Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

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.

Advances in High‐resolution Maldi Mass Spectrometry for Neurobiology

“…Ontissue digestion of proteins has also been pursued with the MALDI-FTMS platforms. Rat brain tissue slices were imaged on MALDI target plates pre-coated with trypsin and α-cyano-4hydroxycinnamic acid (CHCA) for direct enzymatic digestion to determine the spatial distribution of proteins, tryptic peptides, and lipids with increased throughput of analysis (Zubair et al, 2016). Schober and colleagues also performed direct on-tissue digestion of proteins on mouse brain tissue for AP-MALDI-FTMS imaging by depositing trypsin with a pneumatic sprayer (Schober et al, 2012).…”

“…Solvent-free dry matrix application methods that rely on sublimation of powdered matrix were also implemented for the MALDI-FTICR-MSI analysis of dopamine antagonists and other targets in rat brain tissue (Caughlin et al, 2017;Goodwin et al, 2011a). Pre-coating the slide with matrix and derivatization reagents such as trypsin prior to placing the tissue on the slide greatly simplifies the imaging workflow and achieves good results for proteins up to 300 kDa in rat brain (Zubair et al, 2016;Basu et al, 2019). While there are many factors associated with preparing a sample for MALDI-MS or MALDI-MSI analysis, the optimization and consideration that goes into each individual step is important for more sensitive, accurate, and reproducible results.…”

Expansion of immature, nucleated red blood cells by transient low-dose methotrexate immune tolerance induction in mice

“…Mass spectrometry imaging was performed with Bruker Autoflex MALDI-TOF/TOF (Bruker Daltonics, Billerica, MA, USA) mass spectrometers. Protein and peptide identification were achieved by correlating experimental peak list with the theoretical tryptic peptides as described by Zubair et al [20]. Theoretical tryptic peptides were generated using mMass (an open-source mass spectrometry tool freely available at www.mmass.org) and the image data were analyzed with FlexImaging 4.1 (Bruker Daltonics).…”

Next-generation protein analysis in the pathology department

“…For fresh-frozen (FF) tissues, the preparation step consists of ethanol washing for direct profiling of protein with a mass below ±25 kDa. An additional preparation step for high-molecular-weight proteins (>±25 kDa) is trypsin application, since these proteins are not easily detected in matrix-assisted laser desorption/ionisation (MALDI) analysis of FF tissues due to their poor ionisation efficiency in this mass range 19 23…”