HOPE-Fixation of Lung Tissue Allows Retrospective Proteome and

Article pubs.acs.org/jpr

HOPE-Fixation of Lung Tissue Allows Retrospective Proteome and Phosphoproteome Studies Olga Shevchuk,† Nada Abidi,† Frank Klawonn,†,‡ Josef Wissing,† Manfred Nimtz,† Christian Kugler,§ Michael Steinert,∥ Torsten Goldmann,⊥,# and Lothar Jan̈ sch*,†,# †

Research Group Cellular Proteomics, Helmholtz Center for Infection Research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany ‡ Department of Computer Science, Ostafalia University of Applied Science, Salzdahlumer Straße 46/48, 38302 Wolfenbüttel, Germany § Lung Clinic Grosshansdorf, Airway Research Center North, Wöhrendamm 80, 22927 Grosshansdorf, Germany ∥ Institute for Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106 Braunschweig, Germany ⊥ Research Center Borstel, Clinical and Experimental Pathology, Airway Research Center North (ARCN), Wöhrendamm 80, 22927 Grosshansdorf, Germany S Supporting Information *

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, 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 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 snapfrozen 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 in both histology and retrospective proteome analyses of patient samples by proteomics. KEYWORDS: lung tissues, HOPE-fixation technique, proteome, post-translational modifications, phosphorylation, biobank, LC-MS/MS

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INTRODUCTION Biobanks (biorepositories) constitute a highly valuable resource for the identification of disease-related processes and biomarkers, but the suitability of stored samples for retrospective genome-wide studies is actually under debate.1 Proteins obviously can be reanalyzed best from snap-frozen (SF) samples. However, handling of SF samples is expensive and freeze-related morphological artifacts or interfering background did not match the requirements of histology.2 Thus, samples are usually pretreated by formalin, which is covalently cross-linking reactive biomolecules, e.g. lysine residues in proteins by formaldehyde, and increases the rigidity of tissues. This supports microscopy-based histology but in parallel also limits the detection of proteins as well as RNA and DNA and biases their representative characterization in genome, transcriptome, and proteome studies (see also Table 1). Therefore, more and more attention is being paid to the evaluation of alternative fixation methods, which should combine advantages from formalin and SF samples. In the past decade a number of studies demonstrated that the HEPES-glutamic acid buffer© 2014 American Chemical Society

mediated organic solvent protection effect (HOPE) technique is a suitable alternative fixative for both biobank repository and molecular pathology.2−7 The fixation protocol was originally developed for lung slices and further adapted for in vitro cultured cells, bronchoalveolar lavages, and prostate cancer.2,6,8 It includes the incubation of fresh tissues or cells in a protecting solution comprising a mixture of buffered amino acids at concentrations between 10 and 100 nmol, followed by dehydration in acetone at 4 °C. Subsequently, specimens are embedded in paraffin for long-term storage.9 HOPE-fixed specimens maintain morphology similar to that of formalinfixed samples and preserve characteristics required for immunohistochemistry. Thus, they have been successfully applied for diagnostics and research of various pulmonary Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: January 31, 2014 Published: April 7, 2014 5230

dx.doi.org/10.1021/pr500096a | J. Proteome Res. 2014, 13, 5230−5239

Journal of Proteome Research

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Table 1. Applications for HOPE, Formalin, and Snap-Frozen Fixation Techniques

diseases, including cancer, asthma, fibrosis, and microbial infections.6,10,11 Furthermore, HOPE allowed establishment of urgently required ex vivo human lung infection models for analyzing immune responses to Haemophilus inf luenza, Streptococcus pneumonia, Chlamydia pneumonia, and Legionella pneumophila.12−19 Importantly, the quality and quantity of nucleic acids extracted from HOPE-fixed tissue is sufficient and comparable with SF, which allows for the application of standard molecular methods such as PCR, RT-PCR, Northern blotting, and transcriptome microarrays.2,6,7,10,11,20−23 In contrast, usage of formalin-fixed paraffin-embedded (FFPE) tissue remains problematic for RNA/DNA studies. At this stage, wholegenome expression profiling of clinical samples was unsuccessful in 10−70% of cases.8 Beside genomics, proteomics is becoming indispensable for clinical research and our understanding of human diseases. Identification of post-translational modifications, such as protein phosphorylation, can reveal the actual activity status of altered cellular processes.24 Phosphorylation of serine, threonine, and tyrosine residues controls intra- and intermolecular protein interactions and is a key process in each eukaryotic signal pathway. Thus, phosphorylations and phosphorylation-modifying enzymes are prime targets for diagnosis, prognosis, as well as the identification of novel drug targets.25 HOPE-fixation already supported functional studies of individual proteins,6,20,26,27 but so far was not evaluated for proteome-scaled approaches. This defined the objective of the present study, which utilized lung patient slices for comparative investigations of HOPE-fixed and SF tissues by state-of-the-art peptide sequencing strategies. The manual and statistical inspections certified a high reproducibility of results obtained from HOPE-treated technical replicates at the level of SF

material. In general, HOPE-treated human lung tissues could be validated to be fully suitable for standard proteome and phosphoproteome strategies, resulting in representative and patient-specific MS data profiles.

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MATERIALS AND METHODS

Ethics Statement

For human lung tissue explants, all patients gave informed written consent. All procedures were performed according to German national guidelines and approved by the local ethical committee, Ethik-Kommission der Medizinischen Fakultät der Universität Lübeck (03/153). Lung Sample Preparation by HOPE-Fixation and Histology

Lung tissue samples were obtained from surgery patients as described previously.13 Tumor-free lung specimens (1 cm3) were taken at least 5 cm from the tumor front. From each donor, one piece of lung tissue was immediately frozen in liquid nitrogen and the other piece was fixed using the HOPEtechnique (DCS Diagnostics, Hamburg, Germany; Vollmer et al., 2006).4,9,13 In brief, the samples were incubated in HOPE solution I at 4 °C for 18 h, followed by dehydration in acetone at 4 °C for 6 h and storage at 4 °C for subsequent experiments. After HOPE-fixation the paraffin blocks were cut (1 μm), deparaffinized, and stained with hematoxylin/eosin.9 Protein Extraction

The HOPE-fixed and snap-frozen lung samples were briefly washed in cold PBS and homogenized in extraction buffer (1 M NaCl, 50 mM Hepes, 1% Triton, 5 mM EDTA, 10 mM NaF, 2.5 mM Na3VO4, supplemented with Benzonase 50 U/mL, 1% protease, and 1% phosphatases inhibitor cocktails, Sigma) using a blender at 25.000 rpm on ice for 30 s. The homogenates were sonicated with a Bandelin Sonifier 250 using 4 × 30 cycles with 5231



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Scientific) and eluted over 180 min for peptides and 120 min for phosphopeptides using a 75 μm × 25 cm analytical column (Acclaim PepMap RSLC, C18, 2 μm, Thermo Scientific) with a gradient buffer 0−95% acetonitrile in 0.1% formic acid. We employed the data-dependent top10 method with the following standard mass spectrometric conditions: spray voltage 19 kV, ion transfer tube temperature 300 °C, 40% collision energy for MS/MS. The data were acquired using the Xcalibur 2.2 software (Thermo Scientific) and processed with Proteome Discoverer (Version 1.3.0.339, Thermo Scientific). Raw MS/ MS data were searched in the UniProtKB/Swiss-Prot protein database (release 2012_09, with 538,010 entries; taxonomy Homo sapiens with 20,235 entries) using the following Mascot search parameters: enzyme, trypsin; and maximum missed cleavages, 2. For phosphopeptides, variable modifications were phosphorylation (S, T, Y) and oxidation (M); static modification was Methyltio (C); peptide tolerance, 10 ppm; and MS/MS tolerance, 0.4 Da. The peptides were considered as unique if they were identified at least once having an individual Mascot peptide score above 30 for peptides and 20 for phosphopeptides. To distinguish between correct and noncorrect peptide identification, a false discovery rate (FDR) filter lower than 1% was applied in the Decoy database search. Localization and scoring of phosphorylated sites on the peptides were performed using PhosphoRS29 integrated in Proteome Discoverer. All MS-data associated with this manuscript are accessible in the PROteomics IDEntification Database (PRIDE).30

50% duty. Cell debris and insoluble material were removed by centrifugation at 4000g for 10 min at 4 °C, and lysates were further clarified by ultracentrifugation at 100.000g for 1 h at 20 °C. The supernatants were passed through a filter with a pore size of 0.45 μm (Sterivex, SVHL10RC, Millipore), and the protein concentration was estimated with the BCA protein assay (Thermo Scientific Pierce), using BSA dissolved in extraction buffer for calibration. The protein solutions were processed immediately or aliquoted and stored at −80 °C. Protein Digestion

Aliquots of 0.5 mg or 2 mg of protein were precipitated for proteomic and phosphoproteomic analyses according to Wessel and Flügge.28 In brief, one part of sample was mixed with four parts of methanol, one part of chloroform, and three parts of ddH2O, mixed vigorously, and centrifuged at 5000g for 15 min at 4 °C. The pellets were washed with methanol, dried at RT, and dissolved in 1 mL of digestion buffer containing 50 mM TEAB, 10% ACN and supplemented with 10 mM NaF, 2.5 mM Na3VO4, and 1% phosphatase inhibitor cocktail (Sigma) for phosphopeptide analysis. The proteins were reduced with 100 μL of 50 mM TCEP for 1 h at 60 °C, alkylated with 50 μL of 200 mM MMTS for 10 min at RT, and digested with trypsin at a protein to enzyme ratio of 70:1 overnight at 37 °C. The digested samples were centrifuged at 4000g for 10 min at RT, acidified to pH 2−3 with formic acid, desalted with Strata C18E columns (Phenomenex), and eluted with 65% (v/v) MeOH in H2O containing 0.5% formic acid according to the manufacturer’s manual. The peptides were dried completely and then dissolved in 3% ACN, 0.2% TFA for LC-MS/MS or in IMAC-buffer for phosphopeptide enrichment.

Bioinformatical and Statistical Analyses

Results obtained from Proteome Discoverer were further analyzed with scripts written with the open source software “R” to validate amino acid compositions, molecular weights, isoelectric point distribution, and the subcellular localization of proteins.31 The hydrophobicity of peptides was calculated using the algorithm developed by Krokhin et al.32 Classification of phosphorylation sites was done based on their probabilities to be located at the indicated amino acid as referred to by Proteome Discoverer. The sites with probability 95−100% were assigned to the class I-unambiguous phosphosites; remaining sites were assigned to class II phosphosites. To distinguish novel and known phosphosites, the data sets were searched against PhosphoSite Plus (http://www.phosphosite. org) and ELM databases (http://phospho.elm.eu.org). The biological pathway analysis of protein sets was supported by GeneGo MetaCore (https://portal.genego.com/). To calculate the correlation coefficient between proteins and phosphopeptides in technical replicates, the Kendall rank correlation coefficient was used. chi-square test analyses were performed to verify whether the type of fixation influences the results obtained by proteomics and phosphoproteomics.

Phosphopeptide Enrichment

Ga−Fe sepharose was utilized for phosphopeptide enrichment. In brief, 1 mL of IMAC Sepharose High Performance (GE Healthcare) was mixed with 10 mL of 0.1 M Ga(NO3)3 solution and incubated at +4 °C. After that, Ga-sepharose was saturated with 10 mL of 0.1 M FeCl3, washed three times with ultrapure water, packed into Pierce Spin Columns (Thermo Scientific), and pre-equilibrated with IMAC buffer (27% MeOH, 27% ACN, 20% acetic acid, pH 2.5). The desalted peptide pellets were resuspended in 40 μL of the same buffer, loaded onto the spin columns, and incubated for 1 h at RT. The unbound peptides were removed by a series of washing steps: nine times with 300 μL of IMAC buffer and 3 times with 300 μL of IMAC 0.2 M NaCl salt buffer. The samples were briefly centrifuged at 2000g for 10 s after every washing step. Finally, the retained phosphopeptides were eluted with 500 μL of 100 mM ammonium phosphate buffer, pH 4.5, and then with 300 μL of 1 M dipotassium hydrogen phosphate pH 9.0. Eluted peptides were reconstituted in TFA for further desalting and dried in vacuo for further storage. To increase the proportion of phosphopeptides, the eluted peptide fraction was subjected to a second identical IMAC.

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LC-MS/MS Analysis

RESULTS AND DISCUSSION

HOPE Preserves Lung Tissue Morphology and Allows Proteome Extraction

The peptides were analyzed on an UltiMate 3000 RSLCnano LC system connected to an LTQ Orbitrap Velos Pro mass spectrometer (both Thermo Scientific). MS scans were combined with collision induced dissociation (CID) in the linear ion trap or with higher energy collision dissociation (HCD) for peptides and phosphopeptides, respectively. Briefly, the peptide mixture was loaded onto a 75 μm × 2 cm precolumn (Acclaim PepMap 100, C18, 3 μm, Thermo

In this study we evaluated HOPE-fixed lung tissues obtained from four surgery patients by comparison with the gold standard in proteomicssnap-frozen tissues. The overall experimental strategy and investigated aspects are outlined in the abstract graphic. Tumor-free lung tissues of about one cubic centimeter were weighted and either immediately frozen in liquid nitrogen or fixed using the HOPE technique. 5232



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provides further support for propagation of this fixation method among pathologists.2,33 One of the crucial steps in proteomics is the reliable and reproducible extraction of proteins. Our protocol for the extraction of proteins from both types of fixation consists of intensive homogenization and sonication of tissue in buffer containing detergent, protease and phosphatase inhibitors and Benzonase, which degraded DNA and reduced the viscosity of the suspension. Overall, the effectiveness of protein extraction from HOPE-fixed material was lower than that from SF, with 26.2 ± 1.8 μg/mg and 47.5 ± 3.6 μg/mg of tissue, respectively. That might be explained by a kind of washing out effect due to several incubations in acetone. Notably, the amount of extracted proteins in other studies was even better in HOPEtreated material in comparison to SF, but whether the quality of those extracted proteins was sufficient for MS detection has not been tested.34

First, we examined the morphology of the lung sections after hematoxylin-eosin staining (Figure 1). Tissue morphology of

HOPE Supports Representative and Comparative Proteome Studies

Profiling patient tissues immediately or retrospectively by proteomics constitutes an indispensable part of our clinical demands. Mass spectrometry is well established for biotyping and in combination with robust liquid chromatography (LCMS) was introduced for biomarker discovery.35 In this study, we compared extracted proteins by 180 min LC-MS/MS gradients. For each donor and fixative method, we performed technical replicates. The overlap between replicates was similar for HOPE and SF samples and does not exceed 79% of protein. Overall technical replicates correlation coefficients range from 0.76 to 0.79 and from 0.76 to 0.77 for HOPE and SF samples, respectively. In total, we identified 2603 proteins (14485 unique peptides) for HOPE and 2910 proteins (15457 unique peptides) for SFtissue (Table 2, Supporting Information Tables 1 and 2). The overlap of proteins identified in HOPE-treated and SF tissues from all donors was 71%. The ratio of exclusive proteins detected in HOPE or SF-samples was found at the same level as in donor-to-donor variations (Figure 2). It is well-known that the type of sample preparation often results in the detection of similar but not identical sets of proteins. The essential question is whether a certain method biases or even impairs the representative character of a proteome study. Therefore, we have validated parameters that can indicate influences of HOPE on the quality of MS and the nature of detectable proteins. First, we evaluated the quality of the acquired MS data. Ion scores of all identified peptides were plotted donor- and fixation-dependently. We did not observe any significant differences regarding peptide ion score qualities in SF and HOPE material or between different donors. Therefore, the

Figure 1. Comparison of the morphological preservation in representative sections of HOPE-fixed (A, B, C) and SF (D, E, F) tissues. Tissues from the three donors (A/D, B/E, C/F) were stained with hematoxylin-eosin (200× magnifications). The morphological preservation of HOPE-fixed tissues is clearly superior to that of SF tissues with regard to alveolar structure, cellular differentiation, and squeezing artifacts.

HOPE-treated specimen was comparable to the morphology of formalin fixed tissues, with respect to both nuclear and cytoplasmic details and preservation of tissue architecture. In contrast, snap-frozen tissues showed artificial tissue disruption and collapse of alveoli; furthermore, these sections revealed only coarse nuclear detail. Thus, HOPE-treated lung samples generally displayed well-preserved histological details, which were previously designated as “formalin-like morphology”. The morphological preservation of HOPE-fixed tissues is clearly superior to SF with regard to alveolar structure, cellular differentiation, and squeezing artifacts. In combination with previously established microscopical techniques for HOPE, such as fluorescence in situ hybridization and immunohistochemistry, the excellent morphology of HOPE-treated tissue

Table 2. Number of Identified Proteins, Peptides, Phosphopeptides, and Corresponding Phosphosites in HOPE-Fixed and SF Lung Tissuea Proteome

HOPE SF

Proteins

Peptides

2603 2910

14 485 15 457

Phosphoproteome Phosphopeptides

Phosphosite

Class I

Total

1P

2P

≥3P

Total

pSer

pThr

pTyr

PSP

ELM

2676 3108

2336 2701

322 382

18 25

3036 3540

2,752 (90.65%) 3,177 (89.75%)

252 (8.3%) 325 (9.18%)

32 (1.05%) 38 (1.07%)

2025 (80,29%) 2334 (81,38%)

1745 (60,84%) 1536 (60,9%)

a 1P, singly phosphorylated peptides; 2P, doubly phosphorylated; ≥3P, phosphopeptides with three and more phospho groups. pSer, pThr, pTyr, phosphorylation on serine, threonine, and tyrosine residues, respectively. PSP, PhosphoSite Plus database (http://www.phosphosite.org); ELMELM database (http://phospho.elm.eu.org).

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amino acid counts derived from all identified peptides indicated no bias in the extraction of proteins with variable biochemical properties in HOPE- and SF-treated samples (Figure 3B). The data are also in accordance with the calculated amino acid composition of the theoretical human proteome.36 We then evaluated the hydrophobicity of peptides detected in HOPEtreated samples and compared it with those identified in SF by using an algorithm developed by Krokhin et al.32 The calculated peptide hydrophobicity scores plotted against retention time revealed no difference between HOPE and SF peptides, and we did not observe significant biases in molecular weights or isoelectric points of detected proteins (Figure 3C, D and Supporting Information Figure S1). Even though proteins could be detected irrespective of their biochemical properties, we asked the question whether the acetone-aided HOPEfixation affects the detection of proteins from different compartments. Modest differences were observed regarding extracellular and cell surface proteins, which better were covered in HOPE-treated tissues, whereas cytosolic and organelle lumen components were somewhat better covered in SF samples. However, examination of the protein distribution among subcellular compartments showed almost identical patterns for both types of tissue fixation (Figure 4A). In addition, we mapped functionally described proteins on main cellular processes for both types of fixation with the support of the GeneGo MetaCore program package. Notably, those analyses revealed striking similarities indicating that

Figure 2. Proteins identified in HOPE-treated and snap-frozen tissues. (A) Overlap between proteins identified in all HOPE-treated and all SF samples. (B) Overlap in proteins identified in donor 1 and donor 2 of HOPE-treated lungs and (C) donor 1 and donor 2 of snap-frozen lungs.

quality of the MS data seemed not to contribute to the observed variation (Figure 3A). Next, we evaluated whether HOPE is biasing the extraction of proteins with certain biochemical properties. Although the mechanism of HOPEfixation is not completely understood, the procedure does not cause the formation of methylene cross-links likewise after treatment with formaline, and peptides should be detectable irrespective of their amino acid composition.2 Indeed, ratios of

Figure 3. Biochemical characteristics of peptides derived from HOPE and SF proteomes. (A) Ion scores boxplot of HOPE and SF peptides from different donors. (B) Frequency of amino acid residues in peptides extracted from HOPE and SF tissues (C, D). Hydrophobicity of assigned HOPE and SF peptides was calculated using the algorithm developed by Krokhin et al.32 and plotted versus reverse phase liquid chromatography retention times. 5234



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Figure 4. Subcellular distribution of proteins identified in HOPE and SF tissue. (A) The ratio of annotated proteins was calculated with scripts using the open source software “R”, based on annotation obtained from Proteome Discoverer 1.3. (B) Top 10 cellular processes analyzed by GeneGo: each process represents a preset network of protein interactions characteristic for the process and shown as red for HOPE-fixed proteins and blue for SF-tissue proteins. Each category contains a minimum of 45 genes. P-Value represents the threshold for the significance of the process. Both HOPE and SF protein data sets significantly cover the top 10 significant cellular processes.

Figure 5. Scatter plots for phosphopeptides identified in HOPE-treated (A) and SF (B) tissue. The phospho-sites localization scores (pRS scores) were plotted against its ion scores. Top 10 cellular processes analyzed by GeneGo: each process represents a preset network of protein interactions characteristic for the process and shown as red for HOPE-fixed proteins and blue for SF-tissue proteins. Each category contains a minimum of 13 genes. P-Value represents the threshold for the significance of the process. 5235



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HOPE supports representative proteome studies. Only the group of proteins with cell adhesion function, which are mainly corresponding to extracellular and cell surface proteins, were marginally enriched in HOPE-treated samples. We assume that this difference may be due to the higher accessibility of extracellular structures for detergent after treatment of tissue with the HOPE solution. Another possible reason is a destruction of such structures due to the process of snap freezing. Finally, we asked the question whether the proteome results can be related to the type of fixation by means of statistics. To answer this question, we applied statistical chi-square tests (for details see Supporting Information file 1). The hypothesis was that the fixation method used does not influence the selection of proteins at the significance level 0.05. Since the calculated Pvalue was 0.8161, data of this study are clearly not rejecting this hypothesis. This result further supports the assumption that no significant relation between the protein identifications and the method of fixation exists. In conclusion, all tested aspects confirmed HOPE-fixation to be suitable for representative proteome analyses of lung tissues. Thus, it was tempting to speculate that the method is also suitable for phosphoproteome studies, which play a pivotal role in the recognition of disease states as several times demonstrated, e.g. in cancer research.16,37

plotted the phospho-sites localization scores (pRS score) in relation to peptide ion scores. The resulting scatter plots display almost equal MS data qualities with respect to the phosphorylation site determination (Figure 5A, B). Furthermore, we calculated the reproducibility of phosphoproteome results obtained from both types of fixation. Actually, the correlation coefficients between technical replicates, which reflected the reproducibility of data, were even slightly better for HOPE (from 0.56 to 0.57) than for SF (from 0.53 to 0.55). Thus, the selected standard method of IMAC-based phosphopeptide enrichment is compatible for sample-specific phosphoproteome analyses from HOPE-fixed lung tissues. Cellular processes can be studied from HOPE-treated samples as well as from SF samples, which is of great importance to characterize deregulated signal pathways in human diseases (Figure 5C). However, phosphorylations are known to be instable, and further experiments are required to validate which fixation technique is superior in preserving phosphorylation states for longer periods of time in biobanks.45 According to the statistical evaluation of the proteome data in the previous section, we finally validated the phosphoproteome results statistically. Again, we applied a chi-square test (for details see Supporting Information) to prove the hypothesis that the type of fixation does not influence the identification of phosphorylated peptides at the significance level 0.05. Since the calculated P-value was 0.9408, also the phosphoproteome data of this study clearly are not rejecting this hypothesis. Therefore, the assumption that no significant relation exists between phosphoproteins identification and the method of fixation is reasonable, too. In summary, HOPE complements our knowledge about the lung tissue phosphoproteome significantly. We could classify 61.7% of all phosphorylation sites (2522) in HOPE and 61.4% (2868) in SF samples as high quality class I information (based on Proteome Discoverer). The average probability for correct phosphorylation site assignment thereby is 99.4% for HOPE and 99.3% for SF. About 80% of phosphorylation sites in both HOPE and SF data sets were previously reported in PhosphoSite Plus (http://www.phosphosite.org) and more than 60% in the ELM database (http://phospho.elm.eu. org).46−48 Consequently, at least 20% of the identified class I phosphorylation sites in this study have to be considered as novel for the human lung, to which HOPE and SF contributed equally (Table 2).

Characterizing the Phosphoproteome of HOPE-Lung Tissues

Phosphoproteome analysis by mass spectrometry is a crucial step for elucidating the differences between cellular processes of normal and pathological conditions, and consequently the discovery of disease biomarkers and appropriate drug targets.38,39 We assumed that the HOPE-fixation technique preserves phosphorylation at least as well as snap freezing. To test this we analyzed the extractability and detectability of phosphorylated proteins from HOPE-treated lungs and compared them with those from SF. We selected a Ga−Fe sepharose for immobilized metal affinity chromatography (IMAC) and enriched potentially phosphorylated peptides from 2 mg of extracted proteins. The obtained peptides were then analyzed by LC-MS/MS by using HCD-fragmentation.40−42 Only those phosphopeptides with a pRS score above 50 were considered as high quality data and included in this study. By this approach we obtained 54.82 ± 5.6% and 58.32 ± 4.9% of phosphopeptides in the total peptide fraction for HOPE and SF, respectively. As the IMAC phosphopeptide enrichment is based on nonspecific adsorption of negatively charged amino acids, we did not expect a higher proportion of phosphopeptides in samples without further fractionation.43,44 In total, we identified 2676 unique phosphopeptides in HOPE tissue and 3108 in SF (Table 2 and Supporting Information Tables 3 and 4). Among phosphopeptides identified in HOPE samples, 2336 were singly phosphorylated, 322 were doubly phosphorylated and 18 have three or more phosphorylation sites. Similar ratios were observed for SF samples, where we detected 2701 peptides with a single phosphate group, 381 with double phosphorylation, and 25 with three and more phosphate groups. Ratios of phosphorylated serine, threonine, and tyrosine residues were found to be highly similar in both types of fixation as well (Table 2). To compare the quality of the identified phosphopeptides, we focused on the suitability of the underlying MS data to determine the site of phosphorylation precisely. Therefore, we

Perspectives for Donor-Dependent Proteome Profilings

The molecular profiling of donor-to-donor variations constitutes a prime task in clinical and translational research. This study provides data about HOPE as a suitable fixative for histology that in parallel matches the requirements to extract and analyze proteins and their phosphorylation sites. Thus, HOPE should be considered in concepts of patient-specific diagnosis and therapy as well as in biobanks, which support retrospective biomarker studies. Due to the limited number of donors used in this study, we cannot make final conclusions, but our chances of success in that direction can be anticipated based on the identifications of donor-specific proteins. As expected, donor-specific proteins exhibited generally lower identification scores in contrast to the group of nondonorspecific proteins (Supporting Information Figure S2). However, scores and their dynamic range were observed always at the same level irrespective of the type of fixation. This also holds true for phosphoproteome data, but the donor-specific 5236



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ABBREVIATIONS IMAC, immobilized metal ion affinity chromatography; HCD, higher energy collision dissociation; HOPE, HEPES, glutamic acid buffer-mediated organic solvent protection effect; MS/MS, tandem mass spectrometry; PTM, post-translational modification

variations seemed higher. This is in line with our understanding of phosphorylation patterns reflecting any variation in the activity status of cells whereas their protein abundances often remain stable.

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CONCLUSION Our success in biobanking will depend on the suitability of the stored human tissues for their molecular characterization by “-omics” technologies. In this study, we evaluated the feasibility of HOPE-treated human lung tissues for proteome analyses. HOPE is well established as a formalin-free fixative for histology, but so far it was not characterized for proteome analyses. All performed evaluations consistently support the idea that results obtained from HOPE-treated tissues are equivalent to those obtained from freshly isolated and snapfrozen samples, which constitutes the gold standard for tissue proteomics. Notably, the biochemical properties, cellular localizations, and pathways covered by the identified proteins were found in perfect accordance irrespective of the used fixation method. Statistical evaluations further confirm HOPE as suitable for representative proteome studies. That also holds true for phosphoproteomics, analysis of which was accomplished based on standard protocols. In conclusion and under consideration of previous evaluations focusing on histological tissue quality, genome, and transcriptome data, we now can recommend HOPE generally as the “fixative of choice” for studies aiming to describe molecular features of human tissues at the systems level. HOPE-fixation opens promising avenues for a better understanding of human diseases and best matches perspective requirements for personalized clinical analytics.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org Supplemental Table 1, Proteins identified in HOPE-treated lung specimens of four donors; Supplemental Table 2, Proteins identified in SF lung specimens of four donors; Supplemental Table 3, Phosphopeptides identified in HOPE-treated lung specimens of four donors; Supplemental Table 4, Phosphopeptides identified in SF lung specimens of four donors; Supplementary Figures S1 and S2; and Supplementary file 1, Statistics for proteome and phosphoproteome.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. # Shared senior authorship.

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ACKNOWLEDGMENTS The authors thank Uwe Kärst for revision of this manuscript, Jasmin Tiebach and Undine Felgenträger for excellent technical assistance, and Sebastan Marwitz, Jens Jäger, Bernhard Schmitt, and Janine Rasch for support to prepare the tissues. Financial support for this study was provided by Grant 0315831B from the federal ministry of education and research (BMBF). 5237



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HOPE-Fixation of Lung Tissue Allows Retrospective Proteome and

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