Abstract
Many pathological processes are not directly correlated to dramatic alterations in protein levels. The changes in local concentrations of important proteins in a subset of cells or at specific loci are likely to play a significant role in disease etiologies, but the precise location might be unknown, or the concentration might be too small to be adequately sampled for traditional proteomic techniques. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a unique analytical method that combines analysis of multiple molecular species and of their distribution in a single platform. As reproducibility is essential for successful biomarker discovery, it is important to systematically assess data quality in biologically relevant MALDI IMS experiments. In the present study, we applied four simple tools to study the reproducibility for individual sections, within-group variation, and between-group variation of data acquired from brain sections of 21 animals divided into three treatment groups. We also characterized protein changes in distinct regions of the striatum from six-month-old rats treated neonatally (postnatal days 9–10) with the cyanobacterial toxin β-N-methylamino-l-alanine (BMAA), which has been implicated in neurodegenerative diseases. The results showed that optimized experimental settings can yield high-quality MALDI IMS data with relatively low variation (14% to 15% coefficient of variance) that allow the characterization of subtle changes in protein expression in various subregions of the brain. This was further exemplified by the dose-dependent reduction of myelin basic protein in the caudate putamen and the nucleus accumbens of adult rats neonatally treated with BMAA (150 and 460 mg/kg). The reduction in myelin basic protein was confirmed through immunohistochemistry and indicates that developmental exposure to BMAA may induce structural effects on axonal growth and/or directly on the proliferation of oligodendrocytes and myelination, which might be important for the previously shown BMAA-induced long-term cognitive impairments.
Highlights
From the ‡Department of Pharmaceutical Biosciences, Uppsala University, 751 24 Uppsala, Sweden; §Department of Chemistry BMC, Analytical Chemistry, Uppsala University, 751241 Uppsala, Sweden
We used Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) as a discovery tool in a non-hypothesisdriven study to pinpoint brain areas of interest and characterize long-term protein changes in adult rats neonatally treated with BMAA
Imaging and histology-directed tissue-profiling MALDI mass spectrometry (MS) are characterized by identical sources of variation; there are undeniable benefits to the evaluation of MS data quality when it comes to imaging MS, as the evaluation of multiple ion distribution images quickly reveals overnormalization effects and defects in the experimental data [18]
Summary
From the ‡Department of Pharmaceutical Biosciences, Uppsala University, 751 24 Uppsala, Sweden; §Department of Chemistry BMC, Analytical Chemistry, Uppsala University, 751241 Uppsala, Sweden. To obtain high signal quality and reproducible IMS results, we have used robotic spotting in several studies (2, 18 –21) This strategy is time consuming and the image resolution is limited to the size and spacing of the matrix deposits (usually 200 to 300 m), we have repeatedly observed low variance in peak intensities from several brain areas. A lower %CV is observed in the analysis of small proteins using sinapinic acid as a matrix (15% CV, compared with 30% CV using 2,5-dihydroxybenzoic acid for neuropeptide analysis) (2, 18 –21) This is comparable to results for histology-directed profiling MALDI IMS (34% CV [9]).
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