4 research outputs found
Brain Region-Specific Dynamics of On-Tissue Protein Digestion Using MALDI Mass Spectrometry Imaging
In mass spectrometry imaging (MSI),
on-tissue proteolytic digestion
is performed to access larger protein species and to assign protein
identities through matching the detected peaks with those obtained
by LCāMS/MS analyses of tissue extracts. The on-tissue proteolytic
digestion also allows the analysis of proteins from formalin-fixed,
paraffin-embedded tissues. For these reasons, on-tissue digestion-based
MSI is frequently used in clinical investigations, for example, to
determine changes in protein content and distribution associated with
a disease. In this work, we sought to investigate the completeness
and uniformity of the digestion in on-tissue digestion MSI. On the
basis of an extensive experiment investigating three groups with varying
incubation times: (i) 1.5 h, (ii) 3 h, and (iii) 18 h, we have found
that longer incubation times improve the repeatability of the analyses.
Furthermore, we discovered morphology-associated differences in the
completeness of the proteolysis for short incubation times. These
results support the notion that a more complete proteolysis allows
better quantitation
Histology-Guided High-Resolution Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging
Mass spectrometry imaging (MSI) is
widely used for clinical research
because when combined with histopathological analysis the molecular
signatures of specific cells/regions can be extracted from the often-complex
histologies of pathological tissues. The ability of MSI to stratify
patients according to disease, prognosis, and response is directly
attributable to this cellular specificity. MSI developments are increasingly
focused on further improving specificity, through higher spatial resolution
to better localize the signals or higher mass resolution to better
resolve molecular ions. Higher spatial/mass resolution leads to increased
data size and longer data acquisition times. For clinical applications,
which analyze large series of patient tissues, this poses a challenge
to keep data load and acquisition time manageable. Here we report
a new tool to perform histology guided MSI; instead of analyzing large
parts of each tissue section the histology from adjacent tissue sections
is used to focus the analysis on the areas of interest, e.g., comparable
cell types in different patient tissues, thereby minimizing data acquisition
time and data load. The histology tissue section is annotated and
then automatically registered to the MSI-prepared tissue section;
the registration transformation is then applied to the annotations,
enabling them to be used to define the MSI measurement regions. Using
a series of formalin-fixed, paraffin-embedded human myxoid liposarcoma
tissues, we demonstrate an 80% reduction of data load and acquisition
time, thereby enabling high resolution (mass or spatial) to be more
readily applied to clinical research. The software is freely available
for download
Comprehensive Analysis of the Mouse Brain Proteome Sampled in Mass Spectrometry Imaging
On-tissue enzymatic digestion is
performed in mass spectrometry
imaging (MSI) experiments to access larger proteins and to assign
protein identities. Most on-tissue digestion MSI studies have focused
on method development rather than identifying the molecular features
observed. Herein, we report a comprehensive study of the mouse brain
proteome sampled by MSI. Using complementary proteases, we were able
to identify 5337 peptides in the matrix-assisted laser desorption/ionization
(MALDI) matrix, corresponding to 1198 proteins. 630 of these peptides,
corresponding to 280 proteins, could be assigned to peaks in MSI data
sets. Gene ontology and pathway analyses revealed that many of the
proteins are involved in neurodegenerative disorders, such as Alzheimerās,
Parkinsonās, and Huntingtonās disease
Multimodal Mass Spectrometry Imaging of <i>N</i>āGlycans and Proteins from the Same Tissue Section
On-tissue
digestion matrix-assisted laser desorption/ionization mass spectrometry
imaging (MALDI-MSI) can be used to record spatially correlated molecular
information from formalin-fixed, paraffin-embedded (FFPE) tissue sections.
In this work, we present the <i>in situ</i> multimodal analysis
of <i>N</i>-linked glycans and proteins from the same FFPE
tissue section. The robustness and applicability of the method are
demonstrated for several tumors, including epithelial and mesenchymal
tumor types. Major analytical aspects, such as lateral diffusion of
the analyte molecules and differences in measurement sensitivity due
to the additional sample preparation methods, have been investigated
for both <i>N</i>-glycans and proteolytic peptides. By combining
the MSI approach with extract analysis, we were also able to assess
which mass spectral peaks generated by MALDI-MSI could be assigned
to unique <i>N</i>-glycan and peptide identities