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S-acylproteomic analysis of HeLa cells by nGF-SPE

ABSTRACT: S-acylation is an essential lipidation form in eukaryotes, in which various fatty acids can be covalently attached to specific cysteine residues. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of enrichment procedure as well as LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate and octanoic chain respectively, providing a global insight to the endogenously heterogeneous modification state. S-acylproteome were separated at flow rate of 300 nL/min on a nanoElute chromatography system (Bruker Daltonics) with the IonOptick column (AURORA Series column, 75 µm i.d. × 25 cm, C18, 1.6 μm) and then analyzed by a hybrid trapped ion mobility spectrometry quadrupole time of-flight mass spectrometer (timsTOF Pro, Bruker Daltonics) via data-dependent acquisition (DDA) mode. Obtained raw data was analyzed with PEAKS Online.

ORGANISM(S): Homo Sapiens

SUBMITTER: Haojie Lu

PROVIDER: PXD042747 | iProX | Mon Jun 05 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) in human serum is challenging due to the wide dynamic range of serum protein abundances, the lack of a complete serum N-Glycan database and the existence of non-specifically digested peptides and numerous modifications. In this regard, a spectral library search method was presented for serum N-linked intact glycopeptides identification with target-decoy and motif-specific false discovery rate (FDR) control. Low-abundant glycoproteins were firstly separated from high-abundant proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) column and a portion of the enriched N-linked intact glycopeptides were processed by N-Glycosidase F (PNGase F) to generate de-glycopeptides. Both N-linked intact glycopeptides and de-glycopeptides were analyzed by LC-MS/MS. From N-linked de-glycopeptides datasets, 764 N-linked glycoproteins, 1,699 N-linked glycosites and 3,328 unique N-linked de-glycopeptides were identified. The spectra of these N-linked de-glycopeptides were utilized for N-linked de-glycopeptides library construction and identification of N-linked intact glycopeptides. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked de-glycopeptides, and two different N-Glycan mass databases including 27 modified N-Glycan masses and 712 unmodified N-Glycan masses were combined and utilized during spectral library search for identification of N-linked intact glycopeptides. In total, from the N-linked intact glycopeptides datasets, 526 N-linked glycoproteins, 1,036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-Glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level and N-linked de-glycopeptides level.

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S-acylproteomic analysis of HeLa cells by nGF-SPE

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