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Thermal Proteome Prodiling and Glycoproteomics of U937 cells

ABSTRACT: Protein glycosylation regulates essential cellular processes including protein folding, stability, and cell-cell interactions; however, how aberrant glycosylation impacts protein function and interaction networks remains poorly understood. Here we combine mass spectrometry-based proteomics, chemical glycobiology, and molecular dynamics simulations to systematically investigate glycosylation dependent protein stability and function. By perturbing the secretory pathway at defined steps, we generated proteins with distinct glycan structures and analyzed their functional consequences using thermal proteome profiling (TPP). This approach revealed that cells mount convergent stress responses to diverse glycosylation perturbations, characterized by coordinated trafficking reorganization. This reorganization effectively redirects protein flux from secretion toward degradation. We further identified that perturbing terminal glycan modifications, particularly sialylation and fucosylation, exerts the most profound effects on cell surface protein function. We studied in detail how loss of fucosylation restricts the conformational dynamics of key domains in integrin alpha 4, reducing VCAM-1 binding affinity and suggesting a potential therapeutic strategy for multiple sclerosis. Overall, our systematic approach uncovered extensive complexity in how glycosylation regulates protein function beyond simple glycoform identification and provides a resource for dissecting essential glycan-protein relationships.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Johannes Hevler

LAB HEAD: Carolyn Bertozzi

PROVIDER: PXD071666 | Pride | 2025-12-11

REPOSITORIES: Pride

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			Action	DRS
	20231216_SA_JFH_EXP2_vehicle_1_fr1.mzML	Mzml
	20231216_SA_JFH_EXP2_vehicle_1_fr10.mzML	Mzml
	20231216_SA_JFH_EXP2_vehicle_1_fr11.mzML	Mzml
	20231216_SA_JFH_EXP2_vehicle_1_fr12.mzML	Mzml
	20231216_SA_JFH_EXP2_vehicle_1_fr2.mzML	Mzml

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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.

Project description:Glycosylation changes are closely related to various diseases like cancer. However, the quantitative analysis of the site-specific glycans at proteomics scale remains challenging because of the extremely low interpretation rate of glycopeptide spectra. Here, we present a new software tool, GlyPep-Quant, for the sensitive quantification and identification of site-specific glycans. By implementing a two-step elution profile extraction method for precise location of matched profiles, a well-trained machine learning model and target-decoy matching method for confidence assessment, GlyPep-Quant enabled quantification of 21.3%-173.3% more site-specific glycans without any missing values than cutting-edge quantitative tools like pGlycoQuant, MSFragger-Glyco, and Skyline. To fully utilize identified information from previous large-scale dataset, a virtual match-between-runs quantification scheme was developed by matching library MS1 features to new data and enabled the identification and quantification of over 8-fold more site-specific glycans than conventional identification-based quantifications. The enhanced coverage on site-specific glycans prompted the development of a novel glycoproteomic biomarker discovery method, which involves calculating the ratios of site-specific glycan abundances at the same glycosylation site, thereby minimizing the impact of individual variances of glycoprotein expression and experimental conditions on the detection of glycosylation pattern changes. By using this method, two pairs of site-specific glycan ratios on sites P01011-N127 and P08185-N96, were determined to be high-performance novel biomarkers to classify gastric cancer (GC) from healthy controls with areas under the curves exceeding 0.95. Moreover, the selected glycan abundance ratios performed well in distinguishing GC using an independent cohort quantified by the newly developed library-based strategy with diagnostic accuracy up to 85%, demonstrated the robustness of the quantification module and the value of the discovered marker candidates. It is anticipated that GlyPep-Quant is ready to be applied in a broader range of glycoproteomic research scenarios.

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Thermal Proteome Prodiling and Glycoproteomics of U937 cells

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