The modified-bead stretched sample method: Development and application to MALDI-MS imaging of protein localization in the spinal cord

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used to create spatial distribution maps from lipids, peptides, and proteins in a variety of biological tissues. MALDI-MSI often involves trade-offs between the extent of analyte extraction and desired spatial resolution, compromises that can adversely affect detectability. For example, increasing the extraction time can lead to unwanted analyte spatial redistribution. With the stretched sample method (SSM), the extraction period can be extended, resulting in reduced analyte redistribution while suppressing detection of cationic salt adducts. The SSM involves thaw-mounting a thin tissue section onto a substrate of small glass beads embedded in Parafilm M and then stretching the membrane to fragment the tissue into thousands of bead-sized pieces. Here, we applied the SSM method to MALDI-MSI using rat spinal cord as a model. We used surface-modified beads coated with trypsin or chymotrypsin in order to facilitate controlled digestion and detection of proteins. The enzymatic reactions were maintained by repeatedly condensing water on the stretched sample surface. As a result, new peptides formed by tryptic or chymotryptic protein digestion were detected and identified using a combination of MALDI-MSI and offline liquid chromatography tandem mass spectrometric analysis. Localization of these peptides indicated the distribution of their proteins of origin, including myelin basic protein, actin beta, and tubulin alpha chain. Additionally, we used uncoated beads to create distribution maps of many endogenous lipids and small peptides. The extension of the SSM using modified beads resulted in the creation of mosaic bead surfaces where adjacent beads were coated with different enzymes or other reactive chemicals, permitting investigation of the distributions of a wider range of analytes in biological samples within a single experiment.

Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

“…An innovative sample preparation technique, the stretched sample method, was developed by Zimmerman et al to account for the small size of single neurons [22, 53-55]. Briefly, dissected, individual neurons were placed on a bead monolayer array embedded in a stretchable substrate, Parafilm.…”

Stretched Tissue Mounting for MALDI Mass Spectrometry Imaging

“…19,21 The samples were stored at −80 °C until use. Prior to use, all samples were dried under nitrogen gas.…”

“…However, spatial resolution becomes limited by the minimum droplet spacing achievable by the printer (typically >100 μm). 3,19,24 Alternatively, an airbrush- or piezo-assisted spraying device can be used to coat the sample by producing small droplets (~20 μm), forming a thin layer of solution on the sample surface, resulting in small matrix crystal sizes. 26–27 Because of the smaller droplet and matrix crystal sizes, spraying provides relatively high spatial resolution with low analyte redistribution.…”

MALDI imaging mass spectrometry for direct tissue analysis: technological advancements and recent applications

“…The combination of MALDI-IMS with the ''sample stretching'' method proposed by Monroe et al divides the sample tissue into cell-sized fragments, destroying the morphological information and is therefore rather interesting for single cell analyses (Monroe et al 2006;Tucker et al 2011;Zimmerman et al 2008Zimmerman et al , 2010. The oversampling approach moves the laser over the sample in small increments and takes measurements with overlapping laser shots.…”