MALDI imaging of formalin-fixed paraffin-embedded tissues: application to model animals of Parkinson disease for biomarker hunting

A common technique for the long-term storage of tissues in hospitals and clinical laboratories is preservation in formalin-fixed paraffin-embedded (FFPE) blocks. Such tissues stored for more than five years have not been useful for proteomic studies focused on biomarker discovery. Recently, MS-based proteomic analyses of FFPE showed positive results on blocks stored for less than 2 days. However, most samples are stored for more than one year, and thus our objective was to establish a novel strategy using as a model system 6-hydroxydopamine (6-OHDA) treated rat brain tissues stored in FFPE blocks for more than 9 years. We examined MALDI tissue profiling combining the use of automatic spotting of the MALDI matrix with in situ tissue enzymatic digestion. On adjacent sections, the identification of compounds is carried out by tissue digestion followed by nanoLC/MS-MS analysis. The combination of these approaches provides MALDI direct analysis, MALDI/MS imaging, as well as the localization of a large number of proteins. This method is validated since the analyses confirmed that ubiquitin, trans-elongation factor 1, hexokinase, and the Neurofilament M are down-regulated as previously shown in human or Parkinson animal models. In contrast, peroxidoredoxin 6, F1 ATPase, and alpha-enolase are up-regulated. In addition, we uncovered three novel putative biomarkers, the trans-elongation factor 1 (eEF1) and the collapsin response mediator 1 and 2 from protein libraries. Finally, we validate the CRMP-2 protein using immunocytochemistry and MALDI imaging based on the different ions from trypsic digestion of the protein. The access to archived FFPE tissue using MALDI profiling and imaging opens a whole new area in clinical studies and biomarker discovery from hospital biopsy libraries.     

Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues

The heterogeneity of breast cancer requires the discovery of more incisive molecular tools that better define disease progression and prognosis. Proteomic analysis of homogeneous tumor cell populations derived by laser microdissection from formalin-fixed, paraffin-embedded (FFPE) tissues has proven to be a robust strategy for conducting retrospective cancer biomarker investigations. We describe an MS-based analysis of laser microdissected cancerous epithelial cells derived from twenty-five breast cancer patients at defined clinical disease stages with the goal of identifying protein abundance characteristics indicative of disease progression and recurrence. Comparative analysis of stage 0 and stage III patients revealed 113 proteins that significantly differentiated these groups and included known factors associated with disease pathogenesis, such as CDH1 and CTNNB1, as well as those previously implicated in breast cancer, such as TSP-1. Similar analyses of patients presenting with stage II disease that did or did not exhibit recurrence two years postdiagnosis revealed 42 proteins that significantly differentiated these subgroups and included IRS-1 and PARK7. These data provide evidence supporting the utility of FFPE tissues for functional proteomic analyses and protein biomarker discovery and yielded protein candidates indicative of disease stage and recurrence in breast cancer that warrant further investigation for diagnostic utility and biological relevance.     

Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography-mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250-300 proteins per 500 ng of tissue with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10(-15)). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.     

Analysis of the formalin-fixed paraffin-embedded tissue proteome: pitfalls, challenges, and future prospectives

Formalin-fixed paraffin-embedded (FFPE) tissues are a real treasure for retrospective analysis considering the amount of samples present in hospital archives, combined with pathological, clinical, and outcome information available for every sample. Although unlocking the proteome of these tissues is still a challenge, new approaches are being developed. In this review, we summarize the different mass spectrometry platforms that are used in human clinical studies to unravel the FFPE proteome. The different ways of extracting crosslinked proteins and the analytical strategies are pointed out. Also, the pitfalls and challenges concerning the quality of FFPE proteomic approaches are depicted. We also evaluated the potential of these analytical methods for future clinical FFPE proteomics applications.     

Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for both prospective and retrospective biomarker discovery. Unlocking the proteomic profile of clinicopathological FFPE tissues is a critically essential step for annotating clinical findings and predicting biomarkers for ultimate disease prognosis and therapeutic follow-up.     

News in Brief

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.     

Proteomic analysis of formalin-fixed prostate cancer tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.     

Equivalence of Protein Inventories Obtained from Formalin-fixed Paraffin-embedded and Frozen Tissue in Multidimensional Liquid Chromatography-Tandem Mass Spectrometry Shotgun Proteomic Analysis

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 μg of protein yielded ∼400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from ∼17% after one year of storage to ∼25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.Formalin-fixed paraffin-embedded (FFPE)¹ tissue samples are routinely prepared during the pathological characterization of clinical specimens and are abundantly available in pathology archives worldwide. The fixation process yields clinically relevant samples that can be stored at ambient temperature and are suitable for pathological examination by light microscopy even after years in storage. Given the wealth of clinical data associated with specimens collected over a span of decades, such as patient treatment regimens and outcomes, FFPE tissue represents a potentially valuable resource for biomarker discovery through retrospective analysis (1, 2).However, fixation of tissue in formalin leads to significant cross-linking among proteins and other biomolecules, rendering the samples incompatible with many biochemical analyses. Immunohistochemical (IHC) analysis of FFPE tissue has been conducted since the 1970s using either proteolysis or protein denaturants to expose antigenic regions of proteins (3, 4). Since the 1990s, detection of antigens in FFPE tissue has been improved through the development of so-called antigen retrieval techniques (5, 6). These methods involve application of heat in the presence of any of a variety of buffers resulting in the cleavage of methylene bridges formed during the course of fixation (2).Despite their utilization for IHC analysis, FFPE tissue samples have been largely overlooked in proteomics studies, due to the assumption that tissue fixation would make proteomic analysis intractable. Recent work appears to refute this notion. In 2005, Hood et al. (7) first described the successful application of shotgun proteome analysis to FFPE tissue. Using laser capture microdissected cells and an optimized extraction method, hundreds of proteins were identified from a cancerous prostate lesion and benign prostate hyperplasia, thus opening the door to comparative proteomic analyses of FFPE tissue. Moreover, the same study showed that the numbers and identities of proteins observed were remarkably similar when applying the method to frozen and FFPE mouse liver, thus lending support to the use of FFPE tissue in biomarker discovery studies. Since the initial demonstration of its feasibility, FFPE tissues from diverse origins including breast, liver, kidney, lymphoma, and bone successfully have been subjected to proteomic analyses (8–14).Although this work suggests the feasibility of biomarker discovery from FFPE tissue, most of these previous studies have been performed on small amounts of material with one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The use of multidimensional peptide separations can extend the dynamic range of the LC-MS/MS analyses to detect lower abundance proteins. Recently, the use of capillary isotachophoresis as the first dimension in a multidimensional peptide separation strategy for analyzing FFPE tissue was described (8). In this study, thousands of proteins were identified out of <4 μg of digest from FFPE human liver sections. However, the apparatus used was an in-house, custom-designed system, not readily accessible to other laboratories. In several of these studies, proteins identified by a single peptide were accepted as valid identifications. Use of single peptide-based identifications elevates the probability of false positive protein identifications, and these identifications often constitute the majority of protein identifications (15).The equivalence of fresh/frozen and FFPE tissue proteomes is a critical issue in evaluating the suitability of employing FFPE tissues for biomarker discovery by comparative proteomic analyses. Hood et al. (7) and Guo et al. (14) reported comparisons from analyses of paired fresh and frozen tissue specimens. Guo et al. (14) reported an apparent overlap of 83% in protein identifications between FFPE and frozen brain tissue specimens, whereas Hood et al. (7) did not report the degree of overlap, but found that FFPE mouse liver tissue yielded about 88% of the identifications determined for frozen mouse liver tissue. The majority of protein identifications in both studies were based on single peptide assignments. These investigations did not explicitly address the effect of formaldehyde-derived modifications on the inventories of identified peptides.An unexplored question with FFPE tissue specimens is the extent to which normal variability in fixation process and storage duration affect the proteomes observed. The duration of tissue fixation is not highly standardized and may vary from hours to several days. One of the most attractive features of FFPE specimens is the opportunity for retrospective biomarker discovery, but the effects of storage for many years on tissue proteomes remains unknown.Here, we address these questions through detailed comparative studies of the analysis of fresh frozen and FFPE tissues by LC-MS/MS-based shotgun proteomics. We used the same fresh tissue specimens to prepare both frozen and FFPE samples for paired comparisons. We evaluated conditions for tissue lysis and digestion and the effects of fixation time and storage duration on the number of protein IDs obtained during shotgun proteomic analysis of FFPE tissue. We also characterized the differences in peptides observed between fixed and frozen specimens in an effort to understand the effect of fixation from a practical biomarker discovery standpoint. Furthermore, we compared analyses of fresh frozen and FFPE colon adenoma tissue by multidimensional LC-MS/MS using gel-based isoelectric focusing of peptides (Fig. 1). The results demonstrate a remarkable overlap in the number and identities of proteins between the fixed and frozen tissue and indicate that variations in duration of fixation and storage have a minimal effect on protein inventories obtained by shotgun proteomic analysis. The data indicate essential equivalence between protein inventories obtained from fresh frozen and FFPE tissue specimens by shotgun proteomics and validate the use of FFPE tissue specimens for biomarker discovery.Open in a separate windowFig. 1.Strategy for multidimensional LC-MS/MS analysis of FFPE tissue.     

Impact of fixation time on GeLC-MS/MS proteomic profiling of formalin-fixed, paraffin-embedded tissues

Formalin-fixed, paraffin-embedded (FFPE) tissue banks represent an invaluable resource for biomarker discovery. Recently, the combination of full-length protein extraction, GeLC-MS/MS analysis, and spectral counting quantification has been successfully applied to mine proteomic information from these tissues. However, several sources of variability affect these samples; among these, the duration of the fixation process is one of the most important and most easily controllable ones. To assess its influence on quality of GeLC-MS/MS data, the impact of fixation time on efficiency of full-length protein extraction efficiency and on quality of label-free quantitative data was evaluated. As a result, although proteins were successfully extracted from FFPE liver samples fixed for up to eight days, fixation time appeared to negatively influence both protein extraction yield and GeLC-MS/MS quantitative proteomic data. Particularly, MS identification efficiency decreased with increasing fixation times. Moreover, amino acid modifications putatively induced by formaldehyde were detected and characterized. These results demonstrate that proteomic information can be achieved also from tissue samples fixed for relatively long times, but suggest that variations in fixation time need to be carefully taken into account when performing proteomic biomarker discovery studies on fixed tissue archives.     

Quantitative Proteomic Analysis of Formalin-fixed and Paraffin-embedded Nasopharyngeal Carcinoma Using iTRAQ Labeling,Two-dimensional Liquid Chromatography,and Tandem Mass Spectrometry

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method. (J Histochem Cytochem 58:517–527, 2010)     

2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples

Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.Histone post-translational modifications (hPTMs)¹ generate a complex combinatorial code that plays a critical role during the physiological and pathological regulation of gene expression (1). Alterations in histone modification patterns have been linked with various diseases, including cancer, often as a result of the aberrant expression or localization of histone modifying enzymes (2). Therefore, accurately dissecting hPTM patterns in normal and diseased tissues could yield epigenetic biomarkers useful for prognostic, diagnostic, and therapeutic purposes. Immunohistochemistry studies have shown the potential of this strategy (3, 4), but they were limited to the analysis of only a few hPTMs. In addition, despite their sensitivity and ease of use, antibody-based assays are hindered by issues such as the difficulty in detecting adjacent modifications and the limited linearity of the signal. As an alternative to traditional antibody-based methods, in recent years MS has become the elective method to analyze hPTMs, thanks to its unbiased nature, accuracy and its ability to quantitate modifications and detect their combinations. Various MS-based workflows optimized for hPTM analysis have been developed (5), but most of the studies focused on cell lines and animal tissue, whereas the potential offered by the analysis of clinical samples has been left largely unexploited. In particular, the MS-based analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) samples has never been addressed.Paraffin embedding following fixation in buffered formalin is the storage method of choice for clinical specimens, thus representing an invaluable source of clinical samples linked to retrospective patient information. Large formalin-fixed paraffin embedded (FFPE) archives, which are available in many hospitals, have been successfully exploited for DNA and RNA analyses, including chromatin immunoprecipitation (6, 7). However, the extensive protein cross-linking generated by formaldehyde fixation has hindered the proteomic study of this type of tissue. This problem has been addressed and overcome only recently in global proteomic studies by taking advantage of extraction protocols based on heat-induced antigen retrieval techniques derived from immunohistochemistry (8, 9). Moreover, a few studies showed the possibility to globally analyze protein post-translational modifications, such as glycosylation and phosphorylation, from fixed and embedded tissues (10–12).Here, we report for the first time the successful application of MS-based analysis of hPTMs to human clinical samples, focusing in particular on the development and validation of a method (PAT-H-MS) to extract histones from FFPE tissues in yield and purity sufficient to enable the subsequent use of a proteomic workflow optimized for hPTM analysis (13). By using this method we were able to profile in a quantitative manner 24 distinct modified histone peptides from human FFPE breast cancer samples belonging to different subtypes, identifying differences in histone methylation patterns of potential clinical relevance. Thus, PAT-H-MS represents a valid approach for hPTM analysis of clinical samples.     

Tissue proteomics using chemical immobilization and mass spectrometry

Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.     

Proteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitis

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the "shotgun" quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ～50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.     

Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations

Archival formalin-fixed paraffin-embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI-TOF imaging MS (MALDI-IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo-preserved tissues. We have developed an in vitro approach using "tissue surrogate" samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI-TOF MS and nLC-MS(E) analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI-IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.     

Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease.     

Two methods for proteomic analysis of formalin‐fixed,paraffin embedded tissue result in differential protein identification,data quality,and cost

Formalin‐fixed paraffin‐embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC‐MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in‐solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre‐analytical variations and analyzed with three technical replicates by LC‐MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre‐analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.     

激光显微切割/质谱联用技术在肾脏疾病诊断中的应用进展

激光显微切割(Laser microdissection,LMD)质谱(Mass spectrometry,MS)联用技术(LMD/MS)已成功应用于肾活检组织甲醛固定石蜡包埋切片的蛋白质组学研究,提高了某些肾脏病的诊断水平,显示出较好的临床应用前景。文中就LMD/MS蛋白质组学技术的原理、方法及该技术在肾淀粉样变性、膜增殖性肾小球肾炎等肾脏疾病的发病机制及诊断分型的应用进展进行综述。