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Metabolomics in amniotic fluid
Published in Moshe Hod, Vincenzo Berghella, Mary E. D'Alton, Gian Carlo Di Renzo, Eduard Gratacós, Vassilios Fanos, New Technologies and Perinatal Medicine, 2019
Alexandra-Maria Michaelidou, Foteini Tsakoumaki, Maria Fotiou, Charikleia Kyrkou, Apostolos P. Athanasiadis
Virgiliou et al. (17) published a study entitled, “Amniotic fluid and maternal serum metabolic signatures in the 2nd trimester associated with pre-term delivery.” The ultimate goal was to identify markers or concentration patterns toward characterizing PTD and subsequently focus on specific assays for diagnostic and/or prognostic purposes. There were 35 preterm and 35 term deliveries paired based on maternal age and gestational week of amniocentesis. Thirteen AF samples out of 35 of PTD cases together with 14/35 matched controls were analyzed using reversed-phase (RP)-UHPLC by time of flight (TOF)/MS (in both ESI+ and ESI−). These findings indicated that medium polarity and unpolar metabolites contribute to the discrimination of the clinical groups under study. Further, a targeted metabolomics approach by using hydrophilic interaction chromatography (HILIC) MS/MS method was performed on the whole set of samples. It was found that glutamine, pyruvate, and inositol were in lower concentrations in PTD cases, whereas glutamate was slightly lower in controls. These changes, according to pathway analysis, are associated with energy metabolism (glycolysis, gluconeogenesis, pyruvate metabolism) as well as metabolism of glutathione, alanine, aspartate, and glutamate (17).
Synthesis of Bioactive Peptides for Pharmaceutical Applications
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Jaison Jeevanandam, Ashish Kumar Solanki, Shailza Sharma, Prabir Kumar Kulabhusan, Sapna Pahil, Michael K. Danquah
Biopeptide purification employing HPLC utilizes the differences in the size of peptide (size exclusion HPLC), hydrophobicity (RP-HPLC) or net charge (ion exchange HPLC) as a tool for separation. The potential for the separation of an HPLC column can be improved by manipulating the conditions of the mobile phase. Also, a mixed mode approach, named as hydrophilic interaction chromatography (HILIC) exhibited exceptional peptide separation potentials (Arumugam et al., 2018).
Metabolomic Techniques to Discover Food Biomarkers
Published in Dale A. Schoeller, Margriet S. Westerterp-Plantenga, Advances in the Assessment of Dietary Intake, 2017
Pekka Keski-Rahkonen, Joseph A. Rothwell, Augustin Scalbert
Regardless of the type of the MS instrument, metabolomics methods can be broadly divided between direct analysis of the sample and separation-based techniques. In direct analysis, the sample is typically introduced into the mass spectrometer with the help of LC pump and autosampler, relying on the MS for selective compound detection. The main advantages of this technique are its high throughput and robustness due to the lack of chromatographic separation, which commonly takes at least 10 minutes per sample (Draper et al. 2013). However, despite the high mass resolution achieved by current instruments, chromatography has remained important in metabolomics, as separation prior to detection adds retention time as an additional dimension to the acquired data, enabling selective analysis of isobaric compounds and reduction of interferences from the sample matrix. In the case of LC–MS, the chromatographic methods are most commonly based on reversed phase columns, with a number of different column dimensions and selectivities being used (Tulipani et al. 2015). Reversed phase chromatography has an established position in LC–MS metabolomics, but typically cannot provide sufficient retention and separation for highly polar molecules such as carbohydrates, phosphates, amino acids, and other short-chain organic acids that are likely to represent a large portion of dietary metabolites. Fortunately, alternative chromatographic techniques exist, and applying two or more orthogonal methods can significantly extend the number of compounds detected (Boudah et al. 2014; Ramakrishnan et al. 2016). Hydrophilic interaction chromatography (HILIC) in particular has been found to be complementary to reversed phase in metabolomics (Kloos et al. 2013) and has enabled successful analysis of highly polar dietary metabolites such as malic acid (Pekkinen et al. 2014) and amino acid-derived betaines (Pekkinen et al. 2015).
Advances in phosphoproteomics and its application to COPD
Published in Expert Review of Proteomics, 2022
Xiaoyin Zeng, Yanting Lan, Jing Xiao, Longbo Hu, Long Tan, Mengdi Liang, Xufei Wang, Shaohua Lu, Tao Peng, Fei Long
Hydrophilic interaction chromatography (HILIC) is performed by employing a strongly polar stationary phase combined with a mobile phase consisting of a high organic phase/low proportion of the aqueous phase. It typically has a more robust binding capacity for strongly polar compounds, so hydrophobic compounds are detected first upon elution, followed by hydrophilic compounds (Figure 2d). HILIC can provide different selectivity than conventional reversed-phase (RP) chromatography. The separation efficiency of HILIC for strongly polar compounds in aqueous-rich mobile phases may be better than that in reversed-phase chromatography due to its organic-rich and less viscous mobile phases [65]. The qualities of HILIC containing aqueous organic phases facilitate the ionization efficiency of mass spectrometry, improving the sensitivity of mass spectrometry analysis. This improvement makes HILIC suitable for combination with mass spectrometry and has increasing popularity. Zappacosta et al. [66] combined HILIC and Fe3+-IMAC enrichment to identify more than 16,000 phosphorylation sites in rat liver.
Characterizing bacterial glycoproteins with LC-MS
Published in Expert Review of Proteomics, 2018
Kelly M. Fulton, Jianjun Li, Juan M. Tomas, Jeffrey C. Smith, Susan M. Twine
Hydrophilic interaction chromatography (HILIC) uses a hydrophilic stationary phase (like NPLC) and a hydrophobic organic solvent as the mobile phase (like RPLC), which results in improved retention of hydrophilic and polar analytes compared with either NPLC or RPLC. Though the mechanism is not clearly understood, it is believed that an aqueous layer forming between the hydrophilic stationary phase and the hydrophobic mobile phase is responsible for the improved retention of hydrophilic analytes, such as glycans and glycopeptides. Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) employs a stationary phase with both positive and negative charges, and therefore has greater specificity for polar analytes than regular HILIC through electrostatic interactions with both positively and negatively charged ions [70]. Since ZIC-HILIC separates glycopeptides based on the glycan component rather than the peptide portion, it provides greater separation of glycopeptides that would co-elute in RPLC [71]. The ZIC-HILIC enrichment method [72] has been used in combination with a number of MS approaches to improve the characterization of bacterial protein glycosylation [41,60,73,74]. However, at least one study suggests that the ZIC-HILIC approach may favor smaller mass glycopeptides over larger mass glycopeptides [60].
Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives
Published in Journal of Extracellular Vesicles, 2018
Charles Williams, Felix Royo, Oier Aizpurua-Olaizola, Raquel Pazos, Geert-Jan Boons, Niels-Christian Reichardt, Juan M. Falcon-Perez
The structural limitations of lectin microarrays can be avoided by using high-resolution MS techniques [35,50]. Briefly, MS instruments are coupled to a previous separation technique, stratifying a sample of released glycans into fractions and facilitating structural analysis through reduced heterogeneity. Several different methods exist for both separation and MS, with sample preparation defined by the class of glycoconjugate that the researcher wishes to assay. To elaborate, different chemical linkages connect glycans to protein or lipid carriers and the glycan isolation methods that have been developed can target only one of the different glycan classes present. For example, N-glycans are readily removed from denatured glycoproteins through enzymatic digestion with PNGaseF but no efficient enzyme exists for the release of O-glycans. Sample separation is usually achieved by ultra or high-performance liquid chromatography (UPLC or HPLC) or, in some applications, capillary electrophoresis (CE). Different separation columns based on different stationary phases are used in LC, including hydrophilic-interaction chromatography (HILIC) columns or porous graphitized carbon columns, and the separation capabilities of LC allows for isomeric structures to be resolved. Moreover, released glycans are usually labelled to enhance glycan separation and mass-spectrometric detection, and several labels can be used for each analytical technique [51]. Considering those MS methods that are not coupled to a sample separation technique, Matrix-Assisted Laser Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) is the most established approach due to its high-throughput and automation potential [52]. For the assignment of glycan structures tandem mass spectrometry (MS/MS) is a particularly powerful tool. Here, selected precursor ions are fragmented into product ions, usually by the collision with a gas, and the fragmentation pattern employed to gain additional insight into the analyte structure [53].