Developing an iMALDI method

Brinda Shah, Jennifer D. Reid, Michael A. Kuzyk, Carol E. Parker, Christoph H. Borchers

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

8 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationThe Low Molecular Weight Proteome
Subtitle of host publicationMethods and Protocols
Pages97-120
Number of pages24
DOIs
Publication statusPublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1023
ISSN (Print)1064-3745

Keywords

  • EGFR
  • EGFRvIII
  • Francisella
  • iMALDI
  • Immuno-MALDI
  • Immunoaffinity
  • MALDI
  • Quantitation
  • SIS peptides
  • Stable isotope labeling

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