Shah Brinda, Reid Jennifer D, Kuzyk Michael A, Parker Carol E, Borchers Christoph H
University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria, BC, Canada.
Methods Mol Biol. 2013;1023:97-120. doi: 10.1007/978-1-4614-7209-4_6.
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.
免疫基质辅助激光解吸/电离（iMALDI）技术包括从样品的酶解消化物中亲和捕获目标肽段，然后在MALDI靶板上对亲和珠进行直接分析。为了测定肽段浓度（进而推断蛋白质浓度），可以将稳定同位素标记的标准肽段（SIS肽段）添加到消化物中，它们会与天然肽段一起被捕获。该技术通过确定含表位肽段的两个分子特征，可提供尽可能高的特异性：（1）分子量，通常通过MALDI-MS测量，精度可达100 ppm或更高；（2）氨基酸序列，通过进行MALDI-MS/MS分析。该技术已被证明能够检测环境样品和人血浆消化物中低至阿托摩尔水平的目标肽段。本章详细描述了如何进行iMALDI分析，从目标肽段的选择开始。展示了iMALDI在检测一种可能构成生物恐怖主义威胁的生物体以及检测人表皮生长因子受体（EGFR）的两种异构体方面的应用实例。
