Services do NOT include
Shipment and costs thereof of the samples will be the responsibility of the user.
Depending on the requested service, ITV will provide:
A list of identified proteins in the sample, including their absolute concentration.
Identification of mutual interacting proteins, including the amino acid sequences of their intersections.
(Semi) quantitative proteome analysis based on mass spectrometry
Description of service
ITV offers Biomolecular Mass Spectrometry-based analytical services, focusing on:
Quantitative proteome analysis, revealing total protein composition, including APIs and impurities. This assay typically identifies and quantifies absolutely individual proteins (e.g. Host Cell Proteins (HCPs) and antigenic proteins) in semi-finished or final vaccine products.
Identification of transient protein-protein interactions utilizing chemical cross-linking mass spectrometry. This assay can be used to identify an intersection of a transient interaction, for example assessing B cell epitopes in terms of antigen-antibody interactions.
For both analytical services, ITV houses nanoscale Liquid Chromatographic systems, combined with state-or-the-art Mass Spectrometers (Orbitrap Fusion Lumos Tribrid from ThermoScientific).
4-8 weeks, upon the availability of all required samples and reagents
0031 30 7920 475
Chemical cross-linking mass spectrometry provides structural information on transient protein-protein interactions in addition to the kinetics and thermodynamic characteristics of these interactions as determined by SPR-based techniques (this latter being offered by IME).
Sample Requirements - input of users
These analytical services can only be performed on samples that have not been chemically inactivated by e.g. formaldehyde, β-propiolactone (BPL) or similar chemical inactivation procedures: chemical inactivation of proteins affects the amino acids typically being targets in the offered assays (i.e. cleavage sites of the enzymes used for proteolytic cleavages).
In case of the identification of B cell epitopes, both the antigen and the monoclonal antibody must be provided by the user/customer. It is important to note, that this assay cannot be performed utilizing polyclonal antibodies obtained from serum samples. If the primary amino acid sequence of the monoclonal antibody is not known beforehand, ITV can assess this sequence using de novo sequencing strategies as part of the offered analytical service.
Hugo D. Meiring, PhD
The absolute quantitative analysis of e.g. HCPs and pathogenic proteins is based essentially on the strategies described by:
J.C. Silva, M.V. Gorenstein, G-Z. Li, J.P.C. Vissers, S.J. Geromanos (2006). Absolute Quantification of Proteins by LCMSE - A virtue of parallel MS acquisition. Mol Cell Proteomics 5(1), 144-156.
T.L. Williams, J.L. Pirkle, J.R. Barr (2012). Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry. Vaccine 30, 2475–2482.
Chemical cross-linking mass spectrometry, as being offered in this analytical service, is based on cleavable cross-linkers described in detail by:
Kao, C.-L.Chiu, D. Vellucci, Y. Yang, V.R. Patel, S. Guan, A. Randall, P. Baldi, S.D. Rychnovsky, L. Huang (2011). Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes. Mol Cell Proteomics 10(1), M110 002212.
F. Liu, D.T. Rijkers, H. Post, A.J.R. Heck (2015). Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat Methods 12(12), 1179-1184.
A. Sinz (2016). Divide and conquer: cleavable cross-linkers to study protein conformation and protein-protein interactions. Anal Bioanal Chem 409(1), 33-44.
The chromatographic separation of the analytes is detail described in detail by:
H.D. Meiring, E. van der Heeft, G.J. ten Hove, A.P.J.M. de Jong (2002). Nanoscale LC–MS(n): technical design and applications to peptide and protein analysis. J Sep Sci 25, 557–568.