Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation

doi:10.1016/j.jprot.2012.02.019

Journal of Proteomics

Volume 75, Issue 8, 18 April 2012, Pages 2384-2395

https://doi.org/10.1016/j.jprot.2012.02.019 Get rights and content

Abstract

Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions.

As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p ≤ 0.05) in irradiated hearts 24 h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives.

Graphical abstract

Highlights

► Label-free method is suitable for quantitative proteome analysis of FFPE tissue. ► Ionising radiation (IR) induces changes in three main biological pathways. ► IR causes down-regulation of mitochondrial complex proteins. ► Fatty acid metabolism and citric acid cycle were impaired by IR. ► Radiation-induced alteration in the glucose oxidative pathway was observed.

Introduction

For decades, pathologists have been using formalin-fixed, paraffin-embedded (FFPE) tissue for histological analysis due to its excellent performance and suitability for long-term storage. Clinical archives including data on diagnosis and outcome may provide information on biological pathways and cellular processes leading to disease, provided that suitable molecular technologies are available [1], [2], [3].

Proteomics analysis using FFPE material as an alternative to fresh-frozen tissue has recently been investigated [4], [5], [6], [7]. Different methods of extraction and separation of proteins from FFPE samples have been established [8], [9], [10], [11]. However, quantitative proteomic studies on archival material have been considered as an almost impossible task, primarily due to the harsh and irreversible fixation procedures and loss of integrity during prolonged storage. The formaldehyde fixation leads to a methylol modification, tagged mainly on lysine residues [11], [12]. Most chemical labels used in quantitative proteomics also target lysine residues, leading to inefficient labelling of FFPE material [13], [14], [15].

Recently, non-labelling approaches have been suggested as an alternative for quantification of FFPE proteome profiles [16], [17]. The few quantitative proteomic studies published so far have been performed using tumour FFPE tissues where areas of interest are easily distinguished and dissected [18], [19], thereby reducing the complexity of the resultant proteome profile.

The aim of this study was to establish a quantitative proteomics work flow using the label-free approach [20] in a tissue where no discrete target area can be observed. We have shown previously that ionising radiation has both immediate and persistent effects on the murine cardiac proteome without causing any morphological changes in the heart [21], [22]. Therefore, we used the cardiac tissue of sham- and total body irradiated C57BL/6 mice as a model system. We applied the optimised protein extraction and separation conditions for FFPE tissue previously developed by us for qualitative proteomics [11]. The analysis indicated radiation-induced impairment of the cardiac mitochondrial proteome. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation.

Section snippets

Materials

Beta-octylglucoside, SDS, and ammonium bicarbonate were obtained from Sigma (St. Louis, MO); RapiGest from Waters (USA); acetone, acetonitrile, formic acid, and trifluoroacetic acid (TFA) from Roth (Karlsuhe, Germany); dithiothreitol (DTT), iodoacetamide, tris-(hydroxymethyl) aminomethane (Tris) and sequencing grade trypsin were obtained from Promega (Madison, WI); cyano-4-hydroxycinnamic acid was obtained from Bruker Daltonik (Bremen, Germany). All solutions were prepared using HPLC grade

Protein quantification and identification

In order to evaluate the technical variability of mass spectrometry runs of label-free peptide quantifications of FFPE samples, we first analysed an aliquot of one control sample by repetitive LC-MS/MS runs using four replicates. After alignment of all peptide intensities and identification of respective proteins, the cumulative intensities of all proteins were plotted against their counterpart in the technical replicates (Supplementary Fig. 1). The resulting scatter plots showed that all four

Discussion

Recent epidemiological data on exposed individuals clearly show that ionising radiation is a risk factor for cardiovascular disease [28], [29], [30]. However, the molecular mechanisms underlying the development of radiation-induced heart disease are not well understood so far. Proteomics is a powerful technology that can provide novel information about such biological mechanisms. In this study, we performed a quantitative proteomic analysis using FFPE cardiac tissue from exposed and non-exposed

Conclusions

Our study shows that label-free proteomic approach enables to detect and quantify radiation-induced alterations in FFPE tissue. It indicates that archival tissue, that frequently is the only source of biomaterial available, plays a valuable role in retrospective molecular studies. This is particularly valid in cardiovascular diseases where additional collecting and sampling of fresh-frozen biomaterial is for ethical reasons cumbersome. We suggest that the three pathways found

Acknowledgements

This work was supported by a grant from the European Community's Seventh Framework Programme (EURATOM) contract no. 232628 (STORE). We acknowledge Dr. Steven E. Lipshultz for the valuable advice and discussions in the preparation of this manuscript. We thank Dr. Hakan Sarioglu, Sandra Helm and Waldemar Schneider for their technical assistance.

References (61)

A. Tanca et al.
Proteomic analysis of formalin-fixed, paraffin-embedded lung neuroendocrine tumor samples from hospital archives
J Proteomics
(2011)
C.B. Fowler et al.
‘Tissue surrogates’ as a model for archival formalin-fixed paraffin-embedded tissues
Lab Invest
(2007)
B. Metz et al.
Identification of formaldehyde-induced modifications in proteins: reactions with model peptides
J Biol Chem
(2004)
A. Ono et al.
Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues
Hum Pathol
(2009)
B.L. Hood et al.
Proteomic analysis of formalin-fixed prostate cancer tissue
Mol Cell Proteomics
(2005)
S.M. Hauck et al.
Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry
Mol Cell Proteomics
(2010)
A.J. Wyrobek et al.
Low dose radiation response curves, networks and pathways in human lymphoblastoid cells exposed from 1 to 10cGy of acute gamma radiation
Mutat Res
(2011)
G. Paradies et al.
Oxidative stress, mitochondrial bioenergetics, and cardiolipin in aging
Free Radic Biol Med
(2010)
C. Gieffers et al.
Mitofilin is a transmembrane protein of the inner mitochondrial membrane expressed as two isoforms
Exp Cell Res
(1997)
R.S. Rector et al.
Mitochondrial trifunctional protein defects: clinical implications and therapeutic approaches
Adv Drug Deliv Rev
(2008)

O. Azimzadeh et al.

Rapid proteomic remodeling of cardiac tissue caused by total body ionizing radiation

Proteomics

(2011)

Z. Barjaktarovic et al.

Radiation–induced Signaling Results in Mitochondrial Impairment in Mouse Heart at 4 Weeks after Exposure to X-rays

PLoS One

(2011)

Cited by (33)

Quantitative protein profiling of phenobarbital-induced drug metabolizing enzymes in rat liver by liquid chromatography mass spectrometry using formalin-fixed paraffin-embedded samples
2021, Journal of Pharmacological and Toxicological Methods
Administration of a compound can induce drug-metabolizing enzymes (DMEs) in the liver. DME induction can affect various parameters in toxicology studies. Therefore, evaluation of DME induction is important for interpreting test compound-induced biological responses. Several methods such as measurement of hepatic microsomal DME activity using substrates, electron microscopy, or immunohistochemistry have been used; however, these methods are limited in throughput and specificity or are not quantitative. Liquid chromatography mass spectrometry (LC/MS)-based protein analysis can detect and quantify multiple proteins simultaneously per assay. Studies have shown that formalin-fixed paraffin-embedded (FFPE) samples, which are routinely collected in toxicology studies, can be used for LC/MS-based protein analysis. To validate the utility of LC/MS using FFPE samples for quantitative evaluation of DME induction, we treated rats with a DME inducer, phenobarbital, and compared the protein expression levels of 13 phase-I and 11 phase-II DMEs between FFPE and fresh frozen hepatic samples using LC/MS. A good correlation between data from FFPE and frozen samples was obtained after analysis. In FFPE and frozen samples, the expression of 6 phase-I and 8 phase-II DMEs showed a similar significant increase and a prominent rise in Cyp2b2 and Cyp3a1 levels. In addition, LC/MS data were consistent with the measurement of microsomal DME activities. These results suggest that LC/MS-based protein expression analysis using FFPE samples is as effective as that using frozen samples for detecting DME induction.
Proteomic developments in the analysis of formalin-fixed tissue
2015, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
From these papers (excluding those that used a pre-fractionation step), the average number of unique protein identifications was 274 ± 222. In more recent times standalone Ion Traps have been replaced with more advanced instrumentation such as Ion Trap/Orbitrap hybrids, combining the sensitivity of the Ion Trap with the high resolution and mass accuracy of the Orbitrap, as seen in 20 papers since 2010, summarized in Table 2 [31,33,39–41,43,53,61,65,66,80–90]. The average number of unique protein identifications from studies using Ion Trap/Orbitrap hybrid instruments as listed in Table 2 is 996 ± 656 (excluding those that used a pre-fractionation step), indicating a marked improvement in proteome coverage as compared to using RP HPLC Ion Trap MS alone.
Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics.
Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin
2014, Journal of Proteomics
Citation Excerpt :
For this purpose, we employed a GeLC–MS/MS approach combining protein fractionation by 1-D SDS-PAGE (Supplementary Figure S4) with label free quantification by LC–MS/MS. In total, 1532 proteins with at least two unique peptides and a protein probability of > 99% were identified, corresponding to 29% of the total P. putida proteome. Quantification of these proteins was performed by a label free approach using the fractionation workflow implemented in the Progenesis LCMS software [36]. Proteins that showed a 2-fold or higher change in abundance and a p-value ≤ 0.05 were considered as differentially regulated.
Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC–MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440.
The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a proteome wide scale. The high proteome coverage of our proteomics survey allowed us to analyze the regulation of whole protein networks instead of single proteins. We were able to reconstruct the complete degradation pathway of vanillin and to monitor the changes in the energy metabolism of KT2440 induced by vanillin as sole carbon source. Vanillin dehydrogenase (Vdh) was not mandatory for vanillin degradation in KT2440 and may be substituted by other aldehyde dehydrogenases that were up-regulated in a wild-type as well as in a Vdh-deficient strain in the presence of vanillin. Aldehyde dehydrogenases, vanillin specific porins and efflux pump systems identified in study will be interesting targets for optimization of vanillin production in Pseudomonas bacteria. Furthermore, several mechanisms of solvent tolerance were induced by vanillin in KT2440. These include increased abundance of several efflux pump systems, chaperones as well as enzymes involved in cyclopropane fatty acid synthesis and trehalose formation. The present work will deepen the understanding of metabolism of aromatic compounds in P. putida and may lead to a more comprehensive understanding of solvent tolerance mechanisms in Gram-negative bacteria in general. Moreover, it will serve as a basis for further strain developments for a biotechnological production of vanillin in P. putida KT2440 or other Pseudomonas strains, highlighting the role of proteomics surveys as a powerful screening technology.
Protein expression profiles of chemo-resistant mixed phenotype liver tumors using laser microdissection and LC-MS/MS proteomics
2013, EuPA Open Proteomics
Citation Excerpt :
Due to the characteristic heterogeneity of the tumors and the fact that informative areas can be very focal, the use of frozen tissue samples usually of small size could result in a low yield of representative cases and is therefore not ideal. Large scale global proteomic analysis of laser microdissected FFPE tissue has been successfully employed to discover differentially expressed proteins between different histological tissue types and thus successfully discovered novel protein biomarkers of disease [10–13]. Many of these studies utilized label free quantitative strategies, such as spectral counting and signal intensities of peptide precursor ions, both approaches benefiting from reduced spectral complexity and more analytical depth as well as good linear dynamic range (over two orders of magnitude for spectral counting) and high quantitative proteome coverage [11–15].
Hepatocellular carcinoma (HCC) treated with trans-arterial chemoembolization (TACE) can later show areas of hepatocellular or cholangiocarcinoma (CC) differentiation. Biomarkers are required to define details in phenotype/pathogenesis of these HCC/CC regions, and similarities to their normal and classic counterparts. Proteins from laser microdissected FFPE tissue were in-gel digested and peptides analyzed by nano-HPLC–MS/MS. We identified 95 proteins with significant differential expression between HCC vs. CC regions. We also found significant differences in other tissue type comparisons. Some protein biomarkers are being reported here for the first time in the context of liver carcinogenesis and may be clinically useful.
The proteomics of formalin-fixed wax-embedded tissue
2013, Clinical Biochemistry
Proteomics, which is the global analysis of protein expression in cells and tissues, has emerged over the last ten to fifteen years as a key set of technologies to improve our understanding of disease processes and to identify new diagnostic, prognostic and predictive disease biomarkers. Whilst most proteomic studies have been conducted on fresh frozen tissue, the continuous improvements in technical procedures for protein extraction and separation, coupled with increasingly powerful bioinformatics, have provided the opportunity for proteomic analysis to be conducted on formalin-fixed wax-embedded tissue. This potential advance should allow proteomic analysis to be performed on the extensive archives of clinically annotated formalin fixed wax embedded tissue blocks stored in pathology departments worldwide. In this review the main techniques and their limitations involved in proteomic analysis of formalin fixed wax embedded tissue will be outlined and examples of their successful application will be indicated.
Application of radiation omics in the development of adverse outcome pathway networks: an example of radiation-induced cardiovascular disease
2022, International Journal of Radiation Biology

View all citing articles on Scopus

¹: These authors contributed equally to the work.

View full text

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Materials

Protein quantification and identification

Discussion

Conclusions

Acknowledgements

J Proteomics

Lab Invest

J Biol Chem

Hum Pathol

Mol Cell Proteomics

Mol Cell Proteomics

Mutat Res

Free Radic Biol Med

Exp Cell Res

Adv Drug Deliv Rev

Trends Cardiovasc Med

Am J Pathol

J Biol Chem

Radiother Oncol

Molecular information obtained from radiobiological tissue archives: achievements of the past and visions of the future

Radiat Environ Biophys

Use of proteomics in radiobiological research: current state of the art

Radiat Environ Biophys

Progress in updating the European Radiobiology Archives

Int J Radiat Biol

Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within

Proteomics

Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting

J Pathol

Protein profiling of formalin fixed paraffin embedded tissue: identification of potential biomarkers for pediatric brainstem glioma

Proteomics Clin Appl

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry

J Histochem Cytochem

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis

J Pathol

Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics

J Proteome Res

Quantitative proteomic analysis of formalin-fixed and paraffin-embedded nasopharyngeal carcinoma using iTRAQ labeling, two-dimensional liquid chromatography, and tandem mass spectrometry

J Histochem Cytochem

Quantitative proteomic analysis of formalin fixed paraffin embedded oral HPV lesions from HIV patients

Open Proteomics J

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands

Proteome Sci

Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry

J Proteome Res

Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression

Clin Cancer Res

Rapid proteomic remodeling of cardiac tissue caused by total body ionizing radiation

Proteomics

Radiation–induced Signaling Results in Mitochondrial Impairment in Mouse Heart at 4 Weeks after Exposure to X-rays

PLoS One