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. Therefore, there is a growing interest in developing novel analytical methods to get further information about quinoa at the chemical level. In this study, we developed a rapid and simple capillary electrophoresis-ultraviolet absorption diode array detection (CE-UV-DAD) method to obtain characteristic multiwavelength electrophoretic profiles of soluble protein extracts from different quinoa grain varieties. Then, advanced chemometric methods (i.e. multivariate curve resolution alternating least squares, MCR-ALS, followed by principal component analysis, PCA, and partial least squares discriminant analysis, PLS-DA) were applied to deconvolute the components present in the electropherograms and classify the quinoa varieties according to their differential protein composition

A review of sample preparation for purification of microRNAs and analysis by mass spectrometry methods

MicroRNAs (miRNAs) play an important role in regulation of different bioprocesses, including multiple diseases, such as cancer, neurodegenerative and immune-related disorders. Analysis of miRNA biomarkers in biological fluids requires accurate, sensitive, reproducible, and multiplexed methods. This review covers miRNA purification and measurement, which are the core of these analytical methods, and critically affect the output of biomarker research studies. With regard to miRNA measurement, the typical bioanalytical methods (e.g. reverse transcription polymerase chain reaction, RT-PCR), which have been extensively reviewed elsewhere, have been excluded to focus on less conventional methods based on mass spectrometry (MS). This review provides a broad overview of liquid-phase and solid-phase extraction purification methods for miRNA clean-up and enrichment and a critical insight into direct and indirect MS-based methods to disclose the true potential of MS in the field

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 AgaroseTM (UAPA), Dynabeads® Protein A (DyPA) and SiMAG-Protein A (SiPA)) and AffiAmino Ultrarapid AgaroseTM (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 6 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 optimised 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- 25 µg·mL-1 and limit of detection (LOD) was around 1 µg·mL-1 (25 times lower than by CE-MS, 25 µg·mL-1). 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

On-line solid-phase extraction to enhance sensitivity in peptide biomarker analysis by microseparation techniques coupled to mass spectrometry: capillary liquid chromatography versus capillary electrophoresis

In this study, on-line solid-phase extraction capillary liquid chromatography-mass spectrometry (SPE-CapLC-MS) and on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) were compared for the analysis of the opioid peptide biomarkers dynorphin A (1-7) (DynA), endomorphin 1 (End 1), and methionine-enkephalin (Met). First, a capillary liquid chromatography-mass spectrometry (CapLC-MS) method was established, which allowed limits of detection (LODs) of 0.5 μg/mL for Dyn A and Met, and 0.1 μg/mL for End 1. Then, a column switching setup operated by a 2-position/6-port micro-valve with a C18 enrichment column was assembled for SPE-CapLC-MS. Under optimized conditions, the LODs for the three peptides were lowered up to 1000-fold compared to CapLC-MS, until detecting 0.5 ng/mL concentrations. Repeatability (<0.2 % and <11 % RSD for retention times and peak areas, respectively), linearity (0.5-100 ng/mL), and durability (20 runs) of the enrichment column were appropriate, and the method was applied to analyze human plasma samples. Finally, the established SPE-CapLC-MS method was compared with a valve-free C18-SPE-CE-MS method previously described by our group for the analysis of these opioid peptides, using the same mass spectrometer. Both methods presented an evident difference regarding the need of a valve for the operation and allowed high preconcentration factors and quite similar LODs (until 0.5 and 0.1 ng/mL by SPE-CaLC-MS and SPE-CE-MS, respectively). Some other distinctions related to the instrumental set-up, procedure and method performance were also disclosed and discussed in detail

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 multi-enzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the non-specific 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 towards 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 [https://doi.org/doi:10.25345/C5Z50X]

Glicoproteínas biomarcadoras de cáncer: un reto analítico actual

Las alteraciones en la glicosilación de las proteínas pueden ser la causa de enfermedades graves, como es el caso de los desórdenes congénitos de la glicosilación (CDGs) o pueden aparecer en respuesta a la presencia de un proceso patológico, como ocurre con el cáncer y los procesos inflamatorios. Por ello, las glicoproteínas se utilizan ampliamente en el diagnóstico de diversas patologías [1, 2]. Por ejemplo, el antígeno carbohidratado 19-9 (CA 19-9), el antígeno carcio-embrionario humano (CEA) o el antígeno prostático específico (PSA) se emplean en la actualidad como biomarcadores para el diagnóstico del cáncer de páncreas, colon o próstata, respectivamente. Sin embargo, sólo se mide el aumento de sus niveles por encima de unos valores establecidos, con lo que en muchos casos estos biomarcadores no son totalmente específicos ya que otras enfermedades pueden presentar niveles anormalmente elevados de estas glicoproteínas. Por este motivo, el reto analítico actual consiste en mejorar la selectividad y la sensibilidad de estos biomarcadores, llevando a cabo una caracterización exhaustiva de los puntos de glicosilación y de la composición y estructura de los carbohidratos, comúnmente llamados glicanos