Capillary Electrophoresis-mass Spectrometry Research Articles

Analysis of metabolomics and proteomics based on capillary electrophoresis-mass spectrometry

Capillary electrophoresis-mass spectrometry (CE-MS) has the advantages of higher sensitivity, higher efficiency, and less sample consumption. Moreover, it possesses obvious advantages during the analysis of strongly charged and highly polar samples. CE-MS has been widely applied in life sciences, medicine, and pharmacology. In the past ten years, the main factors affecting its application were system stability, reproducibility, and data accuracy. In order to solve the existing problems of CE-MS, researchers have invested significant effort in technology innovation to further expand CE-MS application. In the fields of medicine and analytical chemistry, substantial research indicates that CE-MS is superior compared to other metabolomic and proteomic approaches. This study aims at reviewing the latest methods and applications developed in the fields of medicine and analytical chemistry since 2015. Furthermore, it also aims at enhancing the technology development-related application value of CE-MS and serving as a reference for future development. Further development of the CE-MS technology is discussed from the aspects of coating-sample interaction, interface types, and data processing methods. Concerning the coating types, neutral coatings had been applied extensively in CE-MS and there should be no limitation to the charge of the analyte. The coating decreased sample adsorption on the inner wall by covering the surface charge, greatly reducing the electroosmotic flow (EOF). A charged capillary coating could modify such an EOF direction. The cationic coating could reduce the hydrophobic interaction between the sample and the capillary column, resulting in higher EOF. If it is applied to the sheathless interface, the resolution could be improved by extending the capillary length. Anionic coatings are predominant among the anionic compounds, shortening the separation time by reducing the interaction between the anionic compounds and the capillary. The coating type should be chosen relative to the analyte characteristics. Concerning the interface technology, all interfaces should be simple, practical, and non-dependent on sheath liquid and background electrolytes. As far as data processing methods are concerned, it is necessary to design and develop a practical method for span space data comparison and processing. The optimized experimental conditions have effectively improved separation efficiency and data comparison analysis. Furthermore, they established a solid foundation for its application development. CE-MS analysis of complex samples in the fields of metabolomics and proteomics (e. g., of tissues, cells, body fluids, etc.) could provide a visualization method for future clinical analysis. It contributes to the development of cancer pathological analysis, drug development, disease surveillance, etc. The characteristic analysis of small molecule metabolites and protein biomarkers directly reflects on enzymatic activity in the biological systems. It could be associated with the development of various diseases/complications. Omics analysis also has an important directive to disease detection and surveillance with obvious advantages in disease diagnosis, staged treatment, drug development, and patient treatment progress. CE-MS is useful in detecting complications and promoting personalized medicine. It provides technical support for future clinical developments. In addition to a comprehensive review of the recent advances of CE-MS research, this paper also indicates the development directions of CE-MS. In order to avoid the problem of omics analysis and obtain the optimized analysis results, future analysis should be improved from the following three aspects:(i) The analysis conditions should be optimized concerning sample preparation methods and separation techniques. (ii) The analytic techniques should be supported to adjust to capillary coating and interface technology. (iii) New ideas should be developed in the fields of clinical research and statistical analysis.

Capillary electrophoresis-mass spectrometry and its application to proteomic analysis

Proteomic analysis plays an important role in basic biological studies and precision medicine. However, real samples contain numerous proteins with a wide dynamic distribution range. Such high complexity of the samples has a drastic effect on the identification coverage of proteins. Consequently, with advancements in mass spectrometry (MS) technology, concomitant improvements in separation technologies for simplifying the sample should be critical. With the advantages of small sample loading volume, high separation efficiency, and high speed, capillary electrophoresis (CE) coupled to MS has been gained much attention in the field of proteomics research. A nanoflow sheath liquid interface and a sheathless interface have been developed and commercialized, boosting the development of the CE-MS technology. Capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and capillary electrochromatography (CEC) have been successfully combined with MS, and CZE-MS has widespread application. In proteomic research, the "bottom-up" strategy, which is based on the separation and identification of enzymatic peptides, is widely applied. With the limit of detection as low as 1 zmol for peptides, CE-MS has been successfully applied to single-cell proteomic analysis. Besides, CE is complementary to reversed-phase liquid chromatography (RPLC), providing a new approach for the separation and identification of peptides with similar hydrophobic properties (especially, post-translational peptides). The "top-down" strategy, which is based on the separation and identification of intact proteins, can directly provide more accurate and complete information about proteins. For protein separation, CE is advantageous in terms of the high separation resolution and high protein recovery, thereby improving the sensitivity and coverage of protein identification. Native MS enables successful identification and characterization of protein complexes under nondenaturing conditions. Because of the good compatibility of CE with MS, attempts have been made to use CE coupled with native MS for the separation and identification of protein complexes. In this review, the development of the CE-MS technology is first reported, including a robust and sensitive CE-MS interface, and a separation mode coupled to MS. Then, the application of the CE-MS technology to "bottom-up", "top-down" and native MS analysis is discussed. The superiority of CE-MS in proteomic analysis is also emphasized. Finally, the promising future prospects of CE-MS are discussed.

Determination of the binding of natural products to the human c-myb oncogene promoter G-quadruplex DNA by capillary electrophoresis and electrospray ionization mass spectrometry

The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Recent studies have found that the human oncogene c-myb is overexpressed in cancer tissues such as colorectal cancer. C-myb has become a potential therapeutic target for colorectal cancer, leukemia, and other cancers. A guanine (G)-rich DNA sequence located in the promoter region of c-myb can be spontaneously folded to form an intra-molecular G-quadruplex (G4) with cationic induction. The specific recognition of small molecules can stabilize this G4 folding, thus regulating the transcription and expression of c-myb. In this study, pressure assisted capillary electrophoresis frontier analysis (PACE-FA) combined with electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interactions between the human c-myb promoter G4 and natural product molecules. In PACE-FA, an external pressure (no more than 13.8 kPa) in the same direction of the migration of the analyte was used in capillary electrophoresis frontier analysis (CE-FA), which greatly sped up the analysis while maintaining the accuracy of the results. Meanwhile, the combination of PACE-FA and ESI-MS could rapidly determine the affinity and stoichiometric relationship between binding molecules and targets. First, the intramolecular parallel-stranded G4 formation of the c-myb promoter sequence in the presence of cations (K+, NH4+) was investigated by circular dichroism (CD) and ESI-MS. Then, ESI-MS was used to rapidly screen the natural products for candidate molecules that bound the target G4 DNA. The binding interactions were measured by mixing the c-myb DNA with each natural product separately in a 1:4 molar ratio, and then directly infusing these mixtures into the ESI-MS system. From the ESI-MS spectra, IRa values calculated from the relative intensities of DNA and its complex ions were used to probe the binding affinities of the natural products. This parameter denotes the relative binding affinity for a small molecule with the G4 DNA. Three natural products were identified through the screen, and their binding affinities to G4 DNA were ranked as follows:pseudolaric acid B > scopolamine butylbromide > nuciferine. Considering that both specific and non-specific binding existed in the solution phase, a free solution method using PACE-FA was developed to further test the binding ability of these products to the c-myb promoter G4 DNA. In the PACE-FA experiments, the pre-equilibrated mixture of the c-myb G4 DNA and the selected ligand was injected into the capillary prior to separation. Upon applying voltage and an external pressure (6.9 kPa) to the capillary, different species of analytes in the sample migrated at their own rates due to their different sizes and charges. The results showed that scopolamine butylbromide could bind specifically to target G4 DNA with 1:1 stoichiometry and a binding constant of 1.18×105 L/mol. Nuciferine's binding to G4 DNA showed a linear increasing trend due to nonspecific binding; thus nuciferine is a nonspecific binder. Although pseudolaric acid B showed high affinity for the c-myb G4 DNA and 1:1 and 1:2 G4-bound complex ions were observed in ESI-MS measurements, the PACE-FA results indicated that pseudolaric acid B did not bind to target G4 DNA in free solution. Therefore, scopolamine butylbromide could be the best candidate to regulate the transcription of the c-myb oncogene, and is expected to be a precursor for anticancer drugs. In this work, PACE-FA has allowed for significant improvements to the conventional CE-FA technique. This combination of PACE-FA and ESI-MS not only reduced the time needed for binding analysis, but also improved the accuracy and specificity of the affinity analysis compared to conventional binding approaches. Furthermore, this combination could be used to screen other targeted drug candidates and to evaluate their interaction mechanisms.

Urinary charged metabolite profiling of colorectal cancer using capillary electrophoresis-mass spectrometry

Colorectal cancer (CRC) has increasing global prevalence and poor prognostic outcomes, and the development of low- or less invasive screening tests is urgently required. Urine is an ideal biofluid that can be collected non-invasively and contains various metabolite biomarkers. To understand the metabolomic profiles of different stages of CRC, we conducted metabolomic profiling of urinary samples. Capillary electrophoresis-time-of-flight mass spectrometry was used to quantify hydrophilic metabolites in 247 subjects with stage 0 to IV CRC or polyps, and healthy controls. The 154 identified and quantified metabolites included metabolites of glycolysis, TCA cycle, amino acids, urea cycle, and polyamine pathways. The concentrations of these metabolites gradually increased with the stage, and samples of CRC stage IV especially showed a large difference compared to other stages. Polyps and CRC also showed different concentration patterns. We also assessed the differentiation ability of these metabolites. A multiple logistic regression model using three metabolites was developed with a randomly designated training dataset and validated using the remaining data to differentiate CRC and polys from healthy controls based on a panel of urinary metabolites. These data highlight the changes in metabolites from early to late stage of CRC and also the differences between CRC and polyps.

Enhancing confidence of metabolite annotation in Capillary Electrophoresis-Mass Spectrometry untargeted metabolomics with relative migration time and in-source fragmentation

Capillary electrophoresis coupled to mass spectrometry is a power tool in untargeted metabolomics studies to analyze charged and polar compounds. However, identification is a challenge due to the variability of migration times and the lack of MS/MS spectra in CE-TOF-MS, the type of instruments most frequently employed.We present here a CE-MS search platform incorporated in CEU Mass Mediator to annotate metabolites with a confidence level L2. For its the development we analyzed 226 compounds using two fragmentor voltages: 100 and 200 V. The information obtained, such as relative migration times (RMT) and in-source fragments, were incorporated into the platform. In addition, we validated the CE-MS search functionality using different types of biological samples such as plasma samples (human, rat, and rabbit), mouse macrophages, and human urine. The RMT tolerance percentage for the search of metabolites has been determined, establishing 5% for all compounds, except for the compounds migrating in the electro-osmotic flow, for which the tolerance should be of 10%.It has also been demonstrated the robustness of the in-source fragmentation, which makes possible the annotation of compounds by means of their fragmentation pattern. As an example, 3-methylhistidine and 1-methilhistidine, whose RMT are very close, have been annotated. Studies of the fragmentation mechanisms of acyl-L-carnitines have shown that in-source fragmentation follows the general fragmentation rules and is a suitable alternative to MS/MS.

Altered metabolism of chloroplastic NAD kinase-overexpressing Arabidopsis in response to magnesium sulfate supplementation

ABSTRACT Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADPH) is essential for numerous redox reactions and serve as co-factors in multiple metabolic processes in all organisms. NAD kinase (NADK) is an enzyme involved in the synthesis of NADP+ from NAD+ and ATP. Arabidopsis NADK2 (AtNADK2) is a chloroplast-localizing enzyme that provides recipients of reducing power in photosynthetic electron transfer. When Arabidopsis plants were grown on MS medium supplemented with 5 mM MgSO4, an AtNADK2-overexpressing line exhibited higher glutathione and total sulfur accumulation than control plants. Metabolomic analysis of major amino acids and organic acids using capillary electrophoresis–mass spectrometry demonstrated that overexpression of AtNADK2 affected a range of metabolic processes in response to MgSO4 supplementation.

Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes.

With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the Escherichia coli 70S ribosome. The capabilities of 193 nm ultraviolet photodissociation (UVPD) to yield informative backbone sequence ions are compared to those of higher-energy collisional dissociation (HCD). To further improve sequence coverage values, a multistage MS/MS approach is implemented involving front-end collisional activation to disassemble protein complexes into constituent subunits that are subsequently individually isolated and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique proteoforms, including 22 protein-metal complexes and 10 protein-protein complexes. Additionally, mapping metal-bound holo fragment ions resulting from UVPD of protein-metal complexes offers insight into the metal-binding sites.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as "self". For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis - mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography - mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 µL of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (<20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

CE-MS for metabolomics: Developments and applications in the period 2018-2020.

Capillary electrophoresis‐mass spectrometry (CE‐MS) is now a mature analytical technique in metabolomics, notably for the efficient profiling of polar and charged metabolites. Over the past few years, (further) progress has been made in the design of improved interfacing techniques for coupling CE to MS; also, in the development of CE‐MS approaches for profiling metabolites in volume‐restricted samples, and in strategies that further enhance the metabolic coverage. In this article, which is a follow‐up of a previous review article covering the years 2016–2018 (Electrophoresis 2019, 40, 165–179), the main (technological) developments in CE‐MS methods and strategies for metabolomics are discussed covering the literature from July 2018 to June 2020. Representative examples highlight the utility of CE‐MS in the fields of biomedical, clinical, microbial, plant and food metabolomics. A complete overview of recent CE‐MS‐based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, some general conclusions and perspectives are given.

Evaluation of on-line solid-phase extraction capillary electrophoresis–mass spectrometry with a nanoliter valve for the analysis of peptide biomarkers

On-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) is a powerful technique for high throughput sample clean-up and analyte preconcentration, separation, detection, and characterization. The most typical design due to its simplicity and low cost is unidirectional SPE-CE-MS. However, in this configuration, the sample volumes introduced by pressure depend on the dimensions of the separation capillary and some matrix components could be irreversibly adsorbed in its inner walls. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the background electrolyte necessary for an efficient separation and sensitive MS detection. Here, we present SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS) to overcome these drawbacks while keeping the design simple. The nvSPE-CE-MS system is operated with a single CE instrument and two capillaries for independent and orthogonal SPE preconcentration and CE separation, which are interfaced through an external and electrically isolated valve with a 20 nL sample loop. The instrumental setup is proved for the analysis of opioid and amyloid beta peptide biomarkers in standards and plasma samples. NvSPE-CE-MS allowed decreasing the limits of detection (LODs) 200 times with regard to CE-MS. Compared to unidirectional SPE-CE-MS, peak efficiencies were better and repeatabilities similar, but total analysis times longer and LODs for standards slightly higher due to the heart-cut operation and the limited volume of the valve loop. This small difference on the LODs for standards was compensated for plasma samples by the improved tolerance of nvSPE-CE-MS to complex sample matrices. In view of these results, the presented setup can be regarded as a promising versatile alternative to avoid complicated matrix samples entering the separation capillary in SPE-CE-MS.

Annual review of capillary electrophoresis technology in 2019

This paper reviews the capillary electrophoresis (CE) in 2019. The literatures searched from ISI Web of Science published in 2019 (Jan.1st to Dec.31th) are classified and introduced based on bioanalysis, drug analysis, clinical tests and medical diagnosis, chiral separation, foods tests, other compounds and ion analysis, and the application of capillary electrophoresis-mass spectrometry (CE-MS). International and domestic conferences related to CE and the important research reports are briefly introduced.

Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS

Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd’1, Fd’2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches.

Abstract 3510: The metabolomic profile of urine from prostate cancer patients using capillary electrophoresis mass spectrometry (CEMS)

Abstract Background: Prostate cancer (PCa) pathogenesis is influenced by alterations in cellular metabolism. Metabolomics measures these biochemical changes to create global tissue metabolite profiles. Urinary studies are noninvasive and can potentially identify biomarkers for PCa. Objective: We used Capillary Electrophoresis Mass Spectrometry (CE-MS) to analyze urine from men undergoing prostate biopsy to investigate their metabolomic profiles. Methods: We retrospectively analyzed 40 urine samples (20 from bx demonstrating PCa, 20 from benign) that were prospectively collected from men undergoing prostate biopsy. Analysis of charged metabolites done with Agilent CE-MS system (J Proteome Res. 2:488; 2003). Screening of potential biomarkers was performed with statistical protocols and pathway analyses. Metabolites with levels below the detection limit in all samples were excluded. Relative abundances of metabolites were normalized to levels of creatinine. Pathway analysis with MetaboAnalyst (Metabolites 9:57; 2019). Conclusions: CE-MS analysis identified several metabolic pathways that were altered in urine of men with PCa. These metabolites are involved in steroid, aromatic, microorganism and SCFA processes and warrant targeted studies which are underway in our lab. If validated, they have potential to serve as non-invasive biomarkers for PCa diagnosis and therapeutics Citation Format: Alexander Buko, Leo Cheng, Andrew Gustev, Adam Feldman. The metabolomic profile of urine from prostate cancer patients using capillary electrophoresis mass spectrometry (CEMS) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3510.

Abstract 365: The role of protein fucosylation in lung cancer progression

Abstract Lung cancer is the leading cause of cancer-associated deaths worldwide. Metabolic reprogramming facilitates the growth advantage of cancer cells and is a hallmark of cancer. Although multiple studies have investigated the cancer-specific metabolic status of lung cancer, it remains unclear. Aberrant fucosylation, which results from the deficiency or overexpression of fucosyltransferases, is also associated with a variety of cancers, including lung cancer. However, the role and regulatory mechanism(s) of fucosylation in lung cancer remain largely unknown. In order to elucidate the specific metabolic status of human lung cancer tissues, we performed metabolomics profiling of paired tumor and normal tissues from patients with non-small cell lung cancer (NSCLC) using both capillary electrophoresis mass-spectrometry (CE-MS) and imaging mass-spectrometry (I-MS). We used principle component analysis (PCA) to confirm that human lung cancer has distinctive metabolic patterns. We revealed upregulation of the GDP-L-fucose de novo pathway, which regulates the level of fucosylated glycoproteins, in lung cancer tissues compared to the adjacent non-malignant lung tissues. In order to elucidate the mechanisms by which fucosylation is increased in lung cancer tissues, gene expression of fucosyltransferases (FUT1-9) were evaluated. We confirmed that fucosyltransferase 3 (FUT3) expression was increased in a significant proportion of human primary lung adenocarcinoma samples and was correlated with sLeX expression. A fucosylation inhibitor, 6-alkynyl-fucose, inhibited the proliferation and invasion of lung cancer cell lines with high endogenous FUT3 expression in vitro. Exogenous FUT3-overexpressing lung cancer cells did not show increased proliferation, but showed significantly increased invasion and adhesion activities in vitro. In order to confirm whether FUT3 promoted metastasis of A549 cells in an experimental lung metastatic mouse model, A549 lung cancer cells expressing EGFP (A549-EGFP) and A549 lung cancer cells overexpressing FUT3 (A549-FUT3) were intravenously administered to BALB/C nude mice and the numbers of metastatic nodules in the lungs were counted after 8 weeks. In concordance with our observations in vitro, there was no significant difference in tumor growth between the A549-EGFP and A549-FUT3 treatments in vivo. Interestingly, injection of A549-FUT3 resulted in an increase in the number of lung metastases compared to the injection of A549-EGFP, suggesting that FUT3 is a positive regulator of cancer invasion and metastasis. Furthermore, high expression of FUT3 in lung cancer tissues predicted a poorer prognosis for patients with lung adenocarcinoma. These findings highlight the important roles of protein fucosylation in lung cancer progression and provide the preclinical rationale to target this pathway in lung cancer treatment. Citation Format: Keita Masuzawa, Hiroyuki Yasuda, Atsushi Matsuda, Hideki Terai, Yuki Sugiura, Junko Hamamoto, Daisuke Arai, Shizuko Kagawa, Katsura Emoto, Shinnosuke Ikemura, Ichiro Kawada, Kenzo Soejima, Koichi Fukunaga. The role of protein fucosylation in lung cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 365.

Abstract 4760: Discovery of novel biomarkers against refractory cancer using in situ level mass spectrometry informatics

Abstract Background and Purpose: The five-year survival rate of pancreatic cancer (PC) and diffuse-type gastric cancer (DT-GC) is far below 10%. They are known to have dense stroma unlike other types of tumors. Therefore, in the case, these two types of tumor are important to understand biological features of tumor stroma as well as that of tumor cells. However, conventional omics studies have largely focused on tumor cells. There are fewer studies in which both tumor cells and tumor stroma were examined simultaneously. In addition, among omics analysis, although genome and gene expression analysis using next generation sequencing (NGS) combined with informatics have progressed extensively, metabolomics analysis using mass spectrometry seems to be stagnated with no benefits from informatics. Hence, we have decided to develop in situ level mass spectrometry analysis combined with informatics in order to discover new biomarkers against both PC and DT-GC. Materials and Methods: First, we separated tumor cells, tumor stroma and normal cells using laser microdissection from clinical samples (5 cases of PC, 5 cases of DT-GC) and conducted trace-level metabolomics analysis using ultrasensitive capillary electrophoresis-mass spectrometry (CE-MS). We obtained mass spectrometric raw data with retention time, m/z and peak area of each molecules and then created data matrices. Using these data matrices, multivariate analysis such as PCA (principal component analysis) and PLS-DA (partial least squares regression analysis) was performed. Results: In PCA results, there were no significant differences among three data groups (tumor cells, tumor stroma and normal cells) and scatter plot with grouped data didn't follow any rules. Secondly, we conducted PLS-DA analysis and found that scatter plot was clearly divided into three groups. After calculating loading scores, we selected 20 top-ranked metabolites which is specific to each group (tumor cells, tumor stroma or normal cells) respectively. Moreover, all of them were unknown molecules and most of them had wide peak area and fast retention time. Now, we are identifying these molecules to find out which could be useful in the diagnosis and treatment of PC or DT-GC. Conclusion: We established in situ level mass spectrometry analysis and selected new biomarker candidates by loading plots in PLS-DA analysis. It is also indicated that informatics is a powerful approach to convert mass spectrometric raw data into meaningful information. Citation Format: Tamaki Hirakawa, Masahiro Yasunaga, Takayuki Kawai, Yoshihiro Shimizu, Kohei Shitara, Shigehiro Koganemaru, Yasutoshi Kuboki, Toshihiko Doi, Yasuhiro Matsumura. Discovery of novel biomarkers against refractory cancer using in situ level mass spectrometry informatics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4760.

Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and β-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and β-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the β-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

Serum Metabolic Signatures of Chronic Limb-Threatening Ischemia in Patients with Peripheral Artery Disease.

Peripheral artery disease (PAD) is characterized by the atherosclerotic narrowing of lower limb vessels, leading to ischemic muscle pain in older persons. Some patients experience progression to advanced chronic limb-threatening ischemia (CLTI) with poor long-term survivorship. Herein, we performed serum metabolomics to reveal the mechanisms of PAD pathophysiology that may improve its diagnosis and prognosis to CLTI complementary to the ankle–brachial index (ABI) and clinical presentations. Non-targeted metabolite profiling of serum was performed by multisegment injection–capillary electrophoresis–mass spectrometry (MSI–CE–MS) from age and sex-matched, non-diabetic, PAD participants who were recruited and clinically stratified based on the Rutherford classification into CLTI (n = 18) and intermittent claudication (IC, n = 20). Compared to the non-PAD controls (n = 20), PAD patients had lower serum concentrations of creatine, histidine, lysine, oxoproline, monomethylarginine, as well as higher circulating phenylacetylglutamine (p < 0.05). Importantly, CLTI cases exhibited higher serum concentrations of carnitine, creatinine, cystine and trimethylamine-N-oxide along with lower circulating fatty acids relative to well matched IC patients. Most serum metabolites associated with PAD progression were also correlated with ABI (r = ±0.24−0.59, p < 0.05), whereas the ratio of stearic acid to carnitine, and arginine to propionylcarnitine differentiated CLTI from IC with good accuracy (AUC = 0.87, p = 4.0 × 10−5). This work provides new biochemical insights into PAD progression for the early detection and surveillance of high-risk patients who may require peripheral vascular intervention to prevent amputation and premature death.

The Principle of Nanomaterials Based Surface Plasmon Resonance Biosensors and Its Potential for Dopamine Detection.

For a healthy life, the human biological system should work in order. Scheduled lifestyle and lack of nutrients usually lead to fluctuations in the biological entities levels such as neurotransmitters (NTs), proteins, and hormones, which in turns put the human health in risk. Dopamine (DA) is an extremely important catecholamine NT distributed in the central nervous system. Its level in the body controls the function of human metabolism, central nervous, renal, hormonal, and cardiovascular systems. It is closely related to the major domains of human cognition, feeling, and human desires, as well as learning. Several neurological disorders such as schizophrenia and Parkinson’s disease are related to the extreme abnormalities in DA levels. Therefore, the development of an accurate, effective, and highly sensitive method for rapid determination of DA concentrations is desired. Up to now, different methods have been reported for DA detection such as electrochemical strategies, high-performance liquid chromatography, colorimetry, and capillary electrophoresis mass spectrometry. However, most of them have some limitations. Surface plasmon resonance (SPR) spectroscopy was widely used in biosensing. However, its use to detect NTs is still growing and has fascinated impressive attention of the scientific community. The focus in this concise review paper will be on the principle of SPR sensors and its operation mechanism, the factors that affect the sensor performance. The efficiency of SPR biosensors to detect several clinically related analytes will be mentioned. DA functions in the human body will be explained. Additionally, this review will cover the incorporation of nanomaterials into SPR biosensors and its potential for DA sensing with mention to its advantages and disadvantages.