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

Evaluation of ion mobility for the separation of glycoconjugate isomers due to different types of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level.

The study of protein glycosylation can be regarded as an intricate but very important task, making glycomics one of the most challenging and interesting, albeit under-researched, type of "omics" science. Complexity escalates remarkably when considering that carbohydrates can form severely branched structures with many different constituents, which often leads to the formation of multiple isomers. In this regard, ion mobility (IM) spectrometry has recently demonstrated its power for the separation of isomeric compounds. In the present work, the potential of traveling wave IM (TWIMS) for the separation of isomeric glycoconjugates was evaluated, using mouse transferrin (mTf) as model glycoprotein. Particularly, we aim to assess the performance of this platform for the separation of isomeric glycoconjugates due to the type of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level. Straightforward separation of isomers was achieved with the analysis of released glycans, as opposed to the glycopeptides which showed a more complex pattern. Finally, the developed methodology was applied to serum samples of mice, to investigate its robustness when analyzing real complex samples.Ion mobility mass spectrometry is a promising analytical technique for the separation of glycoconjugate isomers due to type of sialic acid linkage. The impact of such a small modification in the glycan structure is more evident in smaller analytes, reason why the analysis of free glycans was easier compared to the intact protein or the glycopeptides. The established methodology could be regarded as starting point in the separation of highly decorated glycoconjugates. This is an important topic nowadays, as differences in the abundance of some glycan isomers could be the key for the early diagnosis, control or differentiation of certain diseases, such as inflammation or cancer

Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity

This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA(+) or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA(+) mice and normal controls. The data supplied in this work is related to the research article entitled "identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38" [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163

Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Ontopath: A language for retrieving ontology fragments

Abstract. In this work we introduce a novel retrieval language, named OntoPath, for specifying and retrieving relevant ontology fragments. This language is intended to extract customized self-standing ontologies from very large, general-purpose ones. Through OntoPath, users can specify the desired detail level in the concept taxonomies as well as the properties between concepts that are required by the target applications. The syntax and aims of OntoPath resemble XPath’s in that they are simple enough to be handled by non-expert users and they are designed to be included in other XML-based applications (e.g. transformations sheets, semantic annotation of web services, etc.). OntoPath has been implemented on the top of the graph-based database G, for which a Protégé OWL plug-in has been designed to access and retrieve ontology fragments

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. 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.This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (RTI2018-097411-B-I00) and the Cathedra UB Rector Francisco Buscarons Ubeda (Forensic Chemistry and Chemical Engineering). Montserrat Mancera-Arteu acknowledges the University of Barcelona for an ADR fellowship. The authors thank Dr. Marina Gay, from the Mass Spectrometry and Proteomics Unit of the Institute for Research in Biomedicine (IRB Barcelona), for her helpful assistance with data interpretation in the nanoLC-MS/MS analysis

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-Sp) and hsa-let-7g-Sp (let-7g-Sp). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the micro cartridge 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 offline 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-Sp and a 23 nucleotide long 5'-phosphorylated miRNA with 3'-uridylation (iso-miR-16-5p), were only detected in the CLL patients