptide of the second set of polypeptides (a) is truncated from the fusion polypeptide and (b) lacks the barcode fragment,
• wherein each polypeptide of both the first set of polypeptides and the second set of polypeptides retains the reference fragment, the method comprising:
• contacting the sample with a protease to produce a plurality of proteolytic fragments that result from cleavage of the first set of polypeptides and the second set of polypeptides, wherein the plurality of proteolytic fragments comprise:
• a plurality of reference fragments;
• and
• a plurality of barcode fragments, and
• determining a ratio of the amount of barcode fragments to the amount of reference fragments,
• thereby assessing the relative amounts of the first set of polypeptides to the second set of polypeptides representing the truncation of the fusion polypeptide.