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.

Integration of Urinary Peptidome and Fecal Microbiome to Explore Patient Clustering in Chronic Kidney Disease.

Millions of people worldwide currently suffer from chronic kidney disease (CKD), requiring kidney replacement therapy at the end stage. Endeavors to better understand CKD pathophysiology from an omics perspective have revealed major molecular players in several sample sources. Focusing on non-invasive sources, gut microbial communities appear to be disturbed in CKD, while numerous human urinary peptides are also dysregulated. Nevertheless, studies often focus on isolated omics techniques, thus potentially missing the complementary pathophysiological information that multidisciplinary approaches could provide. To this end, human urinary peptidome was analyzed and integrated with clinical and fecal microbiome (16S sequencing) data collected from 110 Non-CKD or CKD individuals (Early, Moderate, or Advanced CKD stage) that were not undergoing dialysis. Participants were visualized in a three-dimensional space using different combinations of clinical and molecular data. The most impactful clinical variables to discriminate patient groups in the reduced dataspace were, among others, serum urea, haemoglobin, total blood protein, urinary albumin, urinary erythrocytes, blood pressure, cholesterol measures, body mass index, Bristol stool score, and smoking; relevant variables were also microbial taxa, including Roseburia, Butyricicoccus, Flavonifractor, Burkholderiales, Holdemania, Synergistaceae, Enterorhabdus, and Senegalimassilia; urinary peptidome fragments were predominantly derived from proteins of collagen origin; among the non-collagen parental proteins were FXYD2, MGP, FGA, APOA1, and CD99. The urinary peptidome appeared to capture substantial variation in the CKD context. Integrating clinical and molecular data contributed to an improved cohort separation compared to clinical data alone, indicating, once again, the added value of this combined information in clinical practice.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 µl of 100 nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

Coupling capillary electrophoresis with mass spectrometry for the analysis of oxidized phospholipids in human high-density lipoproteins.

The oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human high-density lipoproteins (HDLs) were investigated by low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS). To accelerate the optimization, native PAPC (n-PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20 mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20 kV and 23°C. The optimal CE separation buffer was then used for the low-flow CE-MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). No sheath gas was used for MS. The linear range for n-PAPC was 2.5-100.0 µg/mL. The coefficient of determination (R2 ) was 0.9918. The concentration limit of detection was 1.52 µg/mL, and the concentration limit of quantitation was 4.60 µg/mL. The optimal low-flow CE-MS method showed good repeatability and sensitivity. The ox-PAPC products in human HDLs were determined based on the in vitro ox-PAPC products of n-PAPC standard. Twenty-one ox-PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox-PAPC products than healthy subjects.

Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS.

Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip-based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5 min of analysis time. Low levels of dimethyl sulfoxide (4%) in the background electrolyte (BGE) improved MS signal quality and component detection. A sensitivity evaluation revealed consistent detection of VP proteoforms when as little as 2.64 × 106 viral particles (≈26.4 picograms) were injected. Besides the traditional VP proteoforms used for serotype identification, multiple VP3 variants were detected, including truncated VP3 variants most likely generated by leaky scanning as well as unacetylated and un-cleaved VP3 proteoforms. Phosphorylation, known to impact AAV transduction efficiency, was also seen in all serotypes analysed. Additionally, low abundant fragments originating from either N- or C-terminus truncation were detected. As the aforementioned VP components can impact product quality and efficacy, the ZipChip's ability to rapidly characterize them illustrates its strength in monitoring product quality during AAV production.

Exploratory Study Analyzing the Urinary Peptidome of T2DM Patients Suggests Changes in ECM but Also Inflammatory and Metabolic Pathways Following GLP-1R Agonist Treatment.

Type II diabetes mellitus (T2DM) accounts for approximately 90% of all diabetes mellitus cases in the world. Glucagon-like peptide-1 receptor (GLP-1R) agonists have established an increased capability to target directly or indirectly six core defects associated with T2DM, while the underlying molecular mechanisms of these pharmacological effects are not fully known. This exploratory study was conducted to analyze the effect of treatment with GLP-1R agonists on the urinary peptidome of T2DM patients. Urine samples of thirty-two T2DM patients from the PROVALID study ("A Prospective Cohort Study in Patients with T2DM for Validation of Biomarkers") collected pre- and post-treatment with GLP-1R agonist drugs were analyzed by CE-MS. In total, 70 urinary peptides were significantly affected by GLP-1R agonist treatment, generated from 26 different proteins. The downregulation of MMP proteases, based on the concordant downregulation of urinary collagen peptides, was highlighted. Treatment also resulted in the downregulation of peptides from SERPINA1, APOC3, CD99, CPSF6, CRNN, SERPINA6, HBA2, MB, VGF, PIGR, and TTR, many of which were previously found to be associated with increased insulin resistance and inflammation. The findings indicate potential molecular mechanisms of GLP-1R agonists in the context of the management of T2DM and the prevention or delaying of the progression of its associated diseases.

Urine Peptidome Analysis Identifies Common and Stage-Specific Markers in Early Versus Advanced CKD.

Given the pathophysiological continuum of chronic kidney disease (CKD), different molecular determinants affecting progression may be associated with distinct disease phases; thus, identification of these players are crucial for guiding therapeutic decisions, ideally in a non-invasive, repeatable setting. Analyzing the urinary peptidome has been proven an efficient method for biomarker determination in CKD, among other diseases. In this work, after applying several selection criteria, urine samples from 317 early (stage 2) and advanced (stage 3b-5) CKD patients were analyzed using capillary electrophoresis coupled to mass spectrometry (CE-MS). The entire two groups were initially compared to highlight the respective pathophysiology between initial and late disease phases. Subsequently, slow and fast progressors were compared within each group in an attempt to distinguish phase-specific disease progression molecules. The early vs. late-stage CKD comparison revealed 929 significantly different peptides, most of which were downregulated and 268 with collagen origins. When comparing slow vs. fast progressors in early stage CKD, 42 peptides were significantly altered, 30 of which were collagen peptide fragments. This association suggests the development of structural changes may be reversible at an early stage. The study confirms previous findings, based on its multivariable-matched progression groups derived from a large initial cohort. However, only four peptide fragments differed between slow vs. fast progressors in late-stage CKD, indicating different pathogenic processes occur in fast and slow progressors in different stages of CKD. The defined peptides associated with CKD progression at early stage might potentially constitute a non-invasive approach to improve patient management by guiding (personalized) intervention.

A comparison of hydrophilic interaction liquid chromatography and capillary electrophoresis for the metabolomics analysis of human serum.

Cationic, anionic, zwitterionic and, partially polar metabolites are very important constituents of blood serum. Several of these metabolites underpin the core metabolism of cells (e.g., Krebs cycle, urea cycle, proteins synthesis, etc.), while others might be considered ancillary but still important to grasp the status of any organism through blood serum analysis. Due to its wide chemical diversity, modern metabolomics analysis of serum is still struggling to provide a complete and comprehensive picture of the polar metabolome, due to the limitations of each specific analytical method. In this study, two metabolomics-based analytical methods using the most successful techniques for polar compounds separation in human serum samples, namely hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE), are evaluated, both coupled to a high-resolution time-of-flight mass spectrometer via electrospray ionization (ESI-Q-TOF-MS). The performance of the two methods have been compared using five terms of comparison, three of which are specific to metabolomics, such as (1) compounds' detectability (2) Pezzatti score (Pezzatti et al. 2018), (3) intra-day precision (repeatability), (4) ease of automatic analysis of the data (through a common deconvolution alignment and extrapolation software, MS-DIAL, and (5) time & cost analysis. From this study, HILIC-MS proved to be a better tool for polar metabolome analysis, while CE-MS helped identify some interesting variables that gave it interest in completing metabolome coverage in metabolomics studies. Finally, in this framework, MS-DIAL demonstrates for the first time its ability to process CE data for metabolomics, although it is not designed for it.

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.

Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, "spray-capillary," for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.

Metabolic changes associated with dark-induced leaf senescence in Arabidopsis nadk2 mutants.

Arabidopsis NADK2 (NAD kinase 2) is a chloroplast-localized enzyme involved in NADP+ synthesis, which acts as the final electron acceptor in the photosynthetic electron transfer chain. The NADK2-deficient mutant (nadk2) was used to analyze the effect of NAD(P)(H) unbalance in the dark-induced leaf senescence. During senescence, WT plants and nadk2 mutants showed a similar reduction in chlorophyll content. NAD(P)(H) quantification showed that the amount of total NAD(P)(H) decreased on the day 7 in WT but on the day 3 in nadk2. The phosphorylation ratio (i.e. NADP(H)/NAD(H)) decreased on day 1 in WT. In contrast, the nadk2 showed lower phosphorylation ratio at 0 day and no change throughout the aging process. Metabolome analysis showed that the metabolic profiles of both WT plants and nadk2 mutants subjected to dark-induced senescence adopted similar patterns as the senescence progressed. However, the changes in individual metabolites in the nadk2 mutants were different from those of the WT during dark-induced senescence.

Capillary electrophoresis-mass spectrometry as a tool for Caenorhabditis elegans metabolomics research.

INTRODUCTION: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes. OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome. METHOD: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument. RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites. CONCLUSION: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.

Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography (2019-2022).

Capillary electrochromatography (CEC) plays a significant role in chiral separation via the double separation principle, partition coefficient difference between the two phases, and electroosmotic flow-driven separation. Given the distinct properties of the inner wall stationary phase (SP), the separation ability of each SP differs from one another. Particularly, it provides large room for promising applications of open tubular capillary electrochromatography (OT-CEC). We divided the OT-CEC SPs developed over the past four years into six types: ionic liquids, nanoparticle materials, microporous materials, biomaterials, non-nanopolymers, and others, to mainly introduce their characteristics in chiral drug separation. There also added a few classic SPs that occurred within ten years as supplements to enrich the features of each SP. Additionally, we discuss their applications in metabolomics, food, cosmetics, environment, and biology as analytes in addition to chiral drugs. OT-CEC plays an increasingly significant role in chiral separation and may promote the development of capillary electrophoresis (CE) combined with other instruments in recent years, such as CE with mass spectrometry (CE/MS) and CE with ultraviolet light detector (CE/UV).

Aptamer affinity-based microextraction in-line coupled to capillary electrophoresis mass spectrometry using a porous layer/nanoparticle -modified open tubular column.

An aptamer affinity based microextraction column is developed to be directly in-line coupled to capillary electrophoresis-mass spectrometry (CE-MS) for analyzing mycotoxins in food samples. Single-stranded DNA aptamers for selective recognition of aflatoxin B1 (AFB1) and ochratoxin A (OTA) targets are co-immobilized via covalent bonds on the surface of the inlet end of a capillary, which is pre-modified with three-dimensional porous layer and gold nanoparticles to enhance the specific surface area and loading capacity. The outlet of the capillary is designed as a porous tip to serve as the spray source for injection to the mass spectrometry. All the necessary processes for pretreatment and analysis of a sample are accomplished in one injection, including aptamer affinity-based microextraction, CE separation and MS detection of analytes. AFB1 and OTA are simultaneously determined in a wide linear range with sample consumption of only 1 µL and the limit-of-detection as low as 1 pg/mL. The microextraction column exhibits excellent repeatability and stability, which can be used over 45 runs within a month with CE separation efficiency and only MS intensity slightly decreased. Mycotoxins in three kinds of cereal based infant foods are accurately analyzed using the proposed method. The study provides a robust and universal approach that would have potential applications in a variety of analytical fields based on selective molecular recognition coupling to CE-MS analysis.

Determination of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine products in human very low-density lipoproteins by nonaqueous low-flow capillary electrophoresis-mass spectrometry.

A simple and fast low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human very low-density lipoproteins (VLDLs). Native PAPC standard was analyzed to optimize the low-flow CE-MS method. The optimal CE conditions included separation buffer (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 0.5% (v/v) formic acid, 20 mM ammonium acetate), sheath liquid (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 20 mM ammonium acetate), separation voltage (20 kV), separation capillary internal diameter (i.d.) (75 µm), separation capillary temperature (23˚C) and sample injection time (6 s). The selected MS conditions included heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). Sheath gas was not used in this study. The total ion chromatograms (TICs), extracted ion chromatograms (EICs) and MS spectra of native PAPC standard and its in vitro oxidation products showed good repeatability and sensitivity. To determine the ox-PAPC products in human VLDLs, the EICs and MS spectra of VLDLs were compared with the in vitro oxidation products of native PAPC standard. For native PAPC standard, the measured linear range was 2.5 - 100.0 µg/mL, and the coefficients of determination (R2) was 0.9994. The concentration limit of detection (LOD) was 0.44 µg/mL, and the concentration limit of quantitation (LOQ) was 1.34 µg/mL. A total of 21 ox-PAPC products were analyzed for the VLDLs of healthy and uremic subjects. The levels of 7 short-chain and 5 long-chain ox-PAPC products on uremic VLDLs were significantly higher than healthy VLDLs. This simple low-flow CE-MS method might be a good alternative for LC-MS for the analysis of ox-PAPC products. Furthermore, it might also help scientists to expedite the search for uremic biomarkers.

Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography(2019-2022) / 药物分析学报

Capillary electrochromatography(CEC)plays a significant role in chiral separation via the double sepa-ration principle,partition coefficient difference between the two phases,and electroosmotic flow-driven separation.Given the distinct properties of the inner wall stationary phase(SP),the separation ability of each SP differs from one another.Particularly,it provides large room for promising applications of open tubular capillary electrochromatography(OT-CEC).We divided the OT-CEC SPs developed over the past four years into six types:ionic liquids,nanoparticle materials,microporous materials,biomaterials,non-nanopolymers,and others,to mainly introduce their characteristics in chiral drug separation.There also added a few classic SPs that occurred within ten years as supplements to enrich the features of each SP.Additionally,we discuss their applications in metabolomics,food,cosmetics,environment,and biology as analytes in addition to chiral drugs.OT-CEC plays an increasingly significant role in chiral separation and may promote the development of capillary electrophoresis(CE)combined with other instruments in recent years,such as CE with mass spectrometry(CE/MS)and CE with ultraviolet light detector(CE/UV).

Optimization of capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using polyvinyl alcohol coated capillaries. Application to a study on non-small cell lung cancer.

Despite recent developments in separation techniques, the analysis of relatively small highly polar negatively charged analytes (e.g. small organic acids, phosphorylated sugars, and underivatized amino acids) remains challenging. Capillary electrophoresis coupled to mass spectrometry (CE-MS) has been included in the untargeted metabolomics toolbox, although mostly in positive polarity. The aim of this study was to assess the use of CE-MS to analyze highly polar and negatively charged metabolites at physiological levels without the need for derivatization. After a preliminary selection, conditions regarding CE (buffers, applied potential, injection time and applied pressure), electrospray parameters (sheath liquid flow, temperature and drying gas flow, nebulizer, and capillary voltage), and fragmentor voltage were optimized using a capillary coated with polyvinyl alcohol (PVA) for the metabolic profiling of anionic compounds compared to fused silica as the reference capillary. In addition, a database of 240 metabolites with two relative migration times (RMT) obtained against methionine sulfone and 2-morpholinoethanesulfonic acid (MES) as internal standards (IS) has been compiled. Finally, the optimized method has been used to characterize the metabolic profile of blood plasma in patients with non-small cell lung cancer (NSCLC). The identified compounds are mostly amino acids and their derivatives, carboxylic acids and organic compounds from the TCA cycle, and sugars and their phosphoderivates. In addition, we performed a comparative study to find significant differences between non-small cell lung cancer (NSCLC) vs non-cancer individuals, and squamous cell carcinoma (SCC) and adenocarcinoma (ADC) vs non-cancer individuals, respectively, searching for differences between the various types of NSCLC.

Coupling High-Field Asymmetric Ion Mobility Spectrometry with Capillary Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry Improves Protein Identifications in Bottom-Up Proteomic Analysis of Low Nanogram Samples.

In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultrasensitive capillary electrophoresis hyphenated with tandem mass spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng of HeLa protein digest standard identified 1314 ± 30 proteins, 4829 ± 200 peptide groups, and 7577 ± 163 peptide spectrum matches (PSMs) corresponding to a 16, 25, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Label-free quantitation of proteins and peptides was also assessed to determine the precision of replicate analyses from FAIMS methods with increased cycle times. Our results also identified from 1 ng of HeLa protein digest without any prior enrichment 76 ± 9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.

Evaluation of Prototype CE-MS Interfaces.

Capillary electrophoresis-mass spectrometry (CE-MS) coupling is a powerful analytical solution bringing together the separation power of CE and the wealth of chemical information afforded by MS. Nevertheless, interfaces making the hyphenation of both techniques possible have always been the subject of a quest for improvement by their users in search for more sensitive and robust setups. This fact has led to numerous technical developments and new interface designs claiming to outrival existing approaches in different aspects. Nevertheless, the task of evaluating and comparing a new interface to previous solutions is not always straightforward. Issued from our own experience in the field, we herein propose a protocol to optimize the operation parameters of a new CE-MS interface design, assess its analytical performance, and compare it to a reference interface if desired. Electrospray stability, sensitivity, reproducibility, and robustness are practically evaluated as key elements of the process.

Capillary Electrophoresis-Mass Spectrometry (CE-MS) by Sheath-Flow Nanospray Interface and Its Use in Biopharmaceutical Applications.

Both capillary electrophoresis (CE) and mass spectrometry (MS) technologies are powerful analytical tools that have been used extensively in the characterization of biologics in the biopharmaceutical industry. The direct coupling of CE with MS is an attractive approach, in that the high separation capability of CE and the ultrasensitive detection and accurate identification performance of MS can be combined to provide a powerful system for the analysis of complex analytes. In this chapter, we discuss the detailed procedure of carrying out CE-MS analysis using a nano sheath-flow interface and its applications including intact mass analysis of monoclonal antibodies and fusion proteins, and a biotransformation study of two Fc-FGF21 molecules in a single-dose pharmacokinetic mice study. Optimization processes, including the finetuning of CE conditions and MS parameters, are illustrated in this chapter, with focuses on method robustness and assay reproducibility.

Microfabricated Liquid Junction Capillary Electrophoresis-Mass Spectrometry Interface.

Coupling of capillary electrophoresis (CE) with mass spectrometry (MS) represents a powerful combination for performing rapid, efficient, and sensitive analysis of a variety of compounds. Here we describe a construction, operation, and application of a microfabricated liquid junction CE-MS interface. The interface is designed as a microfabricated unit with an integrated liquid junction and electrospray tip made from polyimide, which is positioned in a plastic connection block securing the separation CE capillary and attachable to the CE instrument. The application was demonstrated by CE-MS analysis of dextran oligomers labeled by (2-aminoethyl)trimethylammonium (AETMA) salt.