19 research outputs found
Evaluation of Thin-Layer Chromatography–Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Imaging for Visualization of Crude Oil Interactions
A light oil was separated
into four chromatographic fractions that
serve as proxy for SARA fractions. The fractions were (semi)quantified
on a rod by TLC-flame ionization detection and characterized on a
plate with laser desorption ionization–mass spectrometry imaging
(TLC-LDI-MS). Comparisons of (semi)quantitative TLC-FID and qualitative
TLC-LDI-MS results showed that LDI-MS was most sensitive for detection
of molecules in the polar P1 fraction, and, to some extent, for the
aromatics fraction, while no signal was observed for the most polar
P2 and saturates fractions. Based on these results, limits of the
compositional space, as observed by the laser ionization technique,
were evaluated. The molecular speciation between and within the spots
of the aromatics and the P1 fractions were analyzed and interpreted
in terms of oil–SiO<sub>2</sub> versus oil–solvent interactions,
as a function of molecular characteristics such as DBE, aromaticity
(H/C ratio), heteroatom content, degree of alkylation, and shielding
of heteroatoms. In addition, the high oil loading resulted in an interesting
bifurcation of the aromatics spot, which implies that oil–oil
interactions can be enforced and studied in the TLC model system
Gold Sputtered Fiducial Markers for Combined Secondary Ion Mass Spectrometry and MALDI Imaging of Tissue Samples
Mass
spectrometry imaging (MSI) is a label free technique capable
of providing simultaneous identification and localization of biomolecules.
A multimodal approach is required that allows for the study of the
complexity of biological tissue samples to overcome the limitations
of a single MSI technique. Secondary ion mass spectrometry (SIMS)
allows for high spatial resolution imaging while matrix-assisted laser
desorption (MALDI) offers a significantly wider mass range. The combination
of coregistered SIMS and MALDI images results in detailed and unique
biomolecular information. In this Technical Note, we describe how
gold sputtered/implanted fiducial markers (FM) are created and can
be used to ensure a proper overlay and coregistration of the two-dimensional
images provided by the two MSI modalities
Characterization of Phosphatidylcholine Oxidation Products by MALDI MS<sup><i>n</i></sup>
Phospholipid oxidation has been implicated
in the pathogenesis
and progression of numerous age-related and neurodegenerative diseases.
Despite these implications, this broad class of biomolecules remains
poorly characterized. In this work, the fragmentation patterns of
[M + H]<sup>+</sup> and [M + Na]<sup>+</sup> ions of intact phosphatidylcholine
oxidation products (OxPCs) were characterized by matrix-assisted laser
desorption/ionization tandem mass spectrometry (MALDI MS<sup><i>n</i></sup>, <i>n</i> = 2, 3, and 4). MS<sup>2</sup> of both the [M + H]<sup>+</sup> and [M + Na]<sup>+</sup> ions of
short-chain OxPCs yielded product ions related to the PC headgroup
and the fatty acid substituents. MS<sup>3</sup> of the [M + Na –
N(CH<sub>3</sub>)<sub>3</sub>]<sup>+</sup> ions yielded fragmentation
indicative of the OxPC modification; specifically, a product ion corresponding
to the neutral loss of CO<sub>2</sub> (NL of 44) was observed for
OxPCs containing a terminal carboxylic acid rather than an aldehyde.
Furthermore, MS<sup>4</sup> of the [M + Na – HPO<sub>4</sub>(CH<sub>2</sub>)<sub>2</sub>N(CH<sub>3</sub>)<sub>3</sub>]<sup>+</sup> ions resulted in fragmentation pathways dependent on the <i>sn</i>-2 fatty acid chain length and type of functional group(s).
Specifically, CHO-containing OxPCs with palmitic acid esterified to
the <i>sn</i>-1 position of the glycerol backbone yielded
a NL of 254, 2 u less than the nominal mass of palmitic acid, whereas
the analogous terminal COOH-containing OxPCs demonstrated a NL of
256. Finally, the presence of a γ-ketone relative to the terminal
carboxyl group resulted in C–C bond cleavages along the <i>sn</i>-2 substituent, providing diagnostic product ions for
keto-containing OxPCs. This work illustrates the enhanced selectivity
afforded by MS<sup><i>n</i></sup> on the linear ion trap
and develops a method for the identification of individual products
of PC oxidation
Time-of-Flight Secondary Ion Mass Spectrometry-Based Molecular Distribution Distinguishing Healthy and Osteoarthritic Human Cartilage
Osteoarthritis (OA) is a pathology that ultimately causes
joint destruction. The cartilage is one of the principal affected
tissues. Alterations in the lipid mediators and an imbalance in the
metabolism of cells that form the cartilage (chondrocytes) have been
described as contributors to the OA development. In this study, we
have studied the distribution of lipids and chemical elements in healthy
and OA human cartilage. Time of flight-secondary ion mass spectrometry
(TOF-SIMS) allows us to study the spatial distribution of molecules
at a high resolution on a tissue section. TOF-SIMS revealed a specific
peak profile that distinguishes healthy from OA cartilages. The spatial
distribution of cholesterol-related peaks exhibited a remarkable difference
between healthy and OA cartilages. A distinctive colocalization of
cholesterol and other lipids in the superficial area of the cartilage
was found. A higher intensity of oleic acid and other fatty acids
in the OA cartilages exhibited a similar localization. On the other
hand, CN<sup>–</sup> was observed with a higher intensity in
the healthy samples. Finally, we observed an accumulation of calcium
and phosphate ions exclusively in areas surrounding the chondrocyte
in OA tissues. To our knowledge, this is the first time that TOF-SIMS
revealed combined changes in the molecular distribution in the OA
human cartilage
Active Learning for Convenient Annotation and Classification of Secondary Ion Mass Spectrometry Images
Digital staining for the automated annotation of mass
spectrometry
imaging (MSI) data has previously been achieved using state-of-the-art
classifiers such as random forests or support vector machines (SVMs).
However, the training of such classifiers requires an expert to label
exemplary data in advance. This process is time-consuming and hence
costly, especially if the tissue is heterogeneous. In theory, it may
be sufficient to only label a few highly representative pixels of
an MS image, but it is not known a priori which pixels to select.
This motivates <i>active learning</i> strategies in which
the algorithm itself queries the expert by automatically suggesting
promising candidate pixels of an MS image for labeling. Given a suitable
querying strategy, the number of required training labels can be significantly
reduced while maintaining classification accuracy. In this work, we
propose active learning for convenient annotation of MSI data. We
generalize a recently proposed active learning method to the multiclass
case and combine it with the random forest classifier. Its superior
performance over random sampling is demonstrated on secondary ion
mass spectrometry data, making it an interesting approach for the
classification of MS images
Digestion-Free Analysis of Peptides from 30-year-old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging
Formalin-fixed
neuroendocrine tissues from American cockroaches
(Periplaneta americana) embedded in
paraffin more than 30 years ago were recently analyzed by matrix-assisted
laser desorption/ionization mass spectrometry imaging (MALDI-MSI),
to reveal the histological localization of more than 20 peptide ions.
These represented protonated, and other cationic species of, at least,
14 known neuropeptides. The characterization of peptides in such historical
samples was made possible by a novel sample preparation protocol rendering
the endogenous peptides readily amenable to MSI analysis. The protocol
comprises brief deparaffinization steps involving xylene and ethanol,
and is further devoid of conventional aqueous washing, buffer incubations,
or antigen retrieval steps. Endogenous secretory peptides that are
typically highly soluble are therefore retained in-tissue with this
protocol. The method is fully “top-down”, that is, without
laborious in situ enzymatic digestion that typically disturbs the
detection of low-abundance endogenous peptides by MSI. Peptide identifications
were supported by accurate mass, on-tissue tandem MS analyses, and
by earlier MALDI-MSI results reported for freshly prepared P. americana samples. In contrast to earlier literature
accounts stating that MALDI-MSI detection of endogenous peptides is
possible only in fresh or freshly frozen tissues, or exceptionally,
in formalin-fixed, paraffin-embedded (FFPE) material of less than
1 year old, we demonstrate that MALDI-MSI works for endogenous peptides
in FFPE tissue of up to 30 years old. Our findings put forward a useful
method for digestion-free, high-throughput analysis of endogenous
peptides from FFPE samples and offer the potential for reinvestigating
archived and historically interesting FFPE material, such as those
stored in hospital biobanks
The Use of Mass Spectrometry Imaging to Predict Treatment Response of Patient-Derived Xenograft Models of Triple-Negative Breast Cancer
In recent years,
mass spectrometry imaging (MSI) has been shown
to be a promising technique in oncology. The effective application
of MSI, however, is hampered by the complexity of the generated data.
Bioinformatic approaches that reduce the complexity of these data
are needed for the effective use in a (bio)medical setting. This holds
especially for the analysis of tissue microarrays (TMA), which consist
of hundreds of small tissue cores. Here we present an approach that
combines MSI on tissue microarrays with principal component linear
discriminant analysis (PCA-LDA) to predict treatment response. The
feasibility of such an approach was evaluated on a set of patient-derived
xenograft models of triple-negative breast cancer (TNBC). PCA-LDA
was used to classify TNBC tumor tissues based on the proteomic information
obtained with matrix-assisted laser desorption ionization (MALDI)
MSI from the TMA surface. Classifiers based on two different tissue
microarrays from the same tumor models showed overall classification
accuracies between 59 and 77%, as determined by cross-validation.
Reproducibility tests revealed that the two models were similar. A
clear effect of intratumor heterogeneity of the classification scores
was observed. These results demonstrate that the analysis of MALDI-MSI
data by PCA-LDA is a valuable approach for the classification of treatment
response and tumor heterogeneity in breast cancer
Oxygen-Dependent Lipid Profiles of Three-Dimensional Cultured Human Chondrocytes Revealed by MALDI-MSI
Articular
cartilage is exposed to a gradient of oxygen levels ranging
from 5% at the surface to 1% in the deepest layers. While most cartilage
research is performed in supraphysiological oxygen levels (19–21%),
culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes
the chondrogenic phenotype. Exposure of cells to various oxygen levels
alters their lipid metabolism, but detailed studies examining how
hypoxia affects lipid metabolism in chondrocytes are lacking. To better
understand the chondrocyte’s behavior in response to oxygen,
we cultured 3D pellets of human primary chondrocytes in normoxia (20%
oxygen) and hypoxia (2.5% oxygen) and employed matrix-assisted laser
desorption ionization mass spectrometry imaging (MALDI-MSI) in order
to characterize the lipid profiles and their spatial distribution.
In this work we show that chondrocytes cultured in hypoxia and normoxia
can be differentiated by their lipid profiles. Among other species,
phosphatidylglycerol species were increased in normoxic pellets, whereas
phosphatidylinositol species were the most prominent lipids in hypoxic
pellets. Moreover, spatial mapping revealed that phospahtidylglyycerol
species were less prominent in the center of pellets where the oxygen
level is lower. Additional analysis revealed a higher abundance of
the mitochondrial-specific lipids, cardiolipins, in normoxic conditions.
In conclusion MALDI-MSI described specific lipid profiles that could
be used as sensors of oxygen level changes and may especially be relevant
for retaining the chondrogenic phenotype, which has important implications
for the treatment of bone and cartilage diseases
Integration of Ion Mobility MS<sup>E</sup> after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography
Direct
analysis by mass spectrometry (imaging) has become increasingly
deployed in preclinical and clinical research due to its rapid and
accurate readouts. However, when it comes to biomarker discovery or
histopathological diagnostics, more sensitive and in-depth profiling
from localized areas is required. We developed a comprehensive, fully
automated online platform for high-resolution liquid extraction surface
analysis (HR-LESA) followed by micro–liquid chromatography
(LC) separation and a data-independent acquisition strategy for untargeted
and low abundant analyte identification directly from tissue sections.
Applied to tissue sections of rat pituitary, the platform demonstrated
improved spatial resolution, allowing sample areas as small as 400
μm to be studied, a major advantage over conventional LESA.
The platform integrates an online buffer exchange and washing step
for removal of salts and other endogenous contamination that originates
from local tissue extraction. Our carry over–free platform
showed high reproducibility, with an interextraction variability below
30%. Another strength of the platform is the additional selectivity
provided by a postsampling gas-phase ion mobility separation. This
allowed distinguishing coeluted isobaric compounds without requiring
additional separation time. Furthermore, we identified untargeted
and low-abundance analytes, including neuropeptides deriving from
the pro-opiomelanocortin precursor protein and localized a specific
area of the pituitary gland (i.e., adenohypophysis) known to secrete
neuropeptides and other small metabolites related to development,
growth, and metabolism. This platform can thus be applied for the
in-depth study of small samples of complex tissues with histologic
features of ∼400 μm or more, including potential neuropeptide
markers involved in many diseases such as neurodegenerative diseases,
obesity, bulimia, and anorexia nervosa
Multiorder Correction Algorithms to Remove Image Distortions from Mass Spectrometry Imaging Data Sets
Time-of-flight
secondary ion mass spectrometry imaging is a rapidly
evolving technology. Its main application is the study of the distribution
of small molecules on biological tissues. The sequential image acquisition
process remains susceptible to measurement distortions that can render
imaging data less analytically useful. Most of these artifacts show
a repetitive nature from tile to tile. Here we statistically describe
these distortions and derive two different algorithms to correct them.
Both a generalized linear model approach and the linear discriminant
analysis approach are able to increase image quality for negative
and positive ion mode data sets. Additionally, performing simulation
studies with repetitive and nonrepetitive tiling error we show that
both algorithms are only removing repetitive distortions. It is further
shown that the spectral component of the data set is not altered by
the use of these correction methods. Both algorithms presented in
this work greatly increase the image quality and improve the analytical
usefulness of distorted images dramatically