title: Data quality in tissue analysis using desorption electrospray ionization.
abstract: There has been a recent surge in applications of mass spectrometry (MS) to tissue analysis, particularly lipid-based tissue imaging using ambient ionization techniques. This recent growth highlights the need to examine the effects of sample handling, storage conditions, and experimental protocols on the quality of the data obtained. Variables such as time before freezing after organ removal, storage time at -80 °C, time stored at room temperature, heating, and freeze/thaw cycles were investigated for their effect on the data quality obtained in desorption electrospray ionization (DESI)-MS using mouse brain. In addition, analytical variables such as tissue thickness, drying times, and instrumental conditions were also examined for their impact on DESI-MS data. While no immediate changes were noted in the DESI-MS lipid profiles of the mouse brain tissue after spending 1 h at room temperature when compared to being frozen immediately following removal, minor changes were noted between the tissue samples after 7 months of storage at -80 °C. In tissue sections stored at room temperature, degradation was noted in 24 h by the appearance of fatty acid dimers, which are indicative of high fatty acid concentrations, while in contrast, those sections stored at -80 °C for 7 months showed no significant degradation. Tissue sections were also subjected to up to six freeze/thaw cycles and showed increasing degradation following each cycle. In addition, tissue pieces were subjected to 50 °C temperatures and analyzed at specific time points. In as little as 2 h, degradation was observed in the form of increased fatty acid dimer formation, indicating that enzymatic processes forming free fatty acids were still active in the tissue. We have associated these dimers with high concentrations of free fatty acids present in the tissue during DESI-MS experiments. Analytical variables such as tissue thickness and time left to dry under nitrogen were also investigated, with no change in the resulting profiles at thickness from 10 to 25 μm and with optimal signal obtained after just 20 min in the dessicator. Experimental conditions such as source parameters, spray solvents, and sample surfaces are all shown to impact the quality of the data. Inter-section (relative standard deviation (%RSD), 0.44-7.2%) and intra-sample (%RSD, 4.0-8.0%) reproducibility data show the high quality information DESI-MS provides. Overall, the many variables investigated here showed DESI-MS to be a robust technique, with sample storage conditions having the most effect on the data obtained, and with unacceptable sample degradation occurring during room temperature storage.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Allison L
authors.initials: AL
authors.lastname: Dill
authors.forename: Livia S
authors.initials: LS
authors.lastname: Eberlin
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Costa
authors.forename: Demian R
authors.initials: DR
authors.lastname: Ifa
authors.forename: R Graham
authors.initials: RG
authors.lastname: Cooks
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D005227:Fatty Acids / Q000032:analysis; D005615:Freezing; D008055:Lipids / Q000032:analysis*; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D005227:Fatty Acids; D008055:Lipids