Dataset Information

HiRIEF LC-MS allows for in depth quantitative analysis of the phosphoproteome.

ABSTRACT: In this project we aimed at improving phosphoproteomics analysis by taking advantage of high resolution isoelectric focusing (HiRIEF) fractionation. We developed a workflow that employs titanium dioxide phospho-enrichment, followed by isobaric labeling with Tandem Mass Tags (TMT) and HiRIEF on a broad pI range (immobilized pH gradient, IPG gel strips employed were 2.5-3.7 and 3-10, Phospho HiRIEF LC-MS). We analyzed HeLa cells untreated (four biological replicates), treated with pervanadate or arrested in mitosis (three biological replicates each). Employing a relatively low amount of material (300 μg of peptides), we identify 22,674 phosphorylation sites, of which 19,036 were localized with high confidence. We demonstrate isoelectric point dependent fractionation of the peptides based on the number of phosphate groups that they carry: 18% of the phospho-peptides identified with the IPG 2.5-3.7 gel strip are multiply phosphorylated peptides and they localize predominantly in the most acidic pI fractions. Identified phosphorylation sites include 1,198 tyrosine phosphorylation sites and 1,491 phospho-sites that were not previously reported in the PhosphoSitePlus database. Total protein quantification performed by standard HiRIEF on the same samples identified 9,185 proteins, of which 4,575 overlap with the proteins identified by Phospho HiRIEF LC-MS. Phosphorylation sites corresponding to these proteins were normalized to total protein abundance, resulting in 18,374 quantified phospho-sites. Kinase association analysis on the quantified phospho-sites resulted in identification of a subset that has putative functions during the mitotic phase and protein-protein interaction network analysis shows a high degree of connectivity of these putatively functional novel phospho-sites.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Rui Branca

LAB HEAD: Janne Lehtio

PROVIDER: PXD005410 | Pride | 2017-07-07

REPOSITORIES: Pride

ACCESS DATA

Publications

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome.

Panizza Elena E Branca Rui M M RMM Oliviusson Peter P Orre Lukas M LM Lehtiö Janne J

Scientific reports 20170703 1

Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and hig ...[more]

PMID: 28674419

Similar Datasets

Project description:Part I - SI-NET tumors High Resolution Isoelectric Focusing (HiRIEF) LC-MS and relative quantification by iTRAQ 8-plex was used to analyze 14 small intestinal neuroendocrine tumors (SI-NETs). The data was obtained from two separate TMT10plex sets and linked by a single internal pooled standard. The internal pooled standard was made by combining equal aliquots of the tryptic peptide mixtures from each of the 14 tissue samples. iTRAQ set1 was composed of 7 SI-NET samples, all from different individuals, and internal pooled standard labelled as follows: Channel 113 (sample Screen-1 with liver metastasis), Channel 114 (sample Screen-8 with liver metastasis), Channel 115 (sample Screen-3 with liver metastasis), Channel 116 (sample Screen-2 with liver metastasis), Channel 117 (sample Screen-5 no liver metastasis), Channel 118 (sample Screen-4 no liver metastasis), Channel 119 (sample Screen-10 no liver metastasis), Channel 121 (internal pooled standard). iTRAQ set2 was composed of 7 SI-NET samples, all from different individuals, and internal pooled standard labelled as follows: Channel 113 (sample Screen-7 with liver metastasis), Channel 114 (sample Screen-11 with liver metastasis), Channel 115 (sample Screen-13 with liver metastasis), Channel 116 (sample Screen-6 no liver metastasis), Channel 117 (sample Screen-12 no liver metastasis), Channel 118 (sample Screen-9 no liver metastasis), Channel 119 (sample Screen-14 no liver metastasis), Channel 121 (internal pooled standard). Part II - time course profiling in cell lines High Resolution Isoelectric Focusing (HiRIEF) LC-MS and relative quantification by TMT 10-plex was used to analyze cellular response to the neddylation inhibitor pevonedistat (MLN4924) at different timepoints in two SI-NET (small intestinal neuroendocrine tumors) cell lines. The data was obtained from two separate TMT 10-plex experiments. TMT set1 includes a time course experiment upon pevonedistat treatment of CNDT2 cells with harvests at 2h, 6h, 12h and 24h after treatment as well as of untreated control cells. Isobaric tag labelling of peptide samples with TMT10plex was used as follows. Biological duplicate controls (TMT channels 126, 127N), duplicate 2h (127C, 128N), duplicate 6h (128C, 129N), duplicate 12h (129C, 130N) and duplicate 24h (130C, 131) samples were employed. TMT set2 includes a time course experiment upon pevonedistat treatment of HC45 cells with harvests at 2h, 6h, 12h and 24h after treatment as well as of untreated control cells. Isobaric tag labelling of peptide samples with TMT10plex was used as follows. Biological duplicate controls (TMT channels 126, 127N), duplicate 2h (127C, 128N), duplicate 6h (128C, 129N), duplicate 12h (129C, 130N) and duplicate 24h (130C, 131) samples were employed.

Dataset Information

HiRIEF LC-MS allows for in depth quantitative analysis of the phosphoproteome.

Publications

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure