De-novo sequencing of mAb

Introduction to de-novo sequencing

Mass spectrometric de-novo sequencing consists of two or three steps. 1) Intact mass determination, 2) peptide mapping using de-novo sequencing software and 3) Edman N-terminal sequencing of peptides.

Intact mass analysis

By ESI-MS, we spray antibody sample in liquid phase through a capillary at high voltage into the MS instrument. Here the mass over charge ratio (m/z) is measured. For antibodies the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. We determine the mass of the antibody with a deconvolution algorithm that calculates the mass of the intact non-protonated antibody. The ESI process requires that the sample preparation is quite pure without interfering salts or detergents.

We can also purify the sample by reversed phase HPLC (Agilent 1200 system) using a short C8 column before being analyzed on a Q-TOF mass spectrometer (Bruker Maxis Impact system). The mass of the sample is determined by deconvolution of the obtained raw spectra using the MaxEnt algorithm. The high resolution and accuracy of the Q-Tof instrument result in very accurate mass determination within 0-3 Da of the theoretical mass.

Peptide sequencing

First we reduce the antibody then separate the heavy chain and the light chain by HPLC. Next, we cleave each fraction into smaller peptides using 5 different specific proteases. The peptides are analyzed by mass spectrometry and all the observed peptides having mass/charge ratio within 300 to 2000 dalton are selected for MS/MS sequencing.

The combined data sets are analyzed using PEAKS De-Novo sequencing software collecting without any sequence information the full sequence of both the heavy and the light chains.

Edman N-terminal sequencing

Based on MS peptide data and De-Novo sequencing data, we now select 5-10 peptides for N-terminal Edman sequencing to verify/identify the exact amino acid sequence of the variable regions. Peptide up to 25 amino acid residues can be sequenced.

The Edman degradation chemistry is a cyclic procedure where the PITC reagent couples to the free N-terminal amino group and cleaves off the protein. We transfer the PITC coupled residue to a flask, convert it to a PTH-residue and identify the amino acid by HPLC chromatography. The Edman degradation reaction cycle repeats to determine a sequence of N-terminal amino acids.

Introduction
Antibody analysis by mass spectrometry requires a pure antibody in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

Antibody samples can be submitted in liquid or lyophilized.

1. Purify the antibody

   1. The chromatographic antibody purity should be >90%
   2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents
   3. Minimum amount ~ 0.5 mg

2. Put the antibody sample into microcentrifuge tube

   1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
   2. Lyophilize or speed vac the antibody to a solid sample to ensure stability during shipment
   3. Alternatively, freeze or refrigerate to +4 ^oC for cold shipment of the liquid sample