![]() An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds. This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry (GC-MS). ![]() However, due to its complex composition, little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule. ![]() In particular, it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Project description:The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Project description:Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. This peptide spectral data contributes to a protein library that can be used to identify a wide range of proteins in ovine serum. CONCLUSIONS:These results demonstrated for the first time the feasibility of characterising the ovine circulating acellular proteome using nanoLC-nanoESI-MS/MS. These data are available via ProteomeXchange with identifier PXD004989. Since Mascot software is considered the industry standard and identified the most proteins, these were analysed using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification tool revealing the association of 349 genes with 127 protein pathway hits. RESULTS:ProteinPilot™ and Mascot identified 245 and 379 protein groups (IDs), respectively, and PeptideShaker validated 133 protein IDs from the entire dataset. PeptideShaker (CompOmics, VIB-UGent) searches were used to validate protein identifications from ProteinPilot™ and Mascot. Proteins were identified using ProteinPilot™ (SCIEX) and Mascot (Matrix Science) software based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1% software by searching a subset of the Universal Protein Resource Knowledgebase (UniProtKB) database (). METHODS:Serum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chromatography nano electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF® 5600+, SCIEX). The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum. Project description:BACKGROUND:Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states.
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