List of differentially-expressed proteins in rat brain sections observed on MALDI-TOF/TOF and identified by mass matching.

<p>• The italicized protein sequences have been verified by top-down tandem mass spectrometry. The rest of the identity assignments are solely based on mass matching. The proteins that can be identified by neither approach were shown as unknown.</p><p>• The bolded sequences represent the proteins that were reported from rat brain in a MALDI-MS-based platform for the first time.</p><p>The proteins that were further identified by top-down MS/MS were highlighted in italics and annotated with UniProt entry, accession number, calculated mass based on the m/z observed on Orbitrap Elite, expected mass based on the matched protein sequence and the mass difference between the calculated mass and expected mass. All MALDI-MS profiling spectra have been processed by ClinProTools and summarized here by “peak statistic” function. PTTA represents p-value of t-test (2 classes); Ratio represents the relative abundance changes of these proteins in the brain sections of rats treated with MK801 and vehicle, calculated based on the average intensity of the ion observed in the MK801-treated class over the vehicle-treated class.</p

Top-Down Proteomics with Mass Spectrometry Imaging: A Pilot Study towards Discovery of Biomarkers for Neurodevelopmental Disorders

<div><p>In the developing mammalian brain, inhibition of NMDA receptor can induce widespread neuroapoptosis, inhibit neurogenesis and cause impairment of learning and memory. Although some mechanistic insights into adverse neurological actions of these NMDA receptor antagonists exist, our understanding of the full spectrum of developmental events affected by early exposure to these chemical agents in the brain is still limited. Here we attempt to gain insights into the impact of pharmacologically induced excitatory/inhibitory imbalance in infancy on the brain proteome using mass spectrometric imaging (MSI). Our goal was to study changes in protein expression in postnatal day 10 (P10) rat brains following neonatal exposure to the NMDA receptor antagonist dizocilpine (MK801). Analysis of rat brains exposed to vehicle or MK801 and comparison of their MALDI MS images revealed differential relative abundances of several proteins. We then identified these markers such as ubiquitin, purkinje cell protein 4 (PEP-19), cytochrome c oxidase subunits and calmodulin, by a combination of reversed-phase (RP) HPLC fractionation and top-down tandem MS platform. More in-depth large scale study along with validation experiments will be carried out in the future. Overall, our findings indicate that a brief neonatal exposure to a compound that alters excitatory/inhibitory balance in the brain has a long term effect on protein expression patterns during subsequent development, highlighting the utility of MALDI-MSI as a discovery tool for potential biomarkers.</p></div

List of proteins that displayed consistent abundance levels in MK801-treated and vehicle-treated rat brain sections via MALDI-MS.

<p>These proteins were all identified by means of top-down tandem MS sequencing on a nanoLC-ESI-LTQ-Orbitrap Elite system.</p

Elevated level of the <i>m/z</i> 8566 ion quantified by the strategy combining MS profiling and imaging.

<p>A) The ratios of the intensities of the ion at <i>m/z</i> 8566 in the profiling spectra of specific regions, regions #2, #5, #3, #6, #7 and #14, where the ion was concentrated. *, p<0.05; **, p<0.01, unpaired student's t-test. (B) An optical image of a rat brain section washed after MSI experiments. (C) The <i>m/z</i> 8566 ion was up-regulated in the averaged MSI spectrum of the thalamus (TH) regions, ROI 1 and ROI2, of the rat brain section treated with vehicle and MK801 as shown in (E). (D) A loading plot of ROI 1 and ROI 2 analyzed by ClinProTools. The <i>m/z</i> 8566 ion was shown to contribute significantly in differentiating the two ROIs in Load 1. (E) MS images of the ions at <i>m/z</i> 8566 for the vehicle and the MK801-treated rat brain sections. The <i>m/z</i> 8566 ion was more concentrated in the frontal cortex, accumbens nucleus (Acb), thalamus (TH) and cerebellum regions in the MK801-treated section compared to the vehicle-treated one.</p

Fragmentation spectra and maps of the <i>m/z</i> 6718 and 8566 ions by HCD, CID and ETD.

<p>(A) HCD fragmentation spectrum of the <i>m/z</i> 6718 ion at monoisotopic <i>m/z</i> 1120.05 (z = 6). The ion at <i>m/z</i> 1120.05 (z = 6) was assigned as PEP-19 based on accurate mass and fragmentation pattern obtained on Orbitrap Elite. The boxed insert on the right is the deconvoluted spectrum of the <i>m/z</i> 1120.05 (z = 6) ion. The original HCD tandem MS spectrum containing the y₂₇ fragment ion is also zoomed-in as an insert on the left. (B) Fragmentation map of PEP-19 by three fragmentation techniques, HCD, CID and ETD. (C) HCD fragmentation spectrum of the <i>m/z</i> 8566 ion at monoisotopic <i>m/z</i> 1070.96 (z = 8). The ion at <i>m/z</i> 1070.96 (z = 8) was assigned as ubiquitin based on accurate mass and fragmentation pattern obtained on Orbitrap Elite. The boxed insert on the right is the deconvoluted spectrum of the <i>m/z</i> 1070.96 (z = 8) ion. The original HCD tandem MS spectrum containing the y₄₀ ion is also enlarged as an insert on the left. (D) Fragmentation map of Ubiquitin by HCD, CID and ETD. The number of the fragment ions produced by each of the fragmentation techniques is listed below the sequence. (E) An illustration of how the fragment ions of HCD (b, y ions), CID (b, y ions) and ETD (c, z ions) are produced and annotated in the fragmentation map is shown in (B) and (D).</p

Elevated level of the <i>m/z</i> 6718 ion quantified by the strategy combining MS profiling and imaging.

<p>A) The ratios of the intensities of the ion at <i>m/z</i> 6718 in the profiling spectra of specific regions, regions #1, #2, #5, #9, #7 and #14, where the ion was concentrated. The averaged intensities and p-values were calculated by ClinProTools and manually confirmed. *, p<0.05; **, p<0.01, unpaired student's t-test. (B) An optical image of a rat brain section washed after MSI experiments. (C) The <i>m/z</i> 6718 ion was up-regulated in the averaged MSI spectrum of the olfactory bulbs, ROI 1 and ROI2, of the rat brain section treated with vehicle and MK801 as shown in (E). (D) A loading plot of ROI 1 and ROI 2 analyzed by ClinProTools. The <i>m/z</i> 6718 ion was shown to contribute significantly in differentiating the two ROIs in Load 2. (E) MS images of the ions at <i>m/z</i> 6718 for the vehicle and the MK801-treated rat brain sections. The <i>m/z</i> 6718 ion was more concentrated in the olfactory bulb, cortex, thalamus (TH) and cerebellum regions in the MK801-treated section compared to the vehicle-treated one.</p

The workflow to identify the proteins that displayed changes in the MK801-treated samples.

<p>(A) MALDI-MS screening spectra of the HPLC fractions that contained the concentrated proteins of interest at <i>m/z</i> 6718 and 8566. (B) Representative MS spectrum acquired from a HPLC fraction containing the <i>m/z</i> 6718 ion on an ESI-Orbitrap Elite system, which provides ultra-high mass resolution and accuracy. The insert is the deconvoluted spectrum averaged from several multiply charged ion forms as highlighted in blue and red. (C) HCD, CID and ETD tandem MS fragmentation spectra of the particular ion at <i>m/z</i> 1120.05 (z = 6) highlighted in red.</p

An overall strategy for mapping proteins in brain tissues by combining MS profiling and imaging.

<p>The animals treated with MK801 were sacrificed and handled pairwise (A). The brains were removed and embedded in gelatin (B) followed by sectioning (C). Four sections from each brain were used and 14 regions as annotated in (C) from each section were profiled (D). An entire section was imaged from one brain (E). Four pairs of brains (vehicle <i>vs.</i> MK801) were profiled and imaged (N = 4), respectively, on a MALDI-TOF/TOF instrument (Bruker autoflex III) (F). The profiling spectra were compared pairwise for each specific region (G), and the imaging data were compared as well (H).</p

Regional profiling of the rat brain sections treated with vehicle and MK801.

<p>(A) Representative overall profiling spectra averaged from 14 regions of 4 serial sections from each animal (N = 4, two classes, i.e. vehicle-treated and MK801-treated rats as displayed in green and red, respectively). (B)–(E) are the zoomed-in spectra of several ions displaying differential expression levels in the two groups. The blue shades indicate the peaks picked by ClinProTools (signal to noise threshold higher than 5 in the average spectra) and included for statistical comparison between the two classes.</p

Map of the study cohort.

<p>A. Map of Bihar state, India with the boxed study cohort. B. Detailed map of Raghopur block showing the study cohort with seven villages of interest; details of water bodies, land and inhabitant distribution.</p