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.

Determination of Sialic Acid Isomers from Released N-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). However, MS-based detection often does not provide sufficient information to distinguish glycan isomers. Ion mobility-mass spectrometry (IM-MS)─a technique that separates ions based on their size, charge, and shape─has recently shown great potential to solve this problem by identifying characteristic isomeric glycan features such as the sialylation and fucosylation pattern. However, while both LC-MS and IM-MS have clearly proven their individual capabilities for glycan analysis, attempts to combine both methods into a consistent workflow are lacking. Here, we close this gap and combine hydrophilic interaction liquid chromatography (HILIC) with IM-MS to analyze the glycan structures released from human alpha-1-acid glycoprotein (hAGP). HILIC separates the crude mixture of highly sialylated multi-antennary glycans, MS provides information on glycan composition, and IMS is used to distinguish and quantify α2,6- and α2,3-linked sialic acid isomers based on characteristic fragments. Further, the technique can support the assignment of antenna fucosylation. This feature mapping can confidently assign glycan isomers with multiple sialic acids within one LC-IM-MS run and is fully compatible with existing workflows for N-glycan analysis.

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].

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 [12C6]/[13C6]-aniline was used to unequivocally detect alterations in the glycan profile of CIA mice. In addition, multivariate data analysis was applied to identify the most meaningful glycan isomers for the discrimination between control and pathological samples. Partial least-squares discriminant analysis (PLS-DA) revealed that five out of fifteen mTf glycan isomers could be potential biomarkers of CIA, most of them corresponding to highly sialylated structures (H6N5S3_2, H6N5S3_3, and H5N4S3_2). Moreover, some of these glycan isomers also seemed to be related with the progression of CIA, especially H6N5S2 and H6N5S3_2, as their overexpression increased with the clinical score of the pathology. Hence, the established methodology not only provides valuable information to find glycan-based biomarkers of CIA, but also leaves the door open to evaluate, in the future, glycosylation changes of many other inflammatory diseases, in which transferrin has been described to be altered.

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 (2.1 and 5.4% percent relative standard deviation for migration times and peak areas, respectively) and microcartridge lifetime (around 20 analyses/microcartridge) were good, the method was linear between 0.5 and 10 µg·mL-1, and limit of detection was 0.2 µg·mL-1 (100 times lower than by CE-MS, 20 µg·mL-1). The method was subsequently applied to the analysis of endogenous α-syn from red blood cells lysate of healthy controls and PD patients.

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. Under the optimized conditions with standards, the microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol·L-1, and the limit of detection (LOD) was around 10 nmol·L-1 (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-5p and a 23 nucleotide long 5'-phosphorylated miRNA with 3'-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

Analysis of O-Glycopeptides by Acetone Enrichment and Capillary Electrophoresis-Mass Spectrometry.

Acetone precipitation was evaluated as a rapid, simple, low-cost, and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovine α1-acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3), and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides, and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behavior of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterward, partial least-squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O126-glycopeptide glycoforms of rhEPO because of their applicability in biopharmaceutical quality control and doping analysis.

Analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads.

In this paper, an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using magnetic beads (MBs) is described for the analysis of serum transthyretin (TTR), which is a protein related to different types of amyloidosis. First, purification of TTR from serum was investigated by off-line immunoprecipitation and CE-MS. The suitability of three Protein A (ProA) MBs (Protein A Ultrarapid Agarose(TM) (UAPA), Dynabeads(®) Protein A (DyPA) and SiMAG-Protein A (SiPA) and AffiAmino Ultrarapid Agarose(TM) (UAAF) MBs to prepare an IA sorbent with a polyclonal antibody (Ab) against TTR, was studied. In all cases, results were repeatable and it was possible the identification and the quantitation of the relative abundance of the six most abundant TTR proteoforms. Although recoveries were the best with UAPA MBs, UAAF MBs were preferred for on-line immunopurification because Ab was not eluted from the MBs. Under the optimized conditions with standards in IA-SPE-CE-MS, microcartridge lifetime (>20 analyses/day) and repeatability (2.9 and 4.3% RSD for migration times and peak areas) were good, the method was linear between 5 and 25 µg/mL and LOD was around 1 µg/mL (25 times lower than by CE-MS, ≈25 µg/mL). A simple off-line sample pretreatment based on precipitation of the most abundant proteins with 5% (v/v) of phenol was necessary to clean-up serum samples. The potential of the on-line method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was demonstrated analysing serum samples from healthy controls and FAP-I patients.

Improved tryptic digestion assisted with an acid-labile anionic surfactant for the separation and characterization of glycopeptide glycoforms of a proteolytic-resistant glycoprotein by capillary electrophoresis time-of-flight mass spectrometry.

Certain glycoproteins are rather difficult to digest due to compacted tertiary or quaternary structures. In a previous study, a capillary LC coupled to TOF-MS (µLC-TOF-MS) method was developed for the detection and characterization of the glycopeptide glycoforms of human transferrin (Tf), a proteolytic resistant glycoprotein, in serum samples. After immunoaffinity purification, Tf was digested with trypsin in the presence of RapiGest(®) and µLC-TOF-MS analyses permitted to detect the N413 and N611 glycopeptide glycoforms. Conversely, the use of this surfactant, albeit mandatory to quantitatively digest the isolated Tf, proved detrimental to CE-TOF-MS analysis due to its interaction with the inner surface of the silica capillary walls. As CE is usually regarded as an interesting alternative to other separation techniques (low consumption of reagents, excellent separation efficiency, and reduced analysis times), in this work, the undesirable interferences of the surfactant have been removed to allow the correct separation and detection of Tf glycoforms by CE-TOF-MS. Moreover, the digestion protocol described by the RapiGest(®) manufacturer has been modified to minimize desialylation of Tf glycopeptide glycoforms. The new developed CE-TOF-MS methodology has been then compared with the former µLC-TOF-MS by means of sensitivity and separation efficiency of Tf glycopeptide glycoforms in the standard glycoprotein. Additionally, Tf glycopeptide glycoforms from serum of healthy volunteers and patients with congenital disorders of glycosylation have also been analyzed following the developed methodology.

Metabolic profiling for the identification of Huntington biomarkers by on-line solid-phase extraction capillary electrophoresis mass spectrometry combined with advanced data analysis tools.

In this work, an untargeted metabolomic approach based on sensitive analysis by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild-type (wt) and HD (R6/1) mice of different ages (8, 12, and 30 weeks), were analyzed by C18 -SPE-CE-MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS datasets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow-up the HD progression. The intracellular signaling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors, and changed expression of neurotransmitters.

Analysis of transthyretin in human serum by capillary zone electrophoresis electrospray ionization time-of-flight mass spectrometry. Application to familial amyloidotic polyneuropathy type I.

Transthyretin (TTR) is known to misfold and aggregate, causing different types of amyloidosis. Familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, is associated with a TTR variant that presents a single amino acid substitution of valine for methionine at position 30 (Met 30). To screen for TTR-related amyloidosis rapidly and reliably, we have developed a novel procedure based on the analysis of monomers from the homotetrameric protein (∼56 kDa). First, we established a CZE-ESI-TOF-MS method to detect wild-type (normal) TTR with or without several PTMs, as well as an extra minor isoform in TTR standard solutions. Later, a sample pretreatment based on immunoprecipitation (IP) and centrifugal filtration was optimized to analyze serum samples from healthy controls and FAP-I patients (including an asymptomatic patient, a symptomatic patient, a liver-transplanted patient with the specific mutation, and a patient originally without the mutation who received a liver transplant from an FAP-I patient (iatrogenic FAP-I)). The mutant TTR (Met 30) variant with a relative molecular mass 32.07 higher than the wild-type TTR was found in the asymptomatic, the symptomatic and the iatrogenic FAP-I patients, who interestingly also presented the same concentration ratio between both variants of TTR (abnormal and normal). In contrast, as in the healthy controls, the abnormal TTR variant was not detected in the liver-transplanted patient with the specific mutation, which confirms the effectiveness of the treatment. The proposed procedure could be regarded as a suitable screening system for individuals with suspected TTR amyloidosis, and to gain insight into TTR structure, to understand the mechanism underlying the disease.

Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on-line solid-phase extraction capillary electrophoresis-mass spectrometry.

Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid ß-protein (Aß) (Aß(1-15) and Aß(10-20) peptides) by on-line immobilized metal affinity SPE-CE (IMA-SPE-CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25-fold and 5-fold decrease in the LODs by IMA-SPE-CE-UV for Aß(1-15) and Aß(10-20) peptides (0.1 and 0.5 µg/mL, respectively) with regard to CE-UV (2.5 µg/mL for both peptides). The phosphate BGE was also used in IMA-SPE-CE-MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aß(10-20) peptide, which could be detected until 0.25 µg/mL. Linearity for Aß(10-20) peptide was good in a narrow concentration range (0.25-2.5 µg/mL, R(2) = 0.93). Lastly, the potential of the optimized Ni(II)-IMA-SPE-CE-MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples.

Combination of CE-MS and advanced chemometric methods for high-throughput metabolic profiling.

In this work, an untargeted approach based on capillary electrophoresis-mass spectrometry (CE-MS) in combination with multivariate data analyses is proposed as a high-throughput general methodology for metabolomic studies. First, total ion electropherograms (TIEs) were considered for exploratory and classification purposes by means of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS data sets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. The proposed approach allowed solving additional electrophoretic issues, such as background noise contributions, low signal-to-noise ratios, asymmetric peaks and migration time shifts. The usefulness of the proposed methodology is demonstrated in a comparative study of the metabolic profiles from baker's yeast (Saccharomyces cerevisiae) samples cultured at two temperatures, 30°C and 37°C. A total number of 80 metabolites were relevant to yeast samples differentiation at the two temperatures and almost 50 of them were tentatively identified based on their accurate experimental molecular mass. The results show that changes in amino acid, nucleotide and lipid metabolic pathways participated in the acclimatization of yeast cells to grow at 37°C.

Fast determination of harmala alkaloids in edible algae by capillary electrophoresis mass spectrometry.

The use of algae as a foodstuff is rapidly expanding worldwide from the East Asian countries, where they are also used for medical care. Harmala alkaloids (HAlk) are a family of bioactive compounds found in the extracts of some plants, including wakame (Undaria pinnatifida), an edible marine invasive algae. HAlks are based on a characteristic ß-carboline structure with at least one amino ionizable group. In this work, we report the successful separation of a mixture of six HAlks (harmine, harmaline, harmol, harmalol, harmane, and norharmane) by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) in less than 8 min. Optimum separation in fused-silica capillaries and detection sensitivity in positive-ion mode were achieved using a background electrolyte (BGE) with 25 mmol L(-1) ammonium acetate (pH 7.8) and 10% (v/v) methanol, and a sheath liquid with 60:40 (v/v) isopropanol-water and 0.05% (v/v) formic acid. The separation method was validated in terms of linearity, limits of detection and quantification, repeatability, and reproducibility. Later, a sample pretreatment was carefully optimized to determine HAlks in commercial wakame samples with excellent recovery and repeatability. For the complex wakame extracts, the MS-MS fragmentation patterns of the different HAlks were useful to ensure a reliable identification. The complete procedure was validated using the standard-addition calibration method, determining matrix effects on the studied compounds. Harmalol, harmine, and harmaline were naturally present in the samples and were quantified at very low concentrations, ranging from 7 to 24 µg kg(-1) dry algae.

Interlaboratory study to evaluate the robustness of capillary electrophoresis-mass spectrometry for peptide mapping.

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields.

On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry for the analysis of large biomolecules: a preliminary report.

The analysis of large biomolecules by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) remains unexplored because of the complex issues that need to be addressed. In this preliminary study, we used the human glycoprotein transferrin (Tf) as a model of a large biomolecule. First, we established by CE-UV a novel method compatible with IA-SPE-CE-MS, based on the use of a fused silica capillary coated with an anionic derivative of polyacrylamide (UltraTrol(TM) Dynamic Pre-Coat High Normal, HN) to prevent protein adsorption. The methodology allowed the detection of the most abundant Tf sialoforms. Repeatability studies demonstrated high stability of the coated capillaries, which was required for on-line immunoextraction and MS detection. IA-SPE-CE-UV and IA-SPE-CE-MS methods were optimized for the analysis of Tf standards and human serum samples using a laboratory-made IA sorbent. Three peaks corresponding to Tf were detected with UV detection when on-line immunoextraction was applied to the standards. The use of MS detection, however, reduced the resolution of the electrophoretic separation. Finally, we demonstrated that it was possible to detect Tf in human serum samples, after off-line serum sample de-salting by centrifugal filtration.

Evaluation of fritless solid-phase extraction coupled on-line with capillary electrophoresis-mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid.

Fritless SPE on-line coupled to CE with UV and MS detection (SPE-CE-UV and SPE-CE-MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 µm id was packed with a C18 sorbent (particle size > 50 µm), which was retained between a short inlet capillary and a separation capillary (50 µm id). Several experimental parameters were optimized by SPE-CE-UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine-enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro-organic solution. Using SPE-CE-MS, peak area and migration time repeatabilities for the three opioid peptides were 12-27% and 4-5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.

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. In this study, we present a rapid and straightforward method based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to generate global fingerprints of quinoa proteins from white quinoa varieties, which were cultivated under conventional and organic farming and processed through boiling and extrusion. The mass spectra of the different protein extracts were processed using the MALDIquant software (version 1.19.3), detecting 49 proteins (with 31 tentatively identified). Intensity values from these proteins were then considered protein fingerprints for multivariate data analysis. Our results revealed reliable partial least squares-discriminant analysis (PLS-DA) classification models for distinguishing between farming and processing conditions, and the detected proteins that were critical for differentiation. They confirm the effectiveness of tracing the agricultural origins and technological treatments of quinoa grains through protein fingerprinting by MALDI-TOF-MS and chemometrics. This untargeted approach offers promising applications in food control and the food-processing industry.

Identification of bioactive peptides in hypoallergenic infant milk formulas by CE-TOF-MS assisted by semiempirical model of electromigration behavior.

Biologically active peptides derived from complex bovine milk protein hydrolysates are of particular interest in food science and nutrition because they have been shown to play different physiological roles, providing benefits in human health. In this study, we used CE-TOF-MS for separation and identification of bioactive peptides in three hypoallergenic infant milk formulas. An appropriate sample cleanup using a citrate buffer with DTT and urea followed by SPE with Sep-Pack® C18 and StrataX™ cartridges allowed the detection of a large number of low molecular mass bioactive peptides. This preliminary identification was solely based on the measured experimental monoisotopic molecular mass values (M(exp)). Later, we evaluated the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m(e) vs. q/M(α), α = 1/2 for the classical polymer model) to describe their migration behavior. The assistance of migration prediction proved to be useful to improve reliability of the identification, avoiding misinterpretations and solving some identity conflicts. After revision, the identity of 24, 30, and 38 bioactive peptides was confirmed in each of the three infant milk formulas. A significant number of these peptides were reported as inhibitors of angiotensin-converting enzyme, however, the presence of sequences with other biological activities such as antihypertensive, antithrombotic, hypocholesterolemic, immunomodulation, cytotoxicity, antioxidant, antimicrobial, antigenic, or opioid was also confirmed.

Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using [12C]/[13C] aniline and ZIC-HILIC-ESI-TOF-MS.

Glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ∼1-5 % for major and ∼5-10 % for minor N-glycans). In a second step, zwitterionic-hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (µZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine α1-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples.