10 research outputs found
Shotgun Analysis of Diacylglycerols Enabled by Thiol–ene Click Chemistry
Diacylglycerols (DAGs)
are a subclass of neutral lipids actively
involved in cell signaling and metabolism. Alteration in DAG metabolism
has been associated with onset and progression of several human-related
diseases. The structural diversity of DAGs and their low concentrations
in biological samples call for the development of methods that are
capable of sensitive identification and quantitation of each DAG species
as well as rapid profiling when a biochemical pathway is perturbed.
In this work, the thiol–ene click chemistry has been employed
to introduce a charge-tag, namely, cysteamine (CA), at a carbon–carbon
double bond (Cî—»C) of unsaturated DAGs. This one-pot photochemical
derivatization is fast (within 1 min), universal (monotagging) for
DAGs varying in fatty acyl chain lengths and the number of Cî—»Cs,
and suitable for small sample volume (e.g., 1–50 μL plasma).
Because of the presence of the amine group in CA, tagged DAGs showed
at least 10 times increase in response to electrospray ionization
as compared to conventional ammonium adduct formation. Low-energy
collision-induced dissociation of CA tagged DAGs allowed confident
assignment of fatty acyl composition. A neutral loss scan based on
characteristic 95 Da loss (a combined loss of CA and H<sub>2</sub>O) of tagged DAGs has been established as a sensitive means for unsaturated
DAG detection (limit of detection = 100 pM) and quantitation from
mixtures. The analytical utility of CA tagging was demonstrated by
shotgun analysis of unsaturated DAGs in human plasma, including samples
from type 2 diabetes mellitus patients
Universal Multilayer Assemblies of Graphene in Chemically Resistant Microtubes for Microextraction
Graphene is a new kind of two-dimensional
carbon nanomaterial with
excellent properties and is promising for solid-phase microextraction
(SPME). Plastic microtubes such as polyÂ(tetrafluoroethylene) (PTFE)
and polyÂ(ether ether ketone) are ideal substrates for in-tube SPME.
However, immobilization of graphene layers onto these materials is
still a problem due to their nature of chemical resistance. In order
to solve the problem, we proposed a novel method based on universal
mussel-inspired polydopamine (PD) and layer-by-layer assembly of graphene
in this work. To make a graphene assembly layer inside PTFE, the strategy
includes two major steps. First, a PD layer is made on the PTFE surface
by noncovalent interaction. Second, multilayer graphene is assembled
on the PD layer by covalent interaction. By repeating these two steps,
a functional graphene oxide (FGO)-modified PTFE tube with a controllable
number of layers can be obtained. Morphology of the multilayer structure
of graphene has been confirmed by scanning electronic microscopy.
Formation of the covalent layer has also been characterized by Foourier
transform infrared and X-ray photoelectron spectroscopy. It is very
interesting that (FGO-PD)<sub>3</sub>-PTFE shows exceptional efficiency
for SPME. Enrichment from 1082- to 2331-fold was achieved for six
polyaromatic hydrocarbons (PAHs). An online SPME-HPLC-fluorescent
detection method has been developed on the basis of (FGO-PD)<sub>3</sub>-PTFE. For qualitative analysis of PAHs, the method has low limits
of detection of 0.05–0.1 pg/mL, which is significantly lower
(up to 1000 times) than that reported in literature. The method shows
wide linear range (0.3–200 pg/mL), good linearity (<i>R</i><sup>2</sup> ≥ 0.9968), and good reproducibility
(relative standard deviation < 3.4%). The method has been applied
to determine PAHs in environmental samples. Good recoveries were obtained,
ranging from 85.1% to 96.7%
LipidOA: A Machine-Learning and Prior-Knowledge-Based Tool for Structural Annotation of Glycerophospholipids
The Paternò–Büchi (PB) reaction
is a carbon–carbon
double bond (CC)-specific derivatization reaction that can
be used to pinpoint the location(s) of CC(s) in unsaturated
lipids and quantitate the location of isomers when coupled with tandem
mass spectrometry (MS/MS). As the data of PB-MS/MS are increasingly
generated, the establishment of a corresponding data analysis tool
is highly needed. Herein, LipidOA, a machine-learning and prior-knowledge-based
data analysis tool, is developed to analyze PB-MS/MS data generated
by liquid chromatography–mass spectrometry workflows. LipidOA
consists of four key functional modules to realize an annotation of
glycerophospholipid (GPL) structures at the fatty acyl-specific CC
location level. These include (1) data preprocessing, (2) picking
CC diagnostic ions, (3) de novo annotation,
and (4) result ranking. Importantly, in the result-ranking module,
the reliability of structural annotation is sorted via the use of
a machine learning classifier and comparison to the total fatty acid
database generated from the same sample. LipidOA is trained and validated
by four PB-MS/MS data sets acquired using different PB reagents on
mass spectrometers of different resolutions and of different biological
samples. Overall, LipidOA provides high precision (higher than 0.9)
and a wide coverage for structural annotations of GPLs. These results
demonstrate that LipidOA can be used as a robust and flexible tool
for annotating PB-MS/MS data collected under different experimental
conditions using different lipidomic workflows
MS<sup>3</sup> Imaging Enables the Simultaneous Analysis of Phospholipid CC and <i>sn</i>-Position Isomers in Tissues
Mass spectrometry (MS) imaging of lipids in tissues with
high structure
specificity is challenging in the effective fragmentation of position-selective
structures and the sensitive detection of multiple lipid isomers.
Herein, we develop an MS3 imaging method for the simultaneous
analysis of phospholipid CC and sn-position
isomers by on-tissue photochemical derivatization, nanospray desorption
electrospray ionization (nano-DESI), and a dual-linear ion trap MS
system. A novel laser-based sensing probe is developed for the real-time
adjustment of the probe-to-surface distance for nano-DESI. This method
is validated in mouse brain and kidney sections, showing its capability
of sensitive resolving and imaging of the fatty acyl chain composition,
the sn-position, and the CC location of phospholipids
in an MS3 scan. MS3 imaging of phospholipids
has shown the capability of differentiation of cancerous, fibrosis,
and adjacent normal regions in liver cancer tissues
Photochemical Tagging for Quantitation of Unsaturated Fatty Acids by Mass Spectrometry
Fatty acid (FA) profiling provides
phenotypic information and is
increasingly used in a broad range of biological and biomedical studies.
Quantitation of unsaturated FAs with confident carbon–carbon
double bond (Cî—»C) location assignment is both sample and time
consuming using traditional gas chromatography mass spectrometry analysis.
In this study, we developed a rapid, sensitive, and quantitative method
for profiling unsaturated FAs without using chromatographic separations.
This method was based on a combination of in-solution photochemical
tagging of a Cî—»C in FAs and a subsequent gas-phase detagging
via tandem (neutral loss scan) mass spectrometry. It enabled quantitation
of unsaturated FAs from various biological samples (blood, plasma,
and cell lines). More importantly, quantitative information on FA
Cî—»C location isomers, which was traditionally overlooked, could
now be obtained and applied to studying FA changes between normal
and cancerous human prostate cells
Zero-Order Release of Gossypol Improves Its Antifertility Effect and Reduces Its Side Effects Simultaneously
Gossypol
was considered a promising male contraceptive but finally
failed due to two side effects: hypokalemia and the irreversibility
of its contraceptive effect. Here we demonstrate that sustained zero-order
release could be a solution for these problems. The in vitro release
of gossypol from gossypol/PEG layer-by-layer films follows a perfect
zero-order kinetics. In vivo tests indicate that the films can maintain
the plasma drug concentration constant in male SD rats for ∼20
days for a 30-bilayer film. The plasma drug concentration is 2 orders
of magnitude lower than the peak plasma drug concentration when administered
orally and the daily dose is >50-fold lower than the commonly used
contraceptive oral dose. However, significant antifertility effects
were still observed. Furthermore, hypokalemia was not observed, and
the antifertility effects can be reversed after a recovery period.
The results suggest that zero-order release can significantly improve
the desired antifertility effect of gossypol and, meanwhile, significantly
reduce its side effects. We envision the drug could be developed to
be an effective, safe, and reversible male contraceptive by zero-order
release
Development of a Miniature Mass Spectrometry System for Point-of-Care Analysis of Lipid Isomers Based on Ozone-Induced Dissociation
Disorder of lipid homeostasis is closely associated with
a variety
of diseases. Although mass spectrometry (MS) approaches have been
well developed for the characterization of lipids, it still lacks
an integrated and compact MS system that is capable of rapid and detailed
lipid structural characterization and can be conveniently transferred
into different laboratories. In this work, we describe a novel miniature
MS system with the capability of both ozone-induced dissociation (OzID)
and collision-induced dissociation (CID) for the assignment of sites
of unsaturation and sn-positions in glycerolipids.
A miniature ozone generator was developed, which can be operated at
a relatively high pressure. By maintaining high-concentration ozone
inside the linear ion trap, OzID efficiency was significantly improved
for the identification of CC locations in unsaturated lipids,
with reaction times as short as 10 ms. Finally, the miniature OzID
MS system was applied to the analysis of CC locations and sn-positions of lipids from biological samples. Direct sampling
and fast detection of changes in phospholipid isomers were demonstrated
for the rapid discrimination of breast cancer tissue samples, showing
the potential of the miniature OzID MS system for point-of-care analysis
of lipid isomer biomarkers in complex samples
Efficient Sample Preparation System for Multi-Omics Analysis via Single Cell Mass Spectrometry
Mass
spectrometry (MS) has become a powerful tool for metabolome,
lipidome, and proteome analyses. The efficient analysis of multi-omics
in single cells, however, is still challenging in the manipulation
of single cells and lack of in-fly cellular digestion and extraction
approaches. Here, we present a streamlined strategy for highly efficient
and automatic single-cell multi-omics analysis by MS. We developed
a 10-pL-level microwell chip for housing individual single cells,
whose proteins were found to be digested in 5 min, which is 144 times
shorter than traditional bulk digestion. Besides, an automated picoliter
extraction system was developed for sampling of metabolites, phospholipids,
and proteins in tandem from the same single cell. Also, 2 min MS2 spectra were obtained from 700 pL solution of a single cell
sample. In addition, 1391 proteins, phospholipids, and metabolites
were detected from one single cell within 10 min. We further analyzed
cells digested from cancer tissue samples, achieving up to 40% increase
in cell classification accuracy using multi-omics analysis in comparison
with single-omics analysis. This automated single-cell MS strategy
is highly efficient in analyzing multi-omics information for investigation
of cell heterogeneity and phenotyping for biomedical applications
Metabonomics Indicates Inhibition of Fatty Acid Synthesis, β‑Oxidation, and Tricarboxylic Acid Cycle in Triclocarban-Induced Cardiac Metabolic Alterations in Male Mice
Triclocarban (TCC) has been identified
as a new environmental pollutant
that is potentially hazardous to human health; however, the effects
of short-term TCC exposure on cardiac function are not known. The
aim of this study was to use metabonomics and molecular biology techniques
to systematically elucidate the molecular mechanisms of TCC-induced
effects on cardiac function in mice. Our results show that TCC inhibited
the uptake, synthesis, and oxidation of fatty acids, suppressed the
tricarboxylic acid (TCA) cycle, and increased aerobic glycolysis levels
in heart tissue after short-term TCC exposure. TCC also inhibited
the nuclear peroxisome proliferator-activated receptor α (PPARα),
confirming its inhibitory effects on fatty acid uptake and oxidation.
Histopathology and other analyses further confirm that TCC altered
mouse cardiac physiology and pathology, ultimately affecting normal
cardiac metabolic function. We elucidate the molecular mechanisms
of TCC-induced harmful effects on mouse cardiac metabolism and function
from a new perspective, using metabonomics and bioinformatics analysis
data
Guaianolide Sesquiterpene Lactones, a Source To Discover Agents That Selectively Inhibit Acute Myelogenous Leukemia Stem and Progenitor Cells
Small molecules that can selectively target cancer stem
cells (CSCs)
remain rare currently and exhibit no common structural features. Here
we report a series of guaianolide sesquiterpene lactones (GSLs) and
their derivatives that can selectively eradicate acute myelogenous
leukemia (AML) stem or progenitor cells. Natural GSL compounds arglabin,
an anticancer clinical drug, and micheliolide (MCL), are able to reduce
the proportion of AML stem cells (CD34<sup>+</sup>CD38<sup>–</sup>) in primary AML cells. Targeting of AML stem cells is further confirmed
by a sharp reduction of colony-forming units of primary AML cells
upon MCL treatment. Moreover, DMAMCL, the dimethylamino Michael adduct
of MCL, slowly releases MCL in plasma and in vivo and demonstrates
remarkable therapeutic efficacy in the nonobese diabetic/severe combined
immunodeficiency AML models. These findings indicate that GSL is an
ample source for chemical agents against AML stem or progenitor cells
and that GSL is potentially highly useful to explore anti-CSC approaches