Dataset Information

JPST000090-2 Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn [Reanalysis: JPST000090]

ABSTRACT: We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO2) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11â€¯300 nonredundant phosphopeptides from triplicate analyses of 100 Î¼g of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches. [Original project description]

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: RPXD034764 | JPOST Repository | Sat Apr 01 00:00:00 BST 2023

REPOSITORIES: jPOST

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			Action	DRS
	04-120627mi-GEN-ERLIC-1.pilotFin.txt	Txt
	04-120627mi-GEN-ERLIC-1.txt	Txt
	04-120627mi-GEN-ERLIC-2.pilotFin.txt	Txt
	04-120627mi-GEN-ERLIC-2.txt	Txt
	04-120627mi-GEN-ERLIC-3.pilotFin.txt	Txt

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Project description:Phosphosites cluster, a set of co-regulation sites, extends a variety of protein function via the distinct combination of phosphorylation events. The coordination of proximal multisite phosphorylation is a universal mechanism to regulate protein function, activity, and interaction pattern. The common method for efficiently separating heterogeneous types of phosphopeptides is insufficient thereby hampering the complete mapping of phosphoproteome. By taking the advantages of distinct binding affinities of Ga2O3 and TiO2 toward phosphopeptides under the same buffer condition, we present a simple and rapid strategy, metal-directed metal oxide chromatography (MD-MOC), for sequential separation of singly and multiply phosphorylated peptides. Among the 6,585 phosphopeptides identified from PC9 cells, up to 92% was uniquely enriched in either the first Ga2O3 or subsequent TiO2 fractions and more than 90 % of phosphopeptides in Ga2O3 fraction were multiply phosphorylated peptide, demonstrating the high complementary properties of these two materials. The high separation efficiency (<10% overlapping of identified phosphopeptides) results in a 1.9-fold superior phosphoproteomic coverage compared to conventional TiO2-based MOC in PC9 cells. Finally, the MD-MOC approach was applied to the large-scale phosphoproteomic profiling of ten mouse organ tissues allowed the identification of 42,086 unique phosphopeptides from 6,669 phosphoproteins using single-shot LC/MS/MS analysis. To our knowledge, the identification of 14,854 multiply phosphorylated peptides presented the largest dataset for multiply phosphorylated peptide from rodent tissues to date. Also, around half identified novel phosphosites (n=2,752) were uniquely came from the multiply phosphorylated peptide dataset further emphasizing the need of improving the detection sensitivity of multiply phosphorylated peptides. Given the good separation ability, stable reproducibility, and ease of tip-based preparation, this MD-MOC presents a sensitive approach for deep phosphoproteome identification and quantification from limited sample for shedding light on in vivo phosphoregulations.

Dataset Information

JPST000090-2 Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn [Reanalysis: JPST000090]

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