Comprehensive Characterization of Raw and Processed Quinoa from Conventional and Organic Farming by Label-Free Shotgun Proteomics.

Quinoa is widely recognized for its exceptional nutritional properties, particularly its complete protein content. This study, for the first time, investigates the effects of processing methods (boiling and extrusion) and farming conditions (conventional and organic) on the proteomic profile. Following a label-free shotgun proteomics approach, a total of 1796 proteins were identified and quantified across all quinoa samples.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Combined with Chemometrics for Protein Profiling and Classification of Boiled and Extruded Quinoa from Conventional and Organic Crops.

Quinoa is an Andean crop that stands out as a high-quality protein-rich and gluten-free food. However, its increasing popularity exposes quinoa products to the potential risk of adulteration with cheaper cereals. Consequently, there is a need for novel methodologies to accurately characterize the composition of quinoa, which is influenced not only by the variety type but also by the farming and processing conditions.

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.

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.

Determination of Sialic Acid Isomers from Released -Glycans Using Ion Mobility Spectrometry.

Complex carbohydrates are ubiquitous in nature and represent one of the major classes of biopolymers. They can exhibit highly diverse structures with multiple branched sites as well as a complex regio- and stereochemistry. A common way to analytically address this complexity is liquid chromatography (LC) in combination with mass spectrometry (MS).

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.

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.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis.

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [C]/[C] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS).

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).

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.

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

With 28 potential -glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of -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.

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

On-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) is a powerful technique for high throughput sample clean-up and analyte preconcentration, separation, detection, and characterization. The most typical design due to its simplicity and low cost is unidirectional SPE-CE-MS. However, in this configuration, the sample volumes introduced by pressure depend on the dimensions of the separation capillary and some matrix components could be irreversibly adsorbed in its inner walls.

Ionic matrices for matrix-assisted laser desorption/ionization mass spectrometry analysis of microRNA biomarkers.

The use of ionic matrices (IMs) was evaluated as an alternative to conventional matrices to analyze microRNAs (miRNAs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). 2, 4, 6-Trihydroxyacetophenone (THAP), 6-aza-2-thiothymine (ATT) and 3-hydroxypicolinic acid (3-HPA) and their IMs with pyridine (PYR) and butylamine (BA) were studied to analyze a standard mixture of miRNAs: miR-21, let-7g and iso-miR-16. Among all the studied matrices, ATT-PYR at 75 mg/mL in acetonitrile (MeCN):HO (50:50, v/v) was selected as the optimal.

Classification of quinoa varieties based on protein fingerprinting by capillary electrophoresis with ultraviolet absorption diode array detection and advanced chemometrics.

Quinoa (Chenopodium quinoa Willd.) is an andean grain with exceptional nutritional properties that has been progressively introduced in western countries as a protein-rich super food with a broad amino acid spectrum. Quinoa is consumed as whole grain, but it is also milled to produce high-value flour, which is susceptible to adulteration.

Polymeric monolithic microcartridges with gold nanoparticles for the analysis of protein biomarkers by on-line solid-phase extraction capillary electrophoresis-mass spectrometry.

In this study, polymeric monoliths with gold nanoparticles (AuNP@monolith) were investigated as microcartridges for the analysis of protein biomarkers by on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS). "Plug-and-play" microcartridges (7 mm) were prepared from a glycidyl methacrylate (GMA)-based monolithic capillary column (5 cm x 250 µm i.d.

Alterations in the Glycan Profile of Mouse Transferrin: New Insights in Collagen-Induced Arthritis.

Transferrin purification from mice serum samples by immunoaffinity chromatography (IAC) was optimized in order to study the possible modifications occurring in its glycans in collagen-induced arthritis (CIA) samples. SDS-PAGE and nanoLC-MS/MS were used to monitor the IAC purification performance. Afterward, a relative quantification of mouse transferrin (mTf) glycan isomers using [C]/[C]-aniline was used to unequivocally detect alterations in the glycan profile of CIA mice.

Analysis of glycopeptide biomarkers by on-line TiO solid-phase extraction capillary electrophoresis-mass spectrometry.

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides.

On-line Aptamer Affinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Blood α-Synuclein.

In this paper, an on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry method is described for the purification, preconcentration, separation, and characterization of α-synuclein (α-syn) in blood at the intact protein level. A single-stranded DNA aptamer is used to bind with high affinity and selectivity α-syn, which is a major component of Lewy bodies, the typical aggregated protein deposits found in Parkinson's disease (PD). Under the conditions optimized with recombinant α-syn, repeatability (2.

Improving separation optimization in capillary electrophoresis by using a general quality criterion.

In this paper we extend the use of the quality criterion t to optimize separations in capillary electrophoresis (CE). The theoretical parameter t' takes into account not only the relative separation between a given pair of compounds but also their separation from the neutral species migrating with the electroosmotic flow (EOF). Furthermore, it can be composed for complex mixtures as a global multicriterium optimization function T', for a rapid, simple and reliable selection of optimized separation conditions by mathematical maximization.

A critical retrospective and prospective review of designs and materials in in-line solid-phase extraction capillary electrophoresis.

Several strategies have been developed to decrease the concentration limits of detection (LODs) in capillary electrophoresis (CE). Nowadays, chromatographic-based preconcentration using a microcartridge integrated in the separation capillary for in-line solid-phase extraction capillary electrophoresis (SPE-CE) is one of the best alternatives for high throughput and reproducible sample clean-up and analyte preconcentration. This review covers different designs (geometrical configurations, with frits or fritless, capillary types, compatibility with commercial instrumentation, etc.

On-line protein digestion by immobilized enzyme microreactor capillary electrophoresis-mass spectrometry.

In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting β-lactoglogulin (β-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, on-line digestion, separation and characterization of the protein digests were possible in less than 30 min.

On-Line Immunoaffinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Serum Transthyretin.

The analysis of low abundant proteins in biological fluids by capillary electrophoresis (CE) is particularly problematic due to the typically poor concentration limits of detection of microscale separation techniques. Another important issue is sample matrix complexity that requires an appropriate cleanup. Here, we describe an on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS) method for the immunoextraction, preconcentration, separation, detection, and characterization of serum transthyretin (TTR).

Multivariate data analysis for the detection of human alpha-acid glycoprotein aberrant glycosylation in pancreatic ductal adenocarcinoma.

Relative quantification of human alpha-acid glycoprotein (hAGP) glycan isomers using [C]/[C]-aniline in combination with multivariate data analysis is proposed as an efficient method for the identification of pancreatic ductal adenocarcinoma (PDAC) glycan biomarkers in serum samples. Intact and desialylated glycans from hAGP, purified from serum samples of patients with PDAC and chronic pancreatitis (ChrP), were labeled with aniline and analyzed by μZIC-HILIC-MS. Afterwards, partial least squares discriminant analysis (PLS-DA) was applied to the relative areas obtained for all glycan isomers in the different samples: pathological (ChrP or PDAC) versus healthy samples.

Identification of antihypertensive peptides in nutraceuticals by capillary electrophoresis-mass spectrometry.

We present capillary electrophoresis-mass spectrometry (CE-MS) in combination with advanced chemometric tools for the analysis of bioactive compounds in food, in particular for the identification of antihypertensive peptides in a nutraceutical derived from a bovine milk protein hydrolysate. Different extracts of the nutraceutical were analyzed by CE-MS, and the electropherograms were processed using a novel data analysis workflow that included regions of interest (ROIs) compression and multivariate curve resolution alternating least squares (MCR-ALS). MCR-ALS permitted the description of the nutraceutical extract as ten characteristic components with their electrophoretic profiles and mass spectra.

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction-Capillary Electrophoresis-Mass Spectrometry.

In this paper, an on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges.