Discovery Sulfoglycomics and Identification of the Characteristic Fragment Ions for High-Sensitivity Precise Mapping of Adult Zebrafish Brain–Specific Glycotopes

Mass spectrometry–based high-sensitivity mapping of terminal glycotopes relies on diagnostic MS2 and/or MS3 ions that can differentiate linkage and define the location of substituents including sulfates. Unambiguous identification of adult zebrafish glycotopes is particularly challenging due to the presence of extra β4-galactosylation on the basic building block of Galβ1-4GlcNAc that can be fucosylated and variably sialylated by N-acetyl, N-glycolyl, or deaminated neuraminic acids. Building on previous groundwork that have identified various organ-specific N- and O-glycans of adult zebrafish, we show here that all the major glycotopes of interest can be readily mapped by direct nano-LC-MS/MS analysis of permethylated glycans. Homing in on the brain-, intestine-, and ovary-derived samples, organ-specific glycomic reference maps based on overlaid extracted ion chromatograms of resolved glycan species, and composite charts of summed intensities of diagnostic MS2 ions representing the distribution and relative abundance of each of the glycotopes and sialic acid variants were established. Moreover, switching to negative mode analysis of sample fractions enriched in negatively charged glycans, we show, for the first time, that a full range of sulfated glycotopes is expressed in adult zebrafish. In particular, 3-O-sulfation of terminal Gal was commonly found, whereas terminal sulfated HexNAc as in GalNAcβ1-4GlcNAc (LacdiNAc), and 3-O-sulfated hexuronic acid as in HNK-1 epitope (SO3-3GlcAβ1-3Galβ1-4GlcNAc) were identified only in the brain and not in the intestine or ovaries analyzed in parallel. Other characteristic structural features of sulfated O- and N-glycans along with their diagnostic ions detected in this discovery mode sulfoglycomic work collectively expand our adult zebrafish glycome atlas, which can now allow for a more complete navigation and probing of the underlying sulfotransferases and glycosyltransferases, in search of the functional relevance of zebrafish-specific glycotopes. Of particular importance is the knowledge of glycomic features distinct from those of humans when using adult zebrafish as an alternative vertebrate model, rather than mouse, for brain-related glyco-neurobiology studies.

Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

Comprehensive Study on the chemical profile and anti-tumor activity of secondary metabolites produced by Aspergillus niger

Objective: Chemical investigation of the extract of sponge-derived fungus, Aspergillus niger, was performed by liquid chromatography coupled with QExactive mass spectrometry for the first time. Method: A total number of 444 constituents were detected, 288 of which were identified positively or tentatively by the comprehensive utilization of accurate molecular weight and fragmentation information acquired from quadrupole orbitrap mass spectrometry. The identified compounds were divided into several types, namely, organic acid, alkaloid, saccharide, amino acid and cyclopeptide, terpenoid, polyketone, phenylpropanoid and other types of compounds. Systematic diagnostic ions and featured fragment patterns were summarized for each type, based on which 8 novel compounds belonging to the same type were characterized. Results: This work provided a rapid approach for the research of microconstituents in a complex analyte. Furthermore, the anti-tumor activity of the extract was evaluated on two different cell lines—Bel-7402 and Hela-S3 in vitro. The tumor-inhibitory effect of the Aspergillus niger extract was confirmed, and may be mainly derived from its pro-apoptotic action. Moreover, the extract exerted more significant cytotoxicity in Bel-7402 cells than Hela-S3 cells, indicative of its selectivity on specific tumor cells. Conclusion: The evidence suggested that the Aspergillus niger extract may potentially serve as a remedy for prevention and therapy of hepatic and breast carcinoma.

Characterization and Quantification of Phenolic Constituents in Peach Blossom by UPLC-LTQ-Orbitrap-MS and UPLC-DAD

Peach blossom comes from the flower of Prunus persica (L.) Batsch, which is used as herbal tea and medicine in China and Korea. It could promote defecation and alleviate the abdominal pain. In this paper, the methods, ultra-performance liquid chromatography (UPLC) method coupled with electrospray ionization hybrid linear trap quadrupole orbitrap mass spectrometry (LTQ OrbitrapMS) and UPLC system coupled with a diode array detector (DAD), were developed for the qualitative and quantitative analysis of the flavonoids and phenolic acids in peach blossoms. Eight standards were divided into 3 types according to their basic skeletons: phenolic acids, quercetin-type flavonoids, and kaempferol-type flavonoids. The MSn fragmentation behaviors and diagnostic ions of these 3 types of compounds were proposed to aid the structural identification of components in peach blossom extract. By extracting the diagnostic ions from the mass spectrum in negative mode, a total of 25 compounds, including 8 phenolic acids and 17 flavonoids, were screened out. Among these compounds, 5 compounds (chlorogenic acid, ferulic acid, rutin, hyperoside, and isoquercetrin) were quantitated by UPLC-DAD. The linearity, precision, accuracy, limit of detection, and limit of quantitation were validated for the quantification method. The validated method was applied to assay 9 batches of peach blossoms from different regions. This study was the first report on the systematic qualitative analysis of compounds in peach blossom, providing insights into the quality control of peach blossom.