Dataset Information

Fe3+-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment

ABSTRACT: Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies aim to systematically catalogue, characterize, and comprehend alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics workflows. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but it remained a bottleneck for methods that require higher throughput or a scale that is beyond the maximum capacity of these columns. Here, we compare our well-established spin column-based enrichment strategy with one encompassing magnetic beads. Our data show little difference in both the number and properties of the phosphopeptides identified when using either method. In all, we illustrate how scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA for global phosphoproteome profiling.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Xenopus Laevis (african Clawed Frog)

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD031327 | Pride | 2022-09-02

REPOSITORIES: Pride

ACCESS DATA

Publications

Fe<sup>3+</sup>-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment.

Liu Xinyue X Rossio Valentina V Thakurta Sanjukta Guha SG Flora Amarjeet A Foster Leigh L Bomgarden Ryan D RD Gygi Steven P SP Paulo Joao A JA

Journal of proteomics 20220321

Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies systematically catalogue and characterize alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics experiments. Previously, we found that spin column-based Fe<sup>3+</sup>-NTA enrichment integrated well with our workflow but remained a bottleneck for methods that require higher throughput or a scale that is ...[more]

PMID: 35331916

Similar Datasets

Project description:We have identified loss of deiminated MA-Brent-1 (an RNA and export binding protein) in the retinal ganglion cells (RGCs) in multiple sclerosis and in glaucoma eyes compared to normal controls. Deimination refers to posttranslational modification of protein bound arginine (not free arginine) in citrulline. Our preliminary studies suggest binding of different repertoire of RNA by non-deiminated and deiminated MA-Brent-1. In vitro, in neurites of cultured RGCs and hippocampal neurons, the select mRNA translation is enhanced by addition of deiminated but not non-deiminated MA-Brent-1. These observations suggest that lack of deiminated MA-Brent-1 has consequences for protein synthesis, remodeling and plasticity of RGCs/neurons. Identification of RNA species bound by deiminated and non-deiminated MA-Brent-1 will enable us there further verification and determining the role that deimination plays in biological function of MA-Brent-1 in multiple sclerosis and glaucoma. To summarize identification of RNA species bound by deiminated and non deiminated MA-Brent-1 will enable us to gain further insight into role of deimination in the overall disease process. To identify the RNA species bound by deiminated and non-deiminated MA-Brent-1 The deiminanted MA-Brent-1 binds to a different repertoire of RNA and non-deiminated MA-Brent-1. Corollary to this hypothesis is that the deiminated MA-Brent-1 enhances translation for bound species of RNA. Loss of deimination results in less dendritic protein synthesis and has consequences for remodeling and plasticity in multiple sclerosis and glaucoma RGCs where lack of deimination (compared to control eyes) has been observed. About 10 microgram of purified recombinant and his-tagged non-deiminated MA-Brent-1 or deiminated MA-Brent-1 will be incubated with about 400 microgram of total RNA. The RNA bound his-tagged protein will be incubated with about 100 microliters of Ni-NTA beads and loaded onto a mini column. The column will be washed with 50 volumes of binding buffer. The bound his-tagged protein will be released using 100 mM immidazole in the binding buffer. The MA-Brent-1 bound RNA will be recovered by boiling the protein in 0.25% SDS followed by purification of RNA using columns or using a 4% PAGE-0.8% agarose composite gel followed by gel purification. The composite gel RNA purification is routine practice in the laboratory. RNA binding experiment for each protein variant (deiminated or non-deiminated) MA-Brent-1 will be repeated twice. Identified RNA species common in both binding experiments for a given species will be candidates for further pursuit. The recombinant purified MA-Brent-1 will be subjected to deimination by incubating with recombinant peptidylarginine deiminase type II (PAD2). PAD2 is a C-terminus GST tag fusion protein and will be purified using GST column. Deiminated and non-deiminated MA-Brent-1, C-terminal his-tag fusion proteins will be purified using Ni-NTA beads/column and immidazole containing buffer as eluents.

Dataset Information

Fe3+-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment

Publications

Fe<sup>3+</sup>-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure