35 research outputs found
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
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
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
Fragmentation of Integral Membrane Proteins in the Gas Phase
Integral membrane proteins (IMPs)
are of great biophysical and
clinical interest because of the key role they play in many cellular
processes. Here, a comprehensive top down study of 152 IMPs and 277
soluble proteins from human H1299 cells including 11 087 fragments
obtained from collisionally activated dissociation (CAD), 6452 from
higher-energy collisional dissociation (HCD), and 2981 from electron
transfer dissociation (ETD) shows their great utility and complementarity
for the identification and characterization of IMPs. A central finding
is that ETD is ∼2-fold more likely to cleave in soluble regions
than threshold fragmentation methods, whereas the reverse is observed
in transmembrane domains with an observed ∼4-fold bias toward
CAD and HCD. The location of charges just prior to dissociation is
consistent with this directed fragmentation: protons remain localized
on basic residues during ETD but easily mobilize along the backbone
during collisional activation. The fragmentation driven by these protons,
which is most often observed in transmembrane domains, both is of
higher yield and occurs over a greater number of backbone cleavage
sites. Further, while threshold dissociation events in transmembrane
domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from
the nearest charge site (R, K, H, N-terminus), fragmentation is strongly
influenced by the N- or C-terminal position relative to that site:
the ratio of observed b- to y-fragments is ∼1:3 if the cleavage
occurs >7 residues N-terminal and ∼3:1 if it occurs >7
residues
C-terminal to the nearest basic site. Threshold dissociation products
driven by a mobilized proton appear to be strongly dependent on not
only relative position of a charge site but also N- or C-terminal
directionality of proton movement
The C‑Score: A Bayesian Framework to Sharply Improve Proteoform Scoring in High-Throughput Top Down Proteomics
The
automated processing of data generated by top down proteomics
would benefit from improved scoring for protein identification and
characterization of highly related protein forms (proteoforms). Here
we propose the “C-score” (short for Characterization
Score), a Bayesian approach to the proteoform identification and characterization
problem, implemented within a framework to allow the infusion of expert
knowledge into generative models that take advantage of known properties
of proteins and top down analytical systems (e.g., fragmentation propensities,
“off-by-1 Da” discontinuous errors, and intelligent
weighting for site-specific modifications). The performance of the
scoring system based on the initial generative models was compared
to the current probability-based scoring system used within both ProSightPC
and ProSightPTM on a manually curated set of 295 human proteoforms.
The current implementation of the C-score framework generated a marked
improvement over the existing scoring system as measured by the area
under the curve on the resulting ROC chart (AUC of 0.99 versus 0.78)
'Iter Dalekarlicum'
Mass spectrometry based proteomics generally seeks to
identify
and fully characterize protein species with high accuracy and throughput.
Recent improvements in protein separation have greatly expanded the
capacity of top-down proteomics (TDP) to identify a large number of
intact proteins. To date, TDP has been most tightly associated with
Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry.
Here, we couple the improved separations to a Fourier-transform instrument
based not on ICR but using the Orbitrap Elite mass analyzer. Application
of this platform to H1299 human lung cancer cells resulted in the
unambiguous identification of 690 unique proteins and over 2000 proteoforms
identified from proteins with intact masses <50 kDa. This is an
early demonstration of high throughput TDP (>500 identifications)
in an Orbitrap mass spectrometer and exemplifies an accessible platform
for whole protein mass spectrometry
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 y<sub>27</sub> 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 y<sub>40</sub> 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