Dataset Information

Rat Liver Phosphoproteome - An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome

ABSTRACT: In depth analysis of rat liver phosphoproteome using optimized HILIC IMAC sample preparation

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Rattus Rattus (black Rat)

TISSUE(S): Liver

SUBMITTER: Roland Annan

LAB HEAD: Roland S Annan

PROVIDER: PXD001372 | Pride | 2016-05-04

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	MaxQuant_output.zip	Other
	RatLiver_FASP_IMAC_0411run_16to24_90min_2-15and15-30_041513.raw	Raw
	RatLiver_FASP_IMAC_0411run_25to28_90min_2-15and15-30_041513.raw	Raw
	RatLiver_FASP_IMAC_0411run_29and30_90min_2-15and15-30_041513.raw	Raw
	RatLiver_FASP_IMAC_0411run_31and32_90min_2-15and15-30_041513.raw	Raw

Items per page:

1 - 5 of 43

Publications

An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome.

Zappacosta Francesca F Scott Gilbert F GF Huddleston Michael J MJ Annan Roland S RS

Journal of proteome research 20150126 2

While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectiv ...[more]

PMID: 25575281

Similar Datasets

Project description:Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO(2), Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 ?g to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO(2) in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems.