Dataset Information

HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies

ABSTRACT: Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins constitute a prime resource for drug research and can give valuable insights into the activity status of signaling pathways. In this study, we extracted proteins from human lung tissue and tested HOPE-treated and snap frozen tissues comparatively by proteome and phosphoproteome analyses. High confident data from accurate mass spectrometry allowed the identification of 2603 proteins and 3036 phosphorylation sites. HOPE-fixation did not hinder the representative extraction of proteins and investigating their biochemical properties, covered subcellular localizations and cellular processes revealed no bias caused by the type of fixation. In conclusion, proteome as well as phosphoproteome data of HOPE lung samples were qualitatively equivalent to results obtained from snap frozen tissues. Thus, HOPE-treated tissues match clinical demands both in histology and retrospective proteome analyses of patient samples by proteomics.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

SUBMITTER: Olga Shevchuk

LAB HEAD: Lothar Jänsch

PROVIDER: PXD000836 | Pride | 2014-05-22

REPOSITORIES: Pride

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HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies.

Shevchuk Olga O Abidi Nada N Klawonn Frank F Wissing Josef J Nimtz Manfred M Kugler Christian C Steinert Michael M Goldmann Torsten T Jänsch Lothar L

Journal of proteome research 20140428 11

Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, the suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins ...[more]

PMID: 24702127

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Project description:Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered a hallmark in cellular signal transduction. However, its analysis remains a challenge. The conventional immunopurification (IP) approach using antibodies pan-specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we aim to optimize the approach using SH2 superbinder for tyrosine phosphoproteome analysis. The present work covalently immobilized SH2 superbinders to agrose beads, designed and evaluated four different SH2 superbinder based experimental workflows for phosphotyrosine analysis. After made a head to head comparison of them, we observed that the combined strategy employing Ti4+-IMAC and covalently immobilized SH2 superbinder enrichment in sequence generated the largest pTyr peptide dataset (3519) with the best selectivity (90%). Next the optimal method was applied for pTyr profiling from normal mouse tissues, and resulted in the identification of 197 pTyr sites (of which 73 were novel) from 5 mg protein digests, which validated its high sensitivity. An comparison with antibody 4G10 by isolating pTyr peptides from 5 mg unstimulated Jurkat cell digests were made, our optimal strategy identified 343 while the antibody 4G10 based method identified 242 pTyr sites, respectively, further confirmed the robustness of the method. Finally, the optimal strategy was applied for quantitative pTyr phosphorylation analysis of EGF stimulated/unstimulated HeLa cells, 261 pTyr sites were successfully quantified from 5 mg stable-isotope dimethyl labling protein digests. In general, the combination of Ti4+-IMAC and SH2 superbinder enrichment in sequence generated a facial and robust strategy for qualitative and quantitative analysis of pTyr proteome.

Dataset Information

HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies

Publications

HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies.

Similar Datasets

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