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Advancing immuno-MRM analyses using high-affinity recombinant antibody fragments

Oct 28, 2015

Since the introduction of the two-dimensional gel and mapping of the mouse, guinea pig, and Escherichia coli protein sequences in 1975, the field of proteomics has rapidly evolved. Proteomics refers to large scale, comprehensive analysis of protein expression, protein-protein interactions, and protein modifications. Mass spectrometry is one of the main technologies utilized in proteomics, and similarly, this technique has also evolved. Unlike traditional mass spectrometry, which is non-specific, multiple reaction monitoring (MRM) is an emerging technology that allows quantitation of specific target protein fragments in complex biological samples. Immunoaffinity enrichment of peptides coupled to targeted MRM (immuno-MRM) allows simultaneous analysis of multiple proteins with high sensitivity and specificity. 

Immuno-MRM requires the use of antibodies to short linear peptides. Currently, affinity-purified polyclonal antibodies are predominantly used (Hoofnagle et al. 2008, Neubert et al. 2010); however some studies have utilized monoclonal antibodies derived through hybridoma technology (Schoenherr et al. 2010 and Razavi et al. 2011). Monoclonal antibodies are generally preferable to polyclonal antibodies for this process due to their specificity and renewable nature. However, the long process and high cost associated with generating traditional anti-peptide monoclonal antibodies make them less desirable for immuno-MRM. 

To address this problem, Whiteaker et al. (2014) investigated whether high affinity recombinant antibody fragments (Fab) isolated from naïve libraries could be utilized in immuno-MRM assays. Recombinant antibodies may be a suitable alternative to traditional monoclonal antibodies for immuno-MRM analysis since they offer several advantages such as access to a wider range of antigens (including toxic, conserved, or self-antigens) and a faster process (12 weeks vs. 6-9 months) that can be automated. In addition, in vitro generation of Fabs in E. coli allows control of selection parameters such as affinity. 

Whiteaker et al. demonstrated that Fab fragments with high affinity to target peptides could successfully be generated using the HuCAL PLATINUM® library (Prassler et al. 2011) and the RapMAT® technology (Prassler et al. 2009) along with inhibition screening with free peptide (full details of the process are outlined in the image below). Furthermore, these antibodies were shown to function similarly to traditional mouse- or rabbit-derived monoclonal antibodies. This study is the first to demonstrate that antibody fragments isolated from a naïve antibody library can be utilized in immuno-MRM based quantification. It therefore sets precedence for utilizing recombinant antibodies in immuno-MRM in lieu of traditional monoclonal antibodies that are time consuming to generate. 


Scheme of the antibody generation process with the HuCAL® phage display antibody library and RapMAT® technology (Whiteaker et al. 2014 ).

Proteomics continues to support our understanding of various diseases such as cancer.  As the immuno-MRM platform advances, key research areas such as biomarker discovery will continue to benefit considerably. As for the use of recombinant antibody fragments from naïve libraries in immuno-MRM, further research will be required to test the applicability of high affinity recombinant antibodies against a broader range of protein analytes than evaluated by Whiteaker et al. 



  • Hoofnagle AN et al. (2008). Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry. Clin Chem 11, 1796-1804.
  • Neubert H et al. (2010). Online high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS: assay development for total salivary pepsin/pepsinogen. Clin Chem 9, 1413-1423.
  • Prassler J et al. (2009). In vitro affinity maturation of HuCAL® antibodies: complementary determining region exchange and RapMAT technology. Immunotherapy 4, 571-583.
  • Prassler J et al. (2011). HuCAL PLATINUM®, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems. J Mol Biol 1, 261-278.
  • Razavi M et al. (2011). MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics. J Immunol Methods 1-2, 50-64.
  • Schoenherr RM et al. (2010). Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring mass spectrometry. J Immunol Methods 1-2, 49-61.
  • Whiteaker JR et al. (2014). High-Affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays. J Proteome Res 13, 2187-2196.

HuCAL®, HuCAL PLATINUM® and RapMAT® are registered trademarks of MorphoSys AG.


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