Unambiguous identification of compounds and their accurate quantification are often challenging in matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The goal of this project is to improve compound identification and quantification by developing a comprehensive workflow that combines MALDI-MSI with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Regions of interest (ROIs) are determined from thin tissue sections (mounted on microscope slides) by performing MALDI-MSI using a Bruker UltrafleXtreme time-of-flight (TOF) MALDI-MSI system. MALDI-MSI datasets are analyzed with SCiLS Lab (Bruker, Germany) for cluster analysis to determine ROIs for subsequent laser ablation sampling. Laser ablation of selected ROIs is accomplished using a mid-IR OPO laser system (at a wavelength of 3 µm); using a customized LabVIEW software, discrete ROI selection and sampling can be performed at a precision of 100 µm in oversampling mode at a speed of 1 mm/s. The laser ablated material is captured and subsequently digested with trypsin for bottom-up UPLC-MS/MS proteomics analysis employing an ion mobility (IM) enhanced data independent acquisition strategy (HDMSE). The UPLC-HDMSE analyses of the laser ablated tryptic digests are performed using a Waters Synapt G2-S HDMS quadrupole TOF mass spectrometer equipped with a nano ACQUITY UPLC system. Protein identification is conducted using Protein Lynx Global Server (PGLS, Waters Corporation, Milford, MA). Preliminary analysis of MS data from two regions of rat brain tissue allowed identification and label free quantification of 250 proteins. Our ongoing research efforts are focused on improved correlation of UPLC-HDMSE identified proteins to MALDI-MSI peaks. MSI heat maps will be graphically overlaid on camera imaging of a consecutive slide to help with both co-registration and ROI selection. We aim to enhance protein identification of MALDI-MSI peaks by combined use of UPLC-HDMSE with our in-house built database and software.

MALDI MSI directed-infrared laser microsampling for tissue proteomics

255th National Meeting (2018)

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