Abstract
The iMALDI (immuno-MALDI) technique involves the affinity capture of target peptides from an enzymatic digest of a sample, followed by the direct analysis of the affinity beads while on a MALDI target. For determination of peptide concentration (and, by inference, protein concentration), stable-isotope-labeled standard peptides (SIS peptides) can be added to the digest and will be captured along with the native peptides. This technique can provide the highest possible specificity by determining two molecular characteristics of the epitope-containing peptides: (1) the molecular weight, typically measured to within 100 ppm or better by MALDI-MS, and (2) the amino acid sequence, by performing MALDI-MS/MS. This technique has been shown to be capable of detecting low-attomole levels of target peptides in environmental samples and in digests of human plasma. This chapter provides a detailed description of how to perform iMALDI analyses, starting with the selection of the target peptides. Examples are shown of the application of iMALDI to the detection of an organism that is a possible bioterrorism threat, and to the detection of two isoforms of human EGFR.
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Acknowledgements
The EGRFvIII work was supported by Genome BC, Genome Canada, and graduate student fellowships from the University of Victoria.
The Francisella study was funded by the Southeast Regional Center of Excellence for Emerging Infections and Biodefense (SERCEB, 5U54AI057157-04); the EGFR work was partially supported by NIH grant P30 CA16086-25, CEHS grant P30ES10126, SBIR grant HHSN261200433014C, and P50-CA58223-09A1. We would also like to acknowledge Jian Jiang and Erin Warren, who performed some of the work shown here.
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Shah, B., Reid, J.D., Kuzyk, M.A., Parker, C.E., Borchers, C.H. (2013). Developing an iMALDI Method. In: Bäckvall, H., Lehtiö, J. (eds) The Low Molecular Weight Proteome. Methods in Molecular Biology, vol 1023. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-7209-4_6
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DOI: https://doi.org/10.1007/978-1-4614-7209-4_6
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