Page 29 - Shimadzu Journal vol.9 Issue1

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Untargeted LC-MS/MS-based metabolic phenotyping Expanding capabilities in routine clinical
applied to the CD248 knock out mouse model toxicology screening using HRAM QTOF
Untargeted LC-MS/MS is a powerful tool by which to identify metabolic Low resolution mass spectrometry with triple quadrupole MS/MS systems
phenotypes. Here we applied metabolic phenotyping analysis to the deliver highly sensitive, robust, reproducible and proven technology plat-
CD248 knockout mouse model. CD248 is a transmembrane glycoprotein, forms for targeted clinical toxicology screening and identifying unknown
expression of which is markedly upregulated in a considerable number of compounds in patient samples. However, high resolution accurate mass-
disease models including tumor growth, inflammation and injury-induced spectrometers (HRAM) provide support for targeted and untargeted work-
fibrosis. In human clinical studies, CD248 expression is upregulated in the flows in which no spectral data are lost and retrospective data analysis can
fat cells of patients with diabetes, conversely, CD248 expression reverted to be supported. To reduce false positive reporting a HRAM data-independent-
a normal range when obesityassociated diabetes was reversed through acquisition MS and MS/MS method was used together with a HRAM MS/
weight loss. In this work, an untargeted LC-MS/MS metabolic phenotyping MS clinical toxicology library for routine screening assays.
analysis, using a reverse-phase LC separation and high resolution accurate
mass (HRAM), was applied to a CD248-/-mouse model following high fat
diet (HFD) feeding to study the effects of diet on serum metabolite profiles.

High Sensitivity Analysis of Steroid Hormones with MALDI-nanochip based Screening of
modified ESI to improve desolvation efficiency Exosomal Biomarkers
Development of a high-sensitivity method to assay a steroid panel in serum We previously demonstrated we could rapidly distinguish fluorouracil re-
samples. Thanks to LC-MS/MS with the newly developed ion source sistant cancer sample groups based on protein profiling of extracellular
IonFocus Unit, the sensitivity of steroid hormone was improved. Steroid vesicles using a linear benchtop MALDI TOF instrument [1]. The aim of
hormones play a major role in the control of metabolism, neurotransmis- this follow up work is to identify the proteins that are differentially ex-
sion, intracellular signaling, gene expression, reproduction and cardiovas- pressed in the different sample groups in order to better understand the
cular. Therefore, steroid hormones are very important in elucidating the disease processes and to support the rapid screening approach developed
mechanisms of various diseases. Furthermore, not only do steroids play previously. Here we present the results from this study using a high perfor-
roles in sedation and seizure prevention, they are known to be effective in mance reflectron MS/MS MALDI-TOF platform for the comparative pro-
cancer treatment and regenerative medicine. Therefore, highly sensitive teomic profiling of circulating extracellular vesicles extracted from
analytical technologies to steroid hormones quantitation in biological sam- colorectal cancer plasma samples.
ples are required in clinical research. Here we investigated higher sensitive
analytical methods to steroid hormones by improving desolvation efficiency
in LC-MS/MS ion source.

LC-MS/MS method development of aflibercept using Multi-target screening of toxicological compounds
Fab-selective proteolysis nSMOL technology in blood on a fully-automated platform consisting of
Aflibercept is a biopharmaceutical drug inhibiting of vascular endothelial sample preparation module CLAM and LC-MS/MS
growth factor (VEGF) signaling and composed of the extracellular domains Multi-target screening by LC/MS/MS has been widely adopted in detection
of human VEGF receptors 1 and 2 that are fused to the Fc portion of the and quantitation of drugs of abuse (DoA) in forensic investigation and toxi-
human IgG1 immunoglobulin. It is important to determine an appropriate cological research. Usually, a wide range of targets are screened in such
dose for medical optimization, but little amount of intraocular fluid is able analysis, including illicit drugs, narcotics, psychotropics, antipsychotics,
to collect as a specimen in the treatment of retina. To offer the quantitative pharmaceuticals and other toxic compounds in urine, serum/plasma and
assessment method of aflibercept in biological matrix, the primary structure whole blood samples. Sample preparation is often a bottleneck due to the
was confirmed using a quadrupole time-of-flight (Q-TOF) mass spectrome- tedious steps. It is also a factor responsible for inaccurate or false negative
ter LCMS-9030, and quantitative analysis was performed with a triple results. We describe a solution by using an automated sample preparation
quadrupole mass spectrometer LCMS-8060 by using nSMOL (nano-surface module CLAM-2000 TM connected with LC/MS/MS system (LCMS-8060)
and molecular orientation limited proteolysis) technology. for multi-target screening of 61 drugs in whole blood. A ready-to-use
method package Rapid Toxicology Screening (Shimadzu) was used to set
up the screening method with human whole blood (frozen) spiked sample
without efforts in LC and MRM method development.

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