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An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples

ABSTRACT: We describe here a simple and integrated spintip-based glycoproteomics technology,termed Glyco-SISPROT, for challenging the lowest starting material limitation and achieving a comprehensive view of glycoproteome with shortest sample processing time. By carefully integrating and optimizing SCX, C18 and Concanavalin A packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in 1770 unique deglycopeptides were characterized from mouse liver by using either 100 macrograms of predigested peptides or directly using 100 macrograms of protein lysates, in which about 30 percentage of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT was that whole processing time was dramatically reduced from a few days to only 6 hours with good reproducibility when protein lysates were directly processed by Glyco-SISPROT.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Ruijun Tian

PROVIDER: MSV000083579 | MassIVE | Fri Mar 15 04:52:00 GMT 2019

REPOSITORIES: MassIVE

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An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples

Project description:Glycosylation, as a biologically important protein post-translational modification, often alters on both glycosites and glycans, simultaneously. However, most of current approaches focused on biased profiling of either glycosites or glycans, and limited by time-consuming process and milligrams of starting protein material. We describe here a simple and integrated spintip-based glycoproteomics technology (termed Glyco-SISPROT) for achieving a comprehensive view of glycoproteome with shorter sample processing time and low microgram starting material. By carefully integrating and optimizing SCX, C18 and Concanavalin A (Con A) packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in ~1770 unique deglycopeptides were characterized from mouse liver by using either 100 microgram of predigested peptides or directly using 100 microgram of protein lysates, in which about 30 percentage of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT is that whole processing time is dramatically reduced from a few days to less than 6 hours with good reproducibility when protein lysates were directly processed by Glyco-SISPROT. We expect that this method will be suitable for multi-level glycoproteome analysis of rare biological samples with high sensitivity.

2019-03-25 | MSV000083630 | MassIVE

An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples

Project description:In this study, we aimed to develop a simple and integrated spintip-based glycoproteomics technology, termed Glyco-SISPROT, for challenging the lowest starting material limitation and achieving comprehensive view of glycoproteome with shortest sample processing time. By carefully integrating and optimizing SCX, C18 and Concanavalin A packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approach by using limited protein starting material. One significant benefit of Glyco-SISPROT is that whole processing time was dramatically reduced from a few days to only 6 hours with good reproducibility when protein lysates were directly processed by Glyco-SISPROT.

2019-03-14 | MSV000083570 | MassIVE

Integrated strategy for high-sensitive phosphoproteome analysis from low micrograms of protein samples

Project description:In this study, we aimed to challenge the lowest starting material limit for phosphoproteome profiling. By carefully optimizing the well-established high-pH reversed-phase (RP) fractionation plus Ti4+-IMAC enrichment strategy, we achieved the identification of 15260 and 8936 unique phosphopeptides from only 500 ug and 250 ug predigested peptides, respectively. To further improve the sensitivity of phosphoproteome analysis for low micrograms of protein samples, we developed an integrated strategy, termed Phospho-SISPROT. This technology integrates three tips in tandem for protein digestion by the simple and integrated spintip-based proteomics technology (SISPROT), phosphopeptide enrichment by Ti4+-IMAC tip, and desalting by StageTip, respectively, which could dramatically reduce the phosphoproteome analysis time from a couple of days to only 6 hours and improve the system sensitivity. The flow through of Phospho-SISPROT could be reused for the global protein identification, which is very helpful for accurate phosphoproteome analysis with limited starting materials. More than 5500 and 600 unique phosphopeptides were respectively identified from 20 ug and 1 ug pervanadate treated HEK 293T cell lysates processed by the Phospho-SISPROT. To the best of our knowledge, this performance is the highest record reported to date by using standard LC-MS/MS setup.

2018-06-04 | MSV000082435 | MassIVE

[HPP-2.0] Combinatory strategy using nanoscale proteomics and machine learning for T cell subtyping in peripheral blood of single multiple myeloma patients

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2024-02-04 | PXD049159 | iProX

Affymetrix U133 Plus 2.0 Arrays Hybridized With Varying Amounts of Starting Material

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2006-07-25 | GSE3062 | GEO

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2011-07-21 | GSE26742 | GEO

UMRR vs Liver

Project description:UMRR (Universal Mouse Reference RNA, Catalog #740100) was purchased from Stratagene and used as the starting material. Liver total RNA (Catalog #64042-1) was purchased from Clontech and used as the starting material. UMRR was labeled with Cyanine-3 (Green) while Liver was labeled with Cyanine-5 (Red). cRNA was made by the single round amplification method. Keywords: repeat sample

2003-03-07 | GSE333 | GEO

UMRR vs Spleen

Project description:UMRR (Universal Mouse Reference RNA, Catalog #740100) was purchased from Stratagene and used as the starting material. Spleen total RNA (Catalog #64044-1) was purchased from Clontech and used as the starting material. UMRR was labeled with Cyanine-3 (Green) while spleen was labeled with Cyanine-5 (Red). cRNA was made by the single round amplification method. Keywords: repeat sample

2003-03-07 | GSE332 | GEO

Blockade of Wnt secretion by LGK974 after ischemia reperfusion injury

Project description:The LAD of C57BL6 mice were temporally occluded to induce ischemia-reperfusion injury of the heart. Mice received a daily dose of the porcupine inhibitor LGK974 starting directly after the surgery to prevent Wnt protein secretion. Control animals received a DMSO vehicle control. Hearts were extracted 48h after induction of ischemia-reperfusion injury. The infarcted area was identified and processed for RNAseq analysis.

2022-11-02 | GSE185444 | GEO

Robust, sensitive and automated phosphopeptide enrichment optimized for low sample amounts applied to primary hippocampal neurons

Project description:Due to the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting towards an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive and less prone to variability. Here, we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on a AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive and reproducible, identifying, localizing and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications we monitored EGF-induced signaling in hippocampal neurons, starting with only 200,000 primary cells resulting in approximately 50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

2016-12-14 | PXD005366 | Pride

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