Dataset Information

Metal ion-decorated ZIC-cHILIC StageTip Enables Simultaneous Profiling of N-Glycoproteome and Phosphoproteome in TKI resistant NSCLC cells

ABSTRACT: Cell membrane proteins are densely decorated with surface glycosylation and intracellular phosphorylation whose interplay determines the cell-cell communication and signaling cascades. However, their concomitant characterization remains extreme challenges due to complexity of glycan structures, low abundance of glycopeptides (particularly sialylation), labile, dynamic nature and low detectability of both phosphopeptides and glycopeptides in mass spectrometry. In addition, the interplay and dynamic change between glycosylation and phosphorylation in tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells is still unclear. In this study, we introduce a streamlined metal ion-decorated ZIC-cHILIC strategy, featuring a simple pH control, to allow simultaneous enrichment and stepwise separation of intact (sialo-)glycopeptides and phosphopeptides.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Yi-Ju Chen

LAB HEAD: Yi-Ju Chen

PROVIDER: PXD056077 | Pride | 2025-08-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20240104_Andy_200ugPC9pep_cH_FecH_GPx3f.mzid	Mzid
	20240104_Andy_200ugPC9pep_cH_FecH_GPx3f.pdResult	Other
	20240104_Crick_200ugPC9pep_POOL_PP.mzid	Mzid
	YJC_20230621_4cells_FecH_PP.msf	Msf
	YJC_20230621_4cells_FecH_PP.pdResult	Other

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Publications

Metal Ion-Enhanced ZIC-cHILIC StageTip for N-Glycoproteomic and Phosphoproteomic Profiling in EGFR-Mutated Lung Cancer Cells.

Chen Yi-Ju YJ Chen Yan-Lin YL Chang Kun-Hao KH Cheng Hsiang-Chun HC Chang Chiao-Chun CC Chen Yu-Ju YJ

Molecular & cellular proteomics : MCP 20250326 6

Surface glycosylation and intracellular phosphorylation regulates the cell-cell communication and signaling cascades. Due to complex glycosylation and dynamic phosphorylation, exploring their interplay remains technically challenging. In this study, we reported a tandem ZIC-cHILIC StageTip strategy for streamlined and simultaneous (sialo)glycoproteomic and phosphoproteomic profiling. We first demonstrated that Fe ions expand the utility of ZIC-cHILIC strategy to phosphoproteomic analysis with gr ...[more]

PMID: 40154885

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Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.