Predictive analysis of breast cancer response to neoadjuvant chemotherapy through plasma metabolomics.

PURPOSE: Preoperative chemotherapy is a critical component of breast cancer management, yet its effectiveness is not uniform. Moreover, the adverse effects associated with chemotherapy necessitate the identification of a patient subgroup that would derive the maximum benefit from this treatment. This study aimed to establish a method for predicting the response to neoadjuvant chemotherapy in breast cancer patients utilizing a metabolomic approach. METHODS: Plasma samples were obtained from 87 breast cancer patients undergoing neoadjuvant chemotherapy at our facility, collected both before the commencement of the treatment and before the second treatment cycle. Metabolite analysis was conducted using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS). We performed comparative profiling of metabolite concentrations by assessing the metabolite profiles of patients who achieved a pathological complete response (pCR) against those who did not, both in initial and subsequent treatment cycles. RESULTS: Significant variances were observed in the metabolite profiles between pCR and non-pCR cases, both at the onset of preoperative chemotherapy and before the second cycle. Noteworthy distinctions were also evident between the metabolite profiles from the initial and the second neoadjuvant chemotherapy courses. Furthermore, metabolite profiles exhibited variations associated with intrinsic subtypes at all assessed time points. CONCLUSION: The application of plasma metabolomics, utilizing CE-MS and LC-MS, may serve as a tool for predicting the efficacy of neoadjuvant chemotherapy in breast cancer in the future after all necessary validations have been completed.

High-Sensitivity Glycoproteomic Analysis of Biological Samples by CZE-ESI-MS.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is a powerful tool for the characterization and identification of the macro- and microheterogeneity of a glycoprotein in a bottom-up approach. This chapter describes in detail the sample preparation procedures using a purified biological sample, prostate-specific antigen, as a model protein, including proteolytic digestion (trypsin). In addition, insights are provided into the strengths of using capillary electrophoresis for obtaining isomer separation of differently linked sialic acids. Lastly, approaches and potential pitfalls for the integration and quantitation of glycopeptide signals from the obtained CZE-MS data are discussed.

Urinary Metabolome Analyses of Patients with Acute Kidney Injury Using Capillary Electrophoresis-Mass Spectrometry.

Acute kidney injury (AKI) is defined as a rapid decline in kidney function. The associated syndromes may lead to increased morbidity and mortality, but its early detection remains difficult. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we analyzed the urinary metabolomic profile of patients admitted to the intensive care unit (ICU) after invasive surgery. Urine samples were collected at six time points: before surgery, at ICU admission and 6, 12, 24 and 48 h after. First, urine samples from 61 initial patients (non-AKI: 23, mild AKI: 24, severe AKI: 14) were measured, followed by the measurement of urine samples from 60 additional patients (non-AKI: 40, mild AKI: 20). Glycine and ethanolamine were decreased in patients with AKI compared with non-AKI patients at 6-24 h in the two groups. The linear statistical model constructed at each time point by machine learning achieved the best performance at 24 h (median AUC, area under the curve: 89%, cross-validated) for the 1st group. When cross-validated between the two groups, the AUC showed the best value of 70% at 12 h. These results identified metabolites and time points that show patterns specific to subjects who develop AKI, paving the way for the development of better biomarkers.

Characterization and monitoring of charge variants of a recombinant monoclonal antibody using microfluidic capillary electrophoresis-mass spectrometry.

Rigorous characterization of biotherapeutics, and monoclonal antibodies in particular, is a challenging task in terms of ensuring safety, efficacy, and potency of a therapeutic agent because of structural heterogeneity during cell culture, purification and storage. In this work, we used microfluidic capillary electrophoresis-mass spectrometry to analyze intact monoclonal antibody and assess the root cause of increases in acidic and basic variants under stress at high temperature. The antibody was analyzed at multiple levels, including its intact state under native conditions, and subunit and peptide levels. The normal and degraded antibodies at different time points were characterized and compared with each other. We concluded that the basic variants in the unstressed sample were produced C-terminal amidation, while the acidic variants were produced by deamidation. In stressed samples, change in the acidic and main peaks were caused by deamidation, and changes in the basic peaks were caused by both deamidation and oxidation. These results demonstrate that microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a powerful direct and generic tool for separation and identification of charge heterogeneity of biotherapeutics.

Residue analytical method for the determination of trifluoroacetic acid and difluoroacetic acid in plant matrices by capillary electrophoresis tandem mass spectrometry (CE-MS/MS).

In the past years, the technology for trace residue analysis of plant protection compounds in plant and animal matrices, soil, and water has gradually changed to meet changing regulatory demands. Generally, from the '70s to the '90s of the last century, the active compounds and only a few major metabolites had to be determined in a typical "residue definition". Step by step and within the framework of product safety assessments of the enforcement of residues in dietary matrices and in the environment, further metabolites have come into the authorities focus. Many active substances were formerly determined via gas chromatography (GC) based detection techniques. The introduction of liquid chromatography tandem mass spectrometry (LC-MS/MS) technology in the '90s and the acceptance of this technique, by official bodies at the end of the '90s, has led to a major change for residue analytical laboratories all over the world. Most of the medium to non-polar active compounds as well as many of the more polar metabolites can be detected with this technique, and today LC-MS/MS is the "workhorse" in many residue analytical laboratories in the industry as well as official enforcement labs responsible for analyzing registration-related field studies. With the demand to analyze further breakdown products, more and more polar compounds, or even (permanently) charged target compounds, have now come into the focus of the registration authorities. This now brings standard LC-based techniques to their limits and requires the application of approaches such as hydrophilic interaction chromatography (HILIC) MS/MS or ion chromatography, however these techniques often incur related uncertainties and problems with matrix samples. The aim of this study was to develop a new CE-MS/MS-based approach to reduce the impact of matrix on the separation and detection of trifluoroacetic acid (TFA) and difluoroacetic acid (DFA) in agrochemical field trials. This project used 7 representative examples of fruit, grain and vegetables which had undergone homogenization and extraction with acetonitrile water and filtration before CE-MS/MS analysis. The CE-MS/MS developed reached the limit of quantitation (LOQ) requirement of current legislation for both TFA and DFA (0.01 mg/kg) in all 7 matrices tested. The mean relative standard deviation (RSD) obtained from the repeat analysis of control field trail samples in all matrices, for both TFA and DFA, was less than 10% meeting GLP guidelines. When compared with LC-MS/MS, using on column loading amounts, the CE-MS/MS was 17 - 43 times more sensitive than a standard method and less matrix effects were observed. The developed method was validated under GLP conditions to provide a GLP-validated residue analytical method for the charged metabolites TFA and DFA in matrix samples from GLP field residue trials.

Bile Metabolites and Risk of Carcinogenesis in Patients With Pancreaticobiliary Maljunction: A Pilot Study.

BACKGROUND/AIM: Pancreaticobiliary maljunction (PBM), a disease with reflux of pancreatic and bile juice in the pancreaticobiliary tract, is a high-risk factor for biliary tract cancer. The aim of this study was to investigate the mechanism of carcinogenesis in PBM using a metabolomics analysis of bile sampled during surgery. PATIENTS AND METHODS: Three patients with PBM without biliary tract cancer, four patients with extrahepatic bile duct cancer (EHBC), and three controls with benign disease were enrolled. Metabolomics analysis of bile samples was performed using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to discriminate the amino acid and lipidomic profiles. RESULTS: The principal component analysis in the capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry revealed similar metabolites in patients with PBM and those with EHBC; furthermore, there was a clear difference between patients with PBM or EHBC compared to controls. The amino acid profiles revealed the following 20 potential carcinogenic candidates for PBM: isoleucine, phenylalanine, tyrosine, leucine, tryptophan, arginine, lysine, valine, asparagine, methionine, aspartic acid, serine, threonine, histidine, glutamine, alanine, proline, glutamic acid, and pyruvic acid. The lipidomic profiles revealed the following 11 carcinogenic candidates: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl glycerol, lysophosphatidyl glycerol, triacylglycerol, diacylglycerol, ceramide, sphyngomyeline, fatty acid, hyperforin, and vitamin D. Among these characteristic metabolites, the branched-chain amino acids, methionine and lysophosphatidylcholine are known to be related to carcinogenesis. CONCLUSION: The bile metabolites were extremely similar in patients with PBM and those with EHBC. Furthermore, amino acid and lipid metabolism was markedly different in patients with PBM or EHBC compared to healthy controls.

[Recent progress in capillary electrophoresis-based high-sensitivity proteomics].

In recent years, proteomic techniques have undergone rapid progress in terms of sample pretreatment, separation, and mass spectrometry (MS) detection. The current MS-based proteomic techniques can be used to identify up to 10000 proteins both qualitatively and quantitatively within a few hours. However, the current mainstream proteomic approaches do not fulfill the need to analyze minute amounts of biological samples, especially rare cells and single mammalian cells. Capillary electrophoresis (CE)-based separation offers many advantages, such as narrow peaks, high separation efficiency, and low sample requirement, which make it an ideal separation approach for combination with high-resolution MS. We have reviewed the state-of-the-art development of integrated and online sample preparation methods and nanoscale liquid chromatography-mass spectrometry (nanoLC-MS) for high-sensitivity proteomics, and described the associated challenges. Integrated and online sample preparation methods can minimize sample loss and improve lysis and digestion efficiencies. The simple and integrated spintip-based proteomics technology (SISPROT) developed by our group has shown robust performance for the comprehensive profiling of various types of samples and the sensitive analysis of small numbers of cells, down to a few hundred. A few groups have applied integrated/online sample preparation methods, such as nanodroplet processing in one pot for trace samples (nanoPOTS) and integrated proteome analysis system for one cell (iPAD-1), to achieve the identification of hundreds of proteins from a single HeLa cell. We propose that one of the key technical challenges in this field is that the performance of current nanoLC separation techniques cannot match modern high-resolution MS techniques, with ultrahigh scan rates of over 40 Hz; therefore, the insufficient chromatographic performance results in reduced utilization of MS/MS scan capacity. Wide chromatographic peaks result in insufficient precursors to trigger MS/MS scans and redundant sampling, irrespective of whether dynamic exclusion has been enabled. In view of the above mentioned technical challenges, we have focused on discussing the unique technical advantages and potential opportunities of CE-MS, which mainly include the following. (1) High-performance capillary electrophoresis (HPCE) separation for minute amounts of tryptic peptide samples. Capillary electrochromatography can further improve the column capacity limit of HPCE. (2) CE-MS interfaces for high-sensitivity proteomics. Although sheath liquid interfaces have proven versatile and robust and are currently more commonly used, sheathless interfaces can significantly enhance the signal/noise ratio owing to decreased analyte dilution and background noise. Thus, sheathless interfaces are potentially more suitable for ultrasensitive proteomics. (3) Synergetic utilization of HPCE separation and MS detection at high scan rates. The most promising way to fully utilize the ultrahigh scan rates of modern high-resolution MS is to enhance the quality of peptide separation. Narrower peptide peaks in HPCE separation may greatly reduce redundant sampling and boost sensitivity. Overall, we anticipate that, after further improvement, CE-MS-based proteomics will be more widely applied to proteomic analysis of minute amounts of biological samples, such as single mammalian cells. Furthermore, more sensitive data acquisition modes, such as data-independent acquisition, may be used for global proteomic profiling, and parallel reaction monitoring may be used to target a limited number of important proteins. Matching between runs and machine learning algorithms may improve the accuracy of proteomic analysis of minute amounts of samples.

[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.

Development of complementary CE-MS methods for speciation analysis of pyrithione-based antifouling agents.

In the recent decade, metal pyrithione complexes have become important biocides for antifouling purposes in shipping. The analysis of metal pyrithione complexes and their degradation products/species in environmental samples is challenging because they exhibit fast UV degradation, transmetalation, and ligand substitution and are known to be prone to spontaneous species transformation within a chromatographic system. The environmental properties of the pyrithione species, e.g., toxicity to target and non-target organisms, are differing strongly, and it is therefore inevitable to identify as well as quantify all species separately. To cope with the separation of metal pyrithione species with minimum species transformation during analysis, a capillary electrophoresis (CE)-based method was developed. The hyphenation of CE with selective electrospray ionization- and inductively coupled plasma-mass spectrometry (ESI-, ICP-MS) provided complementary molecular and elemental information for the identification and quantification of pyrithione species. To study speciation of pyrithiones, a leaching experiment of several commercial antifouling paints containing zinc pyrithione in ultrapure and river water was conducted. Only the two species pyrithione (HPT) and dipyrithione ((PT)2) were found in the leaching media, in concentrations between 0.086 and 2.4 µM (HPT) and between 0.062 and 0.59 µM ((PT)2), depending on the paint and leaching medium. The limits of detection were 20 nM (HPT) and 10 nM ((PT)2). The results show that complementary CE-MS is a suitable tool for mechanistical studies concerning species transformation (e.g., degradation) and the identification of target species of metal pyrithione complexes in real surface water matrices, laying the ground for future environmental studies. Graphical abstract Hyphenation of CE with ESI- and ICP-MS provided complementary molecular and elemental information. Metal pyrithione species released from commercial antifouling paints could be identified and quantified in ultrapure and river water matrices.

Synergistic coupling of in-line single-drop microextraction and on-line large-volume sample stacking for capillary electrophoresis/mass spectrometry.

Single-drop microextraction (SDME) and large-volume sample stacking using an electroosmotic flow pump (LVSEP) were coupled with capillary electrophoresis/mass spectrometry (CE/MS) for sample cleanup and preconcentration. Without filtration or centrifugation of a soil sample containing debris, SDME using a pentanol acceptor drop was directly applied to the sample. After SDME, a large volume of the enriched pentanol extract was injected and further concentrated by LVSEP. For the drop formation in SDME and the sample matrix removal in LVSEP, a run buffer vial was temporarily placed to the electrospray tip, without any physical modification of the CE/MS interface. This method enabled the double preconcentration by SDME and LVSEP, achieving 600~1300-fold enrichments of anionic analytes including pesticide and herbicide compounds to provide limits of detection in the range of 0.4~0.8 ppb in soil. Graphical abstract ᅟ.

Saturated fatty acid determination method using paired ion electrospray ionization mass spectrometry coupled with capillary electrophoresis.

A sensitive and selective capillary electrophoresis-mass spectrometry (CE-MS) method for determination of saturated fatty acids (FAs) was developed by using dicationic ion-pairing reagents forming singly charged complexes with anionic FAs. For negative ESI detection, 21 anionic FAs at pH 10 were separated using ammonium formate buffer containing 40% acetonitrile modifier in normal polarity mode in CE by optimizing various parameters. This method showed good separation efficiency, but the sensitivity of the method to short-chain fatty acids was quite low, causing acetic and propionic acids to be undetectable even at 100 mgL-1 in negative ESI-MS detection. Out of the four dicationic ion-pairing reagents tested, N,N'-dibutyl 1,1'-pentylenedipyrrolidium infused through a sheath-liquid ion source during CE separation was the best reagent regarding improved sensitivity and favorably complexed with anionic FAs for detection in positive ion ESI-MS. The monovalent complex showed improved ionization efficiency, providing the limits of detection (LODs) for 15 FAs ranging from 0.13 to 2.88 µg/mL and good linearity (R2 > 0.99) up to 150 µg/mL. Compared to the negative detection results, the effect was remarkable for the detection of short- and medium-chain fatty acids. The optimized CE-paired ion electrospray (PIESI)-MS method was utilized for the determination of FAs in cheese and coffee with simple pretreatment. This method may be extended for sensitive analysis of unsaturated fatty acids.

Effects of Consuming Xylitol on Gut Microbiota and Lipid Metabolism in Mice.

The sugar alcohol xylitol inhibits the growth of some bacterial species including Streptococcus mutans. It is used as a food additive to prevent caries. We previously showed that 1.5-4.0 g/kg body weight/day xylitol as part of a high-fat diet (HFD) improved lipid metabolism in rats. However, the effects of lower daily doses of dietary xylitol on gut microbiota and lipid metabolism are unclear. We examined the effect of 40 and 200 mg/kg body weight/day xylitol intake on gut microbiota and lipid metabolism in mice. Bacterial compositions were characterized by denaturing gradient gel electrophoresis and targeted real-time PCR. Luminal metabolites were determined by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry. Plasma lipid parameters and glucose tolerance were examined. Dietary supplementation with low- or medium-dose xylitol (40 or 194 mg/kg body weight/day, respectively) significantly altered the fecal microbiota composition in mice. Relative to mice not fed xylitol, the addition of medium-dose xylitol to a regular and HFD in experimental mice reduced the abundance of fecal Bacteroidetes phylum and the genus Barnesiella, whereas the abundance of Firmicutes phylum and the genus Prevotella was increased in mice fed an HFD with medium-dose dietary xylitol. Body composition, hepatic and serum lipid parameters, oral glucose tolerance, and luminal metabolites were unaffected by xylitol consumption. In mice, 40 and 194 mg/kg body weight/day xylitol in the diet induced gradual changes in gut microbiota but not in lipid metabolism.

In-line coupling of single-drop microextraction with capillary electrophoresis-mass spectrometry.

Single-drop microextraction (SDME) was in-line coupled with capillary electrophoresis-mass spectrometry to provide sample cleanup and enrichment simultaneously. Since there is no outlet vial in a conventional capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) configuration, it is not easy to hang a single drop in the capillary inlet for extraction. We overcame the difficulty of coupling SDME and CE-MS by using a temporary outlet reservoir. Basic drugs such as methamphetamine, amphetamine, phenethylamine, methoxyphenamine, and mephentermine were extracted from a basic sample solution to an acidic acceptor drop covered with a thin octanol layer formed at the capillary inlet tip. Compared to the CE-MS method in the multiple reaction monitoring (MRM) mode, the in-line SDME-CE-MS/MS technique showed 130∼150-fold enrichment in 10 min. The relative standard deviations (RSDs) of peak height ranged from 9 to 13 %. RSDs can be reduced from 4 to 6 % using mephentermine as an internal standard. We examined the pretreatment of sample with and without SDME from human urine under the full-scan mode, which confirmed that many metabolites were cleaned up by the selective extraction method of SDME. Even if the analytes from human urine were analyzed under the MRM mode used as a mass filter, there was an isobaric compound causing a disturbance to the analysis. However, in-line SDME-CE-MS/MS made it possible to perform a sample cleanup as well as sample enrichment. The research is extremely advantageous in that it is rapid, convenient, and highly sensitive for the analysis of biological samples using a commercially available instrument.

Hydroxyproline, a serum biomarker candidate for gastric ulcer in rats: a comparison study of metabolic analysis of gastric ulcer models induced by ethanol, stress, and aspirin.

Gastrointestinal symptoms are a common manifestation of adverse drug effects. Non-steroid anti-inflammatory drugs (NSAIDs) are widely prescribed drugs that induce the serious side effect of gastric mucosal ulceration. Biomarkers for these side effects have not been identified and ulcers are now only detectable by endoscopy. We previously identified five metabolites as biomarker candidates for NSAID-induced gastric ulcer using capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic analysis of serum and stomach from rats. Here, to clarify mechanism of changes and limitations of indications of biomarker candidates, we performed CE-MS-based metabolomic profiling in stomach and serum from rats with gastric ulcers induced by ethanol, stress, and aspirin. The results suggest that a decrease in hydroxyproline reflects the induction of gastric injury and may be useful in identifying gastric ulcer induced by multiple causes. While extrapolation to humans requires further study, hydroxyproline can be a new serum biomarker of gastric injury regardless of cause.