Methods and systems for characterizing phosphoserine-containing polypeptides

A method, comprising:

• converting a phosphoserine residue of a polypeptide to a dehydroalanine residue to provide a dehydroalanine-containing polypeptide;
• reacting the dehydroalanine-containing polypeptide with a protected thiol compound of Formula (I):
• R1-(L1)m-S—X (I),
• or a salt thereof, to provide a functionalized polypeptide comprising a residue of Formula (II):
• wherein:
• X is a protecting group;
• L1 is a linking group;
• m is an integer from 0 to 36;
• and
• R1 is a moiety comprising a click chemistry handle;
• digesting the functionalized polypeptide to form two or more peptide fragments,
• wherein at least one peptide fragment is a functionalized peptide fragment comprising R1;
• and
• conjugating the functionalized peptide fragment to a linker.

A method, comprising:

• digesting a polypeptide comprising a phosphoserine residue to form two or more peptide fragments, wherein at least one peptide fragment comprises the phosphoserine residue;
• converting the phosphoserine residue of the at least one peptide fragment to a dehydroalanine residue of the at least one peptide fragment to provide a dehydro alanine-containing peptide fragment;
• reacting the dehydroalanine-containing peptide fragment with a protected thiol compound of Formula (I):
• R1-(L1)m-S—X (I),
• or a salt thereof, to provide a functionalized peptide fragment comprising a residue of Formula (II):
• wherein:
• X is a protecting group;
• L1 is a linking group;
• m is an integer from 0 to 36;
• and
• R1 is a moiety comprising a click chemistry handle;
• and
• conjugating the functionalized peptide fragment to a linker.

A kit, comprising:

• a protected thiol compound comprising a click chemistry handle;
• a base;
• and
• a linker.

A method of enriching a sample comprising one or more polypeptides comprising a target residue, comprising:

• selectively conjugating the target residue to a linker;
• immobilizing the linker to a surface;
• and
• removing any non-immobilized polypeptides or portions of polypeptides.

A method of characterizing a peptide, comprising:

• immobilizing the peptide to a surface of a sample well at a target residue, wherein the target residue is not a terminal amino acid of the peptide;
• contacting the peptide with one or more amino acid recognition molecules labeled with one or more detectable labels;
• detecting one or more series of signal pulses indicative of binding events between the one or more amino acid recognition molecules and the peptide;
• and
• determining at least one chemical characteristic of the peptide.

A method of characterizing a phosphoserine residue of a polypeptide, comprising:

• (a) converting the phosphoserine residue of the polypeptide to a dehydroalanine residue to provide a dehydroalanine-containing polypeptide;
• (b) reacting the dehydroalanine-containing polypeptide with a thiol compound comprising a click chemistry handle to provide a functionalized polypeptide comprising a functionalized dehydroalanine residue;
• (c) digesting the functionalized polypeptide to form two or more peptide fragments, wherein at least one peptide fragment is a functionalized peptide fragment comprising a functionalized dehydroalanine residue;
• (d) contacting the functionalized peptide fragment with one or more amino acid recognition molecules;
• (e) detecting a first series of signal pulses indicative of binding events between the one or more amino acid recognition molecules and a first amino acid at a terminus of the functionalized peptide fragment;
• (f) determining at least one chemical characteristic of an amino acid of the functionalized peptide fragment based on at least one characteristic of the first series of signal pulses;
• (g) removing the first amino acid from the terminus of the functionalized peptide fragment;
• and
• (h) repeating steps (d) to (g) until the functionalized dehydroalanine residue is exposed at the terminus of the functionalized peptide fragment.

A method of characterizing a phosphoserine residue of a polypeptide, comprising:

• (a) digesting the polypeptide to form two or more peptide fragments, wherein at least one peptide fragment is a peptide fragment comprising the phosphoserine residue;
• (b) converting the phosphoserine residue of the at least one peptide fragment to a dehydroalanine residue of the at least one peptide fragment to provide a dehydro alanine-containing peptide fragment;
• (c) reacting the dehydroalanine-containing peptide fragment with a thiol compound comprising a click chemistry handle to provide a functionalized peptide fragment comprising a functionalized dehydroalanine residue;
• (d) contacting the functionalized peptide fragment with one or more amino acid recognition molecules;
• (e) detecting a first series of signal pulses indicative of binding events between the one or more amino acid recognition molecules and a first amino acid at a terminus of the functionalized peptide fragment;
• (f) determining at least one chemical characteristic of an amino acid of the functionalized peptide fragment based on at least one characteristic of the first series of signal pulses;
• (g) removing the first amino acid from the terminus of the functionalized peptide fragment;
• and
• (h) repeating steps (d) to (g) until the functionalized dehydroalanine residue is exposed at the terminus of the functionalized peptide fragment.