Methods of processing a sample for peptide mapping analysis

The method of any one of the preceding claims, wherein the digesting occurs for less than about 12 hours, optionally, less than about 6 hours.

The method of any one of the preceding claims, wherein the digesting occurs at a temperature of about 35° C. to about 40° C., optionally, about 37° C.

The method of any one of the preceding claims, wherein the digesting occurs in the presence of calcium chloride.

The method of any one of the preceding claims, wherein only trypsin is used to digest the polypeptide.

The method of any one of the preceding claims, wherein digesting the polypeptide with trypsin at the E:S weight ratio produces said digested sample comprising one or more peptides comprising a tyrosine at the C-terminus.

The method of any one of the preceding claims, comprising denaturing the polypeptide, reducing the polypeptide, and/or alkylating the polypeptide before digesting the polypeptide with the protease.

The method of any one of the preceding claims, comprising a buffer exchange before digesting the polypeptide with the protease and after denaturing, reducing, and/or alkylating the polypeptide.

The method of any one of the preceding claims, wherein the buffer exchange comprises use of a size exclusion cartridge, optionally, wherein the size exclusion cartridge is a NAP5 cartridge with the Sephadex G-25 gel filtration material.

The method of any one of the preceding claims, further comprising incubating the digested sample in the presence of a chaotrope at a mildly acidic pH.

The method of any one of the preceding claims, comprising injecting peptides of the digested sample into a liquid-chromatography-mass spectrometry (LC-MS) system for peptide mapping analysis.

A method of processing a polypeptide, comprising

• a. digesting the polypeptide with a protease to produce a digested sample comprising at least two peptides;
• and
• b. incubating the digested sample in the presence of a chaotrope and/or at a mildly acidic pH.

A method of increasing the recovery of long peptides of a digested sample, comprising incubating a digested sample in the presence of a chaotrope and/or at a mildly acidic pH, wherein the digested sample comprises at least two peptides and is produced by digesting a polypeptide with a protease.

A method of increasing the solubility of peptides of a digested sample, comprising incubating a digested sample in the presence of a chaotrope and/or at a mildly acidic pH, wherein the digested sample comprises at least two peptides and is produced by digesting a polypeptide with a protease.

A method of processing a polypeptide, comprising digesting the polypeptide with a protease which cleaves C-terminal to tryptophan to produce a digested sample comprising at least two peptides, optionally, wherein at least one peptide comprises a C-terminal tryptophan.