Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry |
| |
Authors: | Faizan Zubair Paul E Laibinis William G Swisher Junhai Yang Jeffrey M Spraggins Jeremy L Norris Richard M Caprioli |
| |
Institution: | 1. Mass Spectrometry Research Center (MSRC), Vanderbilt University, Nashville, Tennessee, USA;2. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Tennessee, USA;3. Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA;4. Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA;5. Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA |
| |
Abstract: | Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd. |
| |
Keywords: | trypsin pre‐coated targets bottom‐up proteomics imaging mass spectrometry MALDI enzymatic digestion sample preparation FTICR mass spectrometry |
|
|