ocus on ass Spectrometry & Developm Richard Kay1 , Helen Ludlow2 , Aimee Cossins2 , Andrew Splevins2 and Alberto Martinez2
1. Quotient Bioresearch Ltd, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW UK. Email:
richard.kay@quotientbioresearch.com 2. Syntaxin Ltd. Abingdon Oxon OX14 3YS UK
The quantitation of proteins in blood based matrices using LC-MS/MS as the detection mechanism is challenging due to the high background protein content. Chemical extraction methods, such as solid phase extraction or organic solvent precipitation, can achieve lower limits of quantitation (LLOQ) in the low ng/mL range, and work best for small proteins and peptides. However, in order to quantify large proteins to the sub ng/mL level, a more targeted approach is required. Immunoprecipitation (IP) extraction methods exploit the highly specific nature of the antibody-antigen interaction, enabling a highly targeted enrichment of the protein of interest. The binding of drug specific antibodies to paramagnetic beads facilitates the extraction and concentration of the protein from plasma or related matrices.
SXN101959
SXN101959 is a recombinantly produced 105 kDa Targeted Secretion Inhibitor (TSI). TSI are based on botulinum toxin, a di-peptide protein – comprising light and heavy chains (Figure 1). SXN101959 is designed to specifically target the GHRH receptor on somatotrophs, which allows internalisation and delivery of the endopeptidase (light chain) leading to inhibition of vesicular secretion of GH.
The therapeutic aim of SXN101959 was to help treat acromegaly, through the reduction of GH release from the pituitary [2,3].
Roll overnight at room temp. Spectroscopy an Immunoprecipitation and LC-MS/MS Based Method for Quantifying the 105 kDa Recombinant Protein SXN101959 in Plasma
SXN101959 Extraction Process
Aliquot plasma (100 µL) into 96 well plate. Add 900 µL buffer and 15 µL of Ab-Beads (MAb/PAb).
Collect beads on magnet, remove supernatant, and wash with buffer. Repeat twice
Perform reduction, alkylation and digestion on beads.
Collect beads on magnet, transfer supernatant for LC-MS/MS analysis
Figure 1. Crystal structure of botulinum toxin [1]. Heavy chain (orange and blue). Light chain (red). LC-MS/MS Analysis Antibody-Bead Preparation
Monoclonal (MAb) and polyclonal (PAb) antibodies raised against SXN101959 were attached to tosyl-activated dynabeads. Attachment was confirmed by tryptically digesting the beads and monitoring antibody specific tryptic peptides by LC-MS/MS.
Following tryptic digestion, peptides were separated by uHPLC-MS/MS on a Waters UPLC system. A Waters T3 HSS 100 x 2.1mm column was used, with a flow rate of 0.7mL/minute. The method had a total run time of 5.5 minutes including a column flush.
Two peptides from SXN101959 were monitored using SRM detection, one each from the heavy and light chain. Tryptic peptides were also monitored from the monoclonal and polyclonal antibodies.
INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2013
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