Evidence on the inhibitory effect of Brassica plants against Acinetobacter baumannii lipases: phytochemical analysis, in vitro, and molecular docking studies.

BACKGROUND: Infections caused by Acinetobacter baumannii are becoming a rising public health problem due to its high degree of acquired and intrinsic resistance mechanisms. Bacterial lipases penetrate and damage host tissues, resulting in multiple infections. Because there are very few effective inhibitors of bacterial lipases, new alternatives for treating A. baumannii infections are urgently needed. In recent years, Brassica vegetables have received a lot of attention since their phytochemical compounds have been directly linked to diverse antimicrobial actions by inhibiting the growth of various Gram-positive and Gram-negative bacteria, yeast, and fungi. Despite their longstanding antibacterial history, there is currently a lack of scientific evidence to support their role in the management of infections caused by the nosocomial bacterium, A. baumannii. This study aimed to address this gap in knowledge by examining the antibacterial and lipase inhibitory effects of six commonly consumed Brassica greens, Chinese cabbage (CC), curly and Tuscan kale (CK and TK), red and green Pak choi (RP and GP), and Brussels sprouts (BR), against A. baumannii in relation to their chemical profiles. METHODS: The secondary metabolites of the six extracts were identified using LC-QTOF-MS/MS analysis, and they were subsequently correlated with the lipase inhibitory activity using multivariate data analysis and molecular docking. RESULTS: In total, 99 metabolites from various chemical classes were identified in the extracts. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) revealed the chemical similarities and variabilities among the specimens, with glucosinolates and phenolic compounds being the major metabolites. RP and GP showed the highest antibacterial activity against A. baumannii, followed by CK. Additionally, four species showed a significant effect on the bacterial growth curves and demonstrated relevant inhibition of A. baumannii lipolytic activity. CK showed the greatest inhibition (26%), followed by RP (21%), GP (21%), and TK (15%). Orthogonal partial least squares-discriminant analysis (OPLS-DA) pinpointed 9 metabolites positively correlated with the observed bioactivities. Further, the biomarkers displayed good binding affinities towards lipase active sites ranging from -70.61 to -30.91 kcal/mol, compared to orlistat. CONCLUSION: This study emphasizes the significance of Brassica vegetables as a novel natural source of potential inhibitors of lipase from A. baumannii.

Assessment of the effect of drying on Brassica greens via a multiplex approach based on LC-QTOF-MS/MS, molecular networking, and chemometrics along with their antioxidant and anticancer activities.

Turnip (Brassica rapa var rapa L.) leaves are a rich source of versatile bioactive phytochemicals with great potential in the food and herbal industries. However, the effect of drying on its constituents has never been studied before. Hereto, three drying techniques were compared, namely, lyophilization (LY), vacuum oven (VO), and shade drying (SD). Chemical profiling utilizing liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) combined with chemometrics showed the different impacts of the drying methods on the phytochemical composition of the alcoholic leaf extracts. Unsupervised principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA) of the LC-QTOF-MS/MS data showed distinct distant clustering across the three drying techniques. Loading plots and VIP scores demonstrated that sinapic acid, isorhamnetin glycosides, and sinapoyl malate were key markers for LY samples. Meanwhile, oxygenated and polyunsaturated fatty acids were characteristic for SD samples and oxygenated polyunsaturated fatty acids and verbascoside were characteristic for VO samples. LY resulted in the highest total phenolics (TP) and total flavonoid (TF) contents followed by SD and VO. LY and SD samples had much higher antioxidant activity than VO measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), and iron metal chelation assays. According to the anticancer activity, the drying methods were ranked in descending order as SD > LY â‰« VO when tested against colon, breast, liver, and lung cancer cell lines. Among the identified compounds, flavonoids and omega-3 fatty acids were key metabolites responsible for the anticancer activity as revealed by partial least squares (PLS) regression and correlation analyses. In conclusion, compared to LY, SD projected out as a cost-effective drying method without compromising the phytochemical and biological activities of Brassica greens. The current findings lay the foundation for further studies concerned with the valorization of Brassica greens.

Identification of cyclooxygenase-II inhibitory saponins from fenugreek wastes: Insights from liquid chromatography-tandem mass spectrometry metabolomics, molecular networking, and molecular docking.

INTRODUCTION: This research explores sustainable applications for waste generated from fenugreek (Trigonella foenum-graecum), a plant with both nutritional and medicinal uses. The study specifically targets waste components as potential sources of nutrients and bioactive compounds. OBJECTIVES: The focus is to conduct detailed metabolic profiling of fenugreek waste, assess its anti-inflammatory properties by studying its cyclooxygenase (COX) inhibitory effect, and correlate this effect to the metabolite fingerprint. MATERIALS AND METHODS: Ethanolic extracts of fenugreek fruit pericarp and a combination of leaves and stems were subjected to untargeted metabolic profiling using liquid chromatography-mass spectrometry integrated with online database searches and molecular networking as an effective dereplication strategy. The study also scrutinized the COX inhibitory capabilities of these extracts and saponin-rich fractions prepared therefrom. Molecular docking was employed to investigate the specific interactions between the identified saponins and COX enzymes. RESULTS: The analysis led to the annotation of 81 metabolites, among which saponins were predominant. The saponin-rich fraction of the fruit pericarp extract displayed the strongest COX-II inhibitory activity in the in vitro inhibition assay (IC50 value of 81.64 ± 3.98 µg/mL). The molecular docking study supported the selectivity of the identified saponins towards COX-II. The two major identified saponins, namely, proto-yamogenin 3-O-[deoxyhexosyl (1 → 2)] [hexosyl (1 → 4)] hexoside 26-O-hexoside and trigofenoside A, were predicted to have the highest affinity to the COX-II receptor site. CONCLUSION: In the present study, we focused on the identification of COX-II inhibitory saponins in fenugreek waste through an integrated approach. The findings offer valuable insights into potential anti-inflammatory and cancer chemoprotective applications of fenugreek waste.

Rapid and Highly Efficient Separation of i-Motif DNA Species by CE-UV and Multivariate Curve Resolution.

The i-motif is a class of nonstandard DNA structure with potential biological implications. A novel capillary electrophoresis with an ultraviolet absorption spectrophotometric detection (CE-UV) method has been developed for the rapid analysis of the i-motif folding equilibrium as a function of pH and temperature. The electrophoretic analyses are performed in reverse polarity of the separation voltage with 32 cm long fused silica capillaries permanently coated with hydroxypropyl cellulose (HPC), after an appropriate conditioning procedure was used to achieve good repeatability. However, the electrophoretic separation between the folded and unfolded conformers of the studied cytosine-rich i-motif sequences (i.e., TT, Py39WT, and nmy01) is compromised, especially for Py39WT and nmy01, which result in completely overlapped peaks. Therefore, deconvolution with multivariate curve resolution-alternating least-squares (MCR-ALS) has been required for the efficient separation of the folded and unfolded species found at different concentration levels at pH 6.5 and between 12 and 40 °C, taking advantage of the small dissimilarities in the electrophoretic mobilities and UV spectra levels. MCR-ALS has also provided quantitative information that has been used to estimate melting temperatures (Tm), which are similar to those determined by UV and circular dichroism (CD) spectroscopies. The obtained results demonstrate that CE-UV assisted by MCR-ALS may become a very useful tool to get novel insight into the folding of i-motifs and other complex DNA structures.

A comprehensive analytical framework integrating liquid chromatography-tandem mass spectrometry metabolomics with chemometrics for metabolite profiling of lettuce varieties and discovery of antibacterial agents.

This study comprehensively characterized the metabolite profiles of six lettuce varieties and established the correlation between the elucidated profiles and their antivirulence effects. A total of 195 metabolites were annotated using LC-QTOF-MS/MS metabolomics assisted by molecular networking and integrated with chemometrics. Red varieties (red longifolia and lolla rosa) demonstrated higher chlorogenic and chicoric acids suggesting their antioxidant properties. In parallel, amino acids and disaccharides were enriched in romaine longifolia rationalizing its palatable taste and nutritional potential, while crispa, capitata, and lolla bionda presented a high ß-carboline alkaloid content. The antibacterial and antihemolytic potential of all varieties against methicillin-sensitive and methicillin-resistant Staphylococcus aureus was assessed and validated by prominent downregulation of α-hemolysin transcriptional levels in both strains. Moreover, correlation analysis revealed sesquiterpenes, ß-carboline alkaloids, amino acids, and oxy-fatty acids as the main bioactives. Results emphasize lettuce significance as a functional food and nutraceutical source, and highlight varieties naturally rich in antibacterial agents to adapt breeding programs.

Aptamer-functionalized stir bar sorptive extraction for selective isolation, identification, and determination of concanavalin A in food by MALDI-TOF-MS.

An aptamer-functionalized stir bar sorptive extraction (SBSE) coating is described for the first time devoted to selective isolation and preconcentration of an allergenic food protein, concavanalin A (Con A), followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) determination. For this purpose, the polytetrafluoroethylene surface of commercial magnetic stir bars was properly modified and vinylized to immobilize a thiol-modified aptamer against Con A via straightforward "thiol-ene" click chemistry. The aptamer-functionalized stir bar was employed as SBSE sorbent to isolate Con A, and several parameters that can affect the extraction efficiency were investigated. Under the optimized conditions, Con A was extracted and desorbed during 30 and 45 min, respectively, at 25 °C and 600 rpm. The SBSE MALDI-TOF-MS method provided limits of detection of 0.5 µg mL-1 for Con A. Furthermore, the SBSE coating was highly selective to Con A compared to other lectins. The developed method was successfully applied to the determination of low levels of Con A in several food matrices (i.e., white beans as well as chickpea, lentils, and wheat flours). Recoveries ranged from 81 to 97% with relative standard deviations below 7%. The aptamer-based stir bars presented suitable physical and chemical long-term stability (1 month) and a reusability of 10 and 5 extraction cycles with standards and food extracts, respectively. The developed aptamer-affinity extraction devices open up the possibility of developing novel highly selective SBSE coatings for the extraction of proteins and peptides from complex samples.

On-line aptamer affinity solid-phase extraction direct mass spectrometry for the rapid analysis of α-synuclein in blood.

On-line aptamer affinity solid-phase extraction direct mass spectrometry (AA-SPE-MS) is presented for the rapid purification, preconcentration, and characterization of α-synuclein (α-syn), which is a protein biomarker related to Parkinson's disease. Valve-free AA-SPE-MS is easily implemented using the typical SPE microcartridges and instrumental set-up necessary for on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). The essential requirement is substituting the application of the separation voltage by a pressure of 100 mbar for mobilization of the eluted protein through the capillary towards the mass spectrometer. Under optimized conditions with recombinant α-syn, repeatability is good in terms of migration time and peak area (percent relative standard deviation (%RSD) values (n = 3) are 1.3 and 6.6% at 1 µg mL-1, respectively). The method is satisfactorily linear between 0.025 and 5 µg mL-1 (R2 > 0.986), and limit of detection (LOD) is 0.02 µg mL-1 (i.e. 1000, 500, and 10 times lower than by CE-MS, direct MS, and AA-SPE-CE-MS, respectively). The established AA-SPE-MS method is further compared with AA-SPE-CE-MS, including for the analysis of α-syn in blood. The comparison discloses the advantages and disadvantages of AA-SPE-MS for the rapid and sensitive targeted analysis of protein biomarkers in biological fluids.

Unveiling the functional components and antivirulence activity of mustard leaves using an LC-MS/MS, molecular networking, and multivariate data analysis integrated approach.

Plant extracts have recently received increased attention as alternative sources of antimicrobial agents in the fight against multidrug-resistant bacteria. Non-targeted metabolomics liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, molecular networking, and chemometrics were used to evaluate the metabolic profiles of red and green leaves of two Brassica juncea (L.) varieties, var. integrifolia (IR and IG) and var. rugosa (RR and RG), as well as to establish a relationship between the elucidated chemical profiles and antivirulence activity. In total, 171 metabolites from different classes were annotated and principal component analysis revealed higher levels of phenolics and glucosinolates in var. integrifolia leaves and color discrimination, whereas fatty acids were enriched in var. rugosa, particularly trihydroxy octadecadienoic acid. All extracts demonstrated significant antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, presenting the IR leaves the highest antihemolytic activity against S. aureus (99 % inhibition), followed by RR (84 %), IG (82 %), and RG (37 %) leaves. Antivirulence of IR leaves was further validated by reduction in alpha-hemolysin gene transcription (∼4-fold). Using various multivariate data analyses, compounds positively correlated to bioactivity, primarily phenolic compounds, glucosinolates, and isothiocyanates, were also identified.

Accurate determination of the milk protein allergen ß-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry.

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein ß-lactoglobulin (ß-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against ß-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 µg mL-1 and the limit of detection (LOD) was 0.05 µg mL-1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (<0.5% and 2.4%, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of ß-LG was shown by analyzing milk-free foods (i.e. a 100% cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control.

Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.

A Proteomics Data Mining Strategy for the Identification of Quinoa Grain Proteins with Potential Immunonutritional Bioactivities.

Quinoa proteins are attracting global interest for their wide amino acid profile and as a promising source for the development of biomedical treatments, including those against immune-mediated diseases. However, information about the bioactivity of quinoa proteins is scarce. In this study, a quinoa grain proteome map obtained by label-free mass spectrometry-based shotgun proteomics was investigated for the identification of quinoa grain proteins with potential immunonutritional bioactivities, including those related to cancer. After carefully examining the sequence similarities of the 1211 identified quinoa grain proteins against already described bioactive proteins from other plant organisms, 71, 48, and 3 of them were classified as antimicrobial peptides (AMPs), oxidative stress induced peptides (OSIPs), and serine-type protease inhibitors (STPIs), respectively, suggesting their potential as immunomodulatory, anti-inflammatory, and anticancer agents. In addition, data interpretation using Venn diagrams, heat maps, and scatterplots revealed proteome similarities and differences with respect to the AMPs, OSIPs, and STPIs, and the most relevant bioactive proteins in the predominant commercial quinoa grains (i.e., black, red, white (from Peru), and royal (white from Bolivia)). The presented proteomics data mining strategy allows easy screening for potentially relevant quinoa grain proteins and commercial classes for immunonutrition, as a basis for future bioactivity testing.

Protein profiling and classification of commercial quinoa grains by MALDI-TOF-MS and chemometrics.

Quinoa is an Andean grain that is attracting attention worldwide as a high-quality protein-rich food. Nowadays, quinoa foodstuffs are susceptible to adulteration with cheaper cereals. Therefore, there is a need to develop novel methodologies for protein characterization of quinoa. Here, we first developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method to obtain characteristic mass spectra of protein extracts from 4 different commercial quinoa grains, which group different varieties marketed as black, red, white (from Peru) and royal (white from Bolivia). Then, data preprocessing and peak detection with MALDIquant allowed detecting 47 proteins (being 30 tentatively identified), the intensities of which were considered as fingerprints for multivariate data analysis. Finally, classification by partial least squares-discriminant analysis (PLS-DA) was excellent, and 34 out of the 47 proteins were critical for differentiation, confirming the potential of the methodology to obtain a reliable classification of quinoa grains based on protein fingerprinting.

On-line Immobilized Enzyme Microreactor Capillary Zone Electrophoresis-Mass Spectrometry for Peptide Mapping.

Peptide mapping is a routine procedure for protein characterization in proteomics. This bottom-up analysis requires digestion of proteins into peptides before liquid chromatography- or capillary zone electrophoresis-mass spectrometry (LC-MS or CZE-MS, respectively). Proteins are usually digested off-line using proteolytic enzymes, typically trypsin, in solution or immobilized on appropriate supports. As an alternative, here we describe on-line immobilized enzyme microreactor capillary zone electrophoresis-mass spectrometry (IMER-CZE-MS) for a straightforward, rapid, and efficient protein digestion followed by separation, detection, and characterization of the generated peptides.

Separation and characterization of bovine milk proteins by capillary electrophoresis-mass spectrometry.

Protein profiling of major bovine milk proteins (i.e., whey and casein proteins) is of great interest in food science and technology. This complex set of protein proteoforms may vary with breed, genetics, lactation stage, health, and nutritional status of the animal. Current routine methods for bovine milk protein profiling at the intact level are typically based on capillary electrophoresis-ultraviolet, which does not allow confirming unequivocally the identity of the separated proteins. As an alternative, in this study, we describe for the first time a novel and simple capillary electrophoresis-mass spectrometry method in positive electrospray ionization mode. Under the optimized conditions, capillary electrophoresis-mass spectrometry allowed the separation and identification at the intact level of major bovine milk whey and casein proteins in less than 15 min. Furthermore, high-resolution mass spectrometry confirmed its importance in the reliable characterization of bovine milk protein proteoforms, especially those with slight molecular mass differences, such as ß-casein A1 and A2, which are relevant to unequivocally identify milk with specific ß-casein compositions (e.g., A2A2 milk, which is widely known as A2 milk). This differentiation was not possible by matrix-assisted laser desorption/ionization mass spectrometry, which provided rapidly and easily a rich but less accurate fingerprint of bovine milk proteins due to the lower mass resolution.

On-line Coupling of Aptamer Affinity Solid-Phase Extraction and Immobilized Enzyme Microreactor Capillary Electrophoresis-Mass Spectrometry for the Sensitive Targeted Bottom-Up Analysis of Protein Biomarkers.

In this paper, we present a fully integrated valve-free method for the sensitive targeted bottom-up analysis of proteins through on-line aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (AA-SPE-IMER-CE-MS). The method was developed analyzing α-synuclein (α-syn), which is a protein biomarker related to different neurodegenerative disorders, including Parkinson's disease. Under optimized conditions, on-line purification and preconcentration of α-syn, enzymatic digestion, electrophoretic separation, and identification of the tryptic peptides by mass spectrometry was achieved in less than 35 min. The limit of detection was 0.02 µg mL-1 of digested protein (66.7% of coverage, i.e., 8 out of 12 expected tryptic peptides were detected). This value was 125 and 10 times lower than for independent on-line digestion by IMER-CE-MS (2.5 µg mL-1) and on-line preconcentration by AA-SPE-CE-MS (0.2 µg mL-1). The repeatability of AA-SPE-IMER-CE-MS was adequate (at 0.5 µg mL-1,% RSD ranged from 3.7 to 16.9% for peak areas and 3.5 to 7.7% for migration times of the tryptic peptides), and the modified capillary could be reused up to 10 analyses with optimum performance, similarly to IMER-CE-MS. The method was subsequently applied to the analysis of endogenous α-syn from red blood cell lysates. Ten α-syn tryptic peptides were detected (83.3% of coverage), enabling the characterization and localization of post-translational modifications of blood α-syn (i.e., N-terminal acetylation).

Systematic literature review of trials assessing recommended systemic treatments in hepatocellular carcinoma.

AIM: To identify and evaluate the similarity of all trials assessing recommended treatments for advanced hepatocellular carcinoma. MATERIALS & METHODS: Single arm and randomized trials from any phase and published any time up to February 2021 were systematically searched. RESULTS: From 5677 records reviewed, 50 trials were included in the review, and 24 for assessed for similarity. In the first-line (1L) setting, several trials assessing sorafenib were noted for enrolling patients with more severe disease and/or performance status than other 1L trials; trials within the second-line (2L) setting were generally similar. Median survival was <2 years in all trial arms. CONCLUSIONS: Trials assessing recommended treatments are largely similar and appropriate for quantitative comparisons of several efficacy and safety outcomes.

A hybrid nano-MOF/polymer material for trace analysis of fluoroquinolones in complex matrices at microscale by on-line solid-phase extraction capillary electrophoresis.

A hybrid material (nano-metal organic framework@organic polymer, named as nano-MOF@polymer) was applied for the first time as sorbent for on-line solid-phase extraction capillary electrophoresis with ultraviolet detection (SPE-CE-UV). The resulting material was prepared building layer-by-layer a HKUST-1 (Hong Kong University of Science and Technology-1) nano-MOF onto the polymer surface, which allowed controlling the thickness and maximizing the active surface area. The sorbent was widely characterized at micro- and nano-scale to validate the synthesis and to establish the material properties. Then, fritless microcartridges (2 mm) were assembled by packing only a few micrograms of sorbent particles and investigated for preconcentration of fluoroquinolones (FQs) in several real samples (river water, human urine and whole cow milk). Under the optimized conditions, the sample (ca. 60 µL) was loaded in separation background electrolyte (BGE, 50 mM phosphate (pH 7)), and retained analytes were eluted using a small volume of 2% v/v formic acid in methanol (ca. 50 nL). The SPE-CE-UV method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability, reproducibility and reusability. The developed method showed a LOD decreasing until 1 ng L-1 when larger volumes of sample were loaded (ca. 180 µL), which was 500,000 times lower than by CE-UV. This undescribed sensitivity enhancement would arise from the homogenous and populated MOF nano-domains and the appropriate permeability of the hybrid material, which would promote high extraction efficiency and loading capacity. Furthermore, the sorbent showed appropriate selectivity regardless the analyzed complex environmental, biological or food matrix samples, achieving excellent detectability and recoveries (>90%).

Characterization and differentiation of quinoa seed proteomes by label-free mass spectrometry-based shotgun proteomics.

Quinoa seed proteins are of prime importance in human nutrition and in plant breeding for cultivar identification and improvement. In this study, proteins from seeds of black, red, white quinoa from Peru and white quinoa from Bolivia (also known as royal) were extracted, digested and analyzed by nano-liquid chromatography coupled to Orbitrap tandem mass spectrometry (LC-MS/MS). The raw mass spectra data were processed for identification and label-free quantification (LFQ) using MaxQuant/Andromeda against a specific quinoa database from The National Center for Biotechnology Information (NCBI). In total, 1,211 quinoa proteins (85 were uncharacterized) were identified. Inspection and visualization using Venn diagrams, heat maps and Gene Ontology (GO) graphs revealed proteome similarities and differences between the four varieties. The presented data provides the most comprehensive experimental quinoa seed proteome map existing to date in the literature, as a starting point for more specific characterization and nutritional studies of quinoa and quinoa-containing foodstuff.

Analysis of Intact Glycoproteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be regarded as a key tool to rapidly obtain molecular mass information of intact glycoproteins in glycoproteomic studies and quality control of recombinant biopharmaceuticals. However, MALDI-TOF MS of these glycosylated compounds is a tricky task due to its low ionization efficiency and fragmentation of labile groups such as sialic acids.Here, we offer the reader a practical overview of the available methodologies for the confident analysis of intact glycoproteins with different glycosylation degree by MALDI-TOF MS. The three proposed methods fulfil the requirements of reproducibility and low extent of glycan fragmentation required to successfully analyze intact glycoproteins.

Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry.

With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X].