Methods and compositions for polypeptide analysis

A method for analyzing a polypeptide, comprising the steps of:

• (a) providing the polypeptide optionally associated directly or indirectly with a recording tag;
• and optionally contacting the polypeptide with a proline aminopeptidase under conditions suitable to cleave an N-terminal proline;
• (b) functionalizing the N-terminal amino acid (NTAA) of the polypeptide with a chemical reagent, wherein the chemical reagent comprises a compound selected from the group consisting of
• (i) a compound of Formula (I):
• or a salt or conjugate thereof,
• wherein
• R1 and R2 are each independently H, C1-6alkyl, cycloalkyl, —C(O)Ra, —C(O)ORb, or —S(O)2Rc;
• Ra, Rb, and Rc are each independently H, C1-6alkyl, C1-6haloalkyl, arylalkyl, aryl, or heteroaryl, wherein the C1-6alkyl, C1-6haloalkyl, arylalkyl, aryl, and heteroaryl are each unsubstituted or substituted;
• R3 is heteroaryl, —NRdC(O)ORe, or —SRf, wherein the heteroaryl is unsubstituted or substituted;
• Rd, Re, and Rf are each independently H or C1-6alkyl;
• and
• optionally wherein when R3 is
• R1 and R2 are not both H;
• (ii) a compound of Formula (II):
• or a salt or conjugate thereof,
• wherein
• R4 is H, C1-6alkyl, cycloalkyl, —C(O)Rg, or —C(O)ORg;
• and
• Rg is H, C1-6alkyl, C2-6alkenyl, C1-6haloalkyl, or arylalkyl, wherein the C1-6alkyl, C2-6alkenyl, C1-6 haloalkyl, and arylalkyl are each unsubstituted or substituted;
• (iii) a compound of Formula (III):
• R5—N═C═S (III)
• or a salt or conjugate thereof,
• wherein
• R5 is C1-6alkyl, C2-6 alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
• wherein the C1-6alkyl, C2-6 alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl are each unsubstituted or substituted with one or more groups selected from the group consisting of halo, —NRhRi, —S(O)2Rj, or heterocyclyl;
• Rh, Ri, and Rare each independently H, C1-6alkyl, C1-6haloalkyl, arylalkyl, aryl, or heteroaryl, wherein the C1-6alkyl, C1-6haloalkyl, arylalkyl, aryl, and heteroaryl are each unsubstituted or substituted;
• (iv) a compound of Formula (IV):
• or a salt or conjugate thereof,
• wherein
• R6 and R7 are each independently H, C1-6alkyl, —CO2C1-4 alkyl, —ORk, aryl, or cycloalkyl, wherein the C1-6alkyl, —CO2C1-4 alkyl, —ORk, aryl, and cycloalkyl are each unsubstituted or substituted;
• and
• Rk is H, C1-6alkyl, or heterocyclyl, wherein the C1-6alkyl and heterocyclyl are each unsubstituted or substituted;
• (v) a compound of Formula (V):
• or a salt or conjugate thereof,
• wherein
• R8 is halo or —ORm;
• Rm is H, C1-6alkyl, or heterocyclyl;
• and
• R9 is hydrogen, halo, or C1-6haloalkyl;
• (vi) a metal complex of Formula (VI):
• MLm (VI)
• or a salt or conjugate thereof,
• wherein
• M is a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn, and Ni;
• L is a ligand selected from the group consisting of —OH, —OH2, 2,2′-bipyridine (bpy), 1,5 dithiacyclooctane (dtco), 1,2-bis(diphenylphosphino)ethane (dppe), ethylenediamine (en), and triethylenetetramine (trien);
• and
• n is an integer from 1-8, inclusive;
• wherein each L can be the same or different;
• and
• (vii) a compound of Formula (VII):
• or a salt or conjugate thereof,
• wherein
• G1 is N, NR13, or CR13R14;
• G2 is N or CH;
• p is 0 or 1;
• R10, R11, R12, R13, and R14 are each independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C1-6alkylamine, and C1-6 alkylhydroxylamine, wherein the C1-6alkyl, C1-6haloalkyl, C1-6alkylamine, and C1-6alkylhydroxylamine are each unsubstituted or substituted, and R10 and R11 can optionally come together to form a ring;
• and
• R15 is H or OH;
• (c) contacting the polypeptide with a first binding agent comprising a first binding portion capable of binding to the functionalized NTAA and
• (c1) a first coding tag with identifying information regarding the first binding agent, or
• (c2) a first detectable label;
• (d) (d1) transferring the information of the first coding tag to the recording tag to generate an extended recording tag and analyzing the extended recording tag, or
• (d2) detecting the first detectable label;
• wherein step (b) is conducted before step (c), after step (c) and before step (d), or after step (d).