Plant Phosphopeptides Enrichment by Immobilized Metal Ion Affinity Chromatography

Huang, Xiahe; Zhang, Yuanya; Ge, Haitao; Lu, Dongping; Wang, Yingchun

doi:10.1007/978-1-0716-1625-3_10

Cited by 4 publications

(10 citation statements)

References 22 publications

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“…Arabidopsis (Col-0) seedlings were ground with liquid nitrogen in a mortar before extraction with 4% SDS lysis buffer. The proteins were digested, and enrichment of the phosphopeptides was performed as previously reported …”

Section: Methodsmentioning

confidence: 99%

“…Phosphopeptides were enriched from the tryptic digest (before desalting) using titanium dioxide beads (TiO 2 ; GL Sciences, 5010-21315) according to the protocol described by Humphrey et al . Plant phosphopeptides were enriched according to the protocol described by Huang et al …”

Section: Methodsmentioning

confidence: 99%

“…The proteins were digested, and enrichment of the phosphopeptides was performed as previously reported. 4 Phosphopeptide Enrichment. Phosphopeptides were enriched from the tryptic digest (before desalting) using titanium dioxide beads (TiO 2 ; GL Sciences, 5010-21315) according to the protocol described by Humphrey et al 18 Plant phosphopeptides were enriched according to the protocol described by Huang et al 4 Nanoflow LC Tandem MS.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…4 Phosphopeptide Enrichment. Phosphopeptides were enriched from the tryptic digest (before desalting) using titanium dioxide beads (TiO 2 ; GL Sciences, 5010-21315) according to the protocol described by Humphrey et al 18 Plant phosphopeptides were enriched according to the protocol described by Huang et al 4 Nanoflow LC Tandem MS. The trap column was prepared by first creating a Kasil frit at one end of a 10 cm length capillary with 200 μm inner diameter/360 μm outer diameter.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…First, phosphoproteins are generally in low stoichiometry compared to their unphosphorylated counterparts, necessitating their pre-enrichment biochemically before being subjected to LC–MS analysis . Second, phosphopeptides are usually not as efficient as unphosphorylated peptides in ionization during LC–MS analysis, resulting in reduced sensitivity in their MS detection . Third, the labile character of the phosphoester bond usually alters the fragmentation pattern of phosphopeptides, resulting in poor spectrum quality and increased difficulty in spectral annotation .…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of the Potential Risk of Analyzing Phosphopeptides with Easy-nLC 1200-Coupled Mass Spectrometers

et al. 2022

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Liquid chromatography−mass spectrometry (LC−MS) is a major tool for the large-scale qualitative and/or quantitative analysis of protein phosphorylation in cells or tissues. The performance of LC is pivotal for the success of phosphoproteomics in both sensitivity and reproducibility. Here, we report that the widely used Easy-nLC 1200 has poor performance in analyzing phosphopeptides, particularly in terms of sensitivity and reproducibility, whereas its predecessor, Easy-nLC 1000, has a much better performance. Therefore, we suggest that Easy-nLC 1200 is not appropriate for LC−MS-based proteomics analysis for samples with a limited amount, particularly phosphopeptides from plants.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%