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A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples
... After the optimization and selection of the best tissue disaggregation method (protocol 3), the deparaffinization efficiency of xylene (a toxic polar solvent) and mineral oil was compared using three different protein extraction buffers: buffer 2 (urea LB), buffer 4 (citrate-SDS LB) and buffer 5 (SDS LB) (see Figure 3A). Better results were observed after the combination of the deparaffinization with xylene at 65 • C and the buffer 4, and the deparaffinization with mineral oil at 95 • C and buffer 2 or buffer 5. To improve the deparaffinization with xylene, we tested the effect of temperature in this process following a method described previously by Araújo et al. (42). As Figure 3B shows, the deparaffinization of the FFPE tissues samples with xylene at RT in combination with all extraction buffers (buffer 1 to 6) and protocol 3 (disaggregation with the TissueLyser) improved the protein extraction showing visible bands in all cases. ...
... Importantly, from these common proteins, more than 80% were found to be mainly expressed in kidney tissue in databases as Uniprot (https://www.uniprot.org/) and were also identified in previously reported articles with FFPE kidney samples (25,26,42,43). A functional analysis of these 468 common proteins were developed using the software FunRich (http://www.funrich.org/). ...
... While the poor results obtained with mineral oil al 95 • C could be due to an incomplete deparaffinization with this solvent that impeded the protein extraction buffer penetration, the bad results obtained with xylene at 65 • C were probably due to the degradation of proteins at this temperature. These drawbacks were avoided after using xylene at RT following a method previously reported method (42,52). As it as mentioned above, the protein extraction efficiency could be modulated by some factors such as temperature, incubation time, extraction buffer composition, application of an ultrasonic method or other methods for tissue disaggregation (7,14). ...
Most tissue biopsies from patients in hospital environments are formalin-fixed and paraffin-embedded (FFPE) for long-term storage. This fixation process produces a modification in the proteins called “crosslinks”, which improves protein stability necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical analysis, since these modifications do not suppose a drawback for this technique; however, crosslinks difficult the protein extraction process. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. The present work aims to establish a deparaffinization and protein extraction protocol from FFPE kidney samples to obtain protein enough of high quality for the subsequent proteomic analysis. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. The optimized conditions will be applied in the identification by LC-MS/MS analysis of proteins extracted from 5, 10, and 15 glomeruli obtained through the microdissection of FFPE renal samples.
... Because FA easily polymerizes at high concentrations and after long storage periods, it is often used commercially as a 37-40 % solution of FA (formalin) [3]. Formalin allows long-term tissue stability and it preserves tissue architecture [4]. The term "formalin" describes aqueous solutions that usually contain both FA and an alcohol stabilizer [5,6]. ...
... The slope factor (SF) turns the expected value of a daily intake of a substance throughout one's lifetime directly into the risk of developing cancer. If we assume that the slope factor is constant, the risk is directly related to the intake: CR = CDI × SF (4) According to the IRIS system, the slope factor in this study of FA is 0.0455 (mg/kg/day) −1 [40][41][42]. ...
Dissecting a human cadaver is an irreplaceable practice in general training of medical students. Cadavers in anatomy laboratories are usually preserved in formalin, an embalming fluid whose basic component is formaldehyde (FA). The aim of this study is to assess the cancer risk of employees and students that are exposed to FA based on the results of three monitoring campaigns, as well as to suggest permanent solutions to the problem of FA exposure based on the results obtained. Three sampling campaigns of formaldehyde concentration in indoor environments were conducted at five different locations at the Anatomy Department of the Faculty of Medicine with the purpose of assessing permanent employees’ and medical faculty first year students’ exposure to FA. Indoor air was continuously sampled during 8 h of laboratory work and analyzed in accordance with the NIOSH Method 3500. Exceeding of the 8 h time-weighted average (8 h TWA) values recommended by Occupational Safety and Health Administration (OSHA) of 0.75 ppm was recorded in 37% of the samples during the three-month monitoring campaign. Cancer risk assessment levels for permanent employees were in the range from 6.43 × 10−3 to 8.77 × 10−4, while the cancer risk assessment levels for students ranged from 8.94 × 10−7 to 1.83 × 10−6. The results of the research show that cancer risk assessment for employees is several thousand times higher than the limit recommended by the EPA (10−6) and point to the importance of reducing exposure to formaldehyde through the reconstruction of the existing ventilation system, continual monitoring, the use of formaldehyde-free products, and plastination of anatomical specimens.
... Hence, to ensure a better performance in the sample preparation, some statistical methodologies are in use today to provide a more refined knowledge on the system, and, more precise results. Among these methodologies the use of experimental designs for exploratory and optimization purposes have been reported in the literature by Seo et al. (2015), Araújo et al. (2014), L'Hocine and Pitre (2016), Oberoi and Sogi (2017), and Tsiaka et al. (2015), for example, in researches that optimized methodologies studying proteins, isoflavanoids and carotenoids using experimental designs and the response surface methodology in the matrices of bovine plasma, renal tissue, nuts, watermelon and shrimps. ...
... Fig. S1 also points out the strong secondary interactions of these three factors. According to Araújo et al. (2014), a first-order effect must be considered individually for interpreting the results only when there are no interactions, as interactions prevail. From this premise, the cubic graph presented in Fig. 1 eases the joint interpretation of these effects. ...
... Different techniques have been reported to remove paraffin residues and reverse formaldehyde crosslinks using various protein extraction buffers [34][35][36][37][38]. Among the different techniques, the application of thermal energy via antigen retrieval and extraction buffers corrected for ionic strength differences were determined to be the key elements for efficient protein extraction [39][40][41][42]. Considering that FFPE tissues can provide the advantage of linking seeding behaviour with neuropathologically characterized brain regions and allow the investigation of larger cohorts, we aimed to establish a streamlined protein extraction protocol specific for the evaluation of αSyn seeding in FFPE human brain tissue. ...
Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.
... Carcinogenic and non-carcinogenic risk values in these occupations were in the range of 5.76 × 10 − 6 -6.43 × 10 − 3 and of 0.004-156, respectively. This was related with the fact that FA is widely used in pathology or histology departments in the autopsy rooms for sterilization purposes, as well as a preservative (formalin) or dehydrating agent during mixture preparation, tissue processing, and staining (Araújo et al., 2014;Corradi et al., 2012). Immersing the corpses in this substance causes that in addition to the exposure of the laboratory technician, the medical students, and their instructors also face the vapors released from the corpses during the dissection courses. ...
Formaldehyde is one of the most widely used substances in a variety of industries, although it was classified as a human carcinogen by the International Agency for Research on Cancer (IARC). The present systematic review was conducted to retrieve studies related to occupational exposure to formaldehyde until November 2, 2022. Aims of the study were to identify workplaces exposed to formaldehyde, to investigate the formaldehyde concentrations in various occupations and to evaluate carcinogenic and non-carcinogenic risks caused by respiratory exposure to this chemical among workers. A systematic search was done in Scopus, PubMed and Web of Science databases to find the studies done in this field. In this review, studies that did not meet the criteria specified by Population, Exposure, Comparator, and Outcomes (PECO) approach were excluded. In addition, the inclusion of studies dealing with the biological monitoring of FA in the body and review studies, conference articles, books, and letters to the editors were avoided. The quality of the selected studies was also evaluated using the Joanna Briggs Institute (JBI) checklist for analytic-cross-sectional studies. Finally, 828 studies were found, and after the investigations, 35 articles were included in this study. The results revealed that the highest formaldehyde concentrations were observed in waterpipe cafes (1,620,000 μg/m3) and anatomy and pathology laboratories (4237.5 μg/m3). Carcinogenic and non-carcinogenic risk indicated the potential health effects for employees due to respiratory exposure as acceptable levels of CR = 1.00 × 10-4 and HQ = 1, respectively were reported to be exceeded in more than 71% and 28.57% of the investigated studies. Therefore, according to the confirmation of formaldehyde's adverse health effects, it is necessary to adopt targeted strategies to reduce or eliminate exposure to this compound from the occupational usage.
... Different techniques have been reported to remove paraffin residues and reverse formaldehyde crosslinks using various protein extraction buffers [34][35][36][37][38]. Among the different techniques, the application of thermal energy via antigen 3 of 18 retrieval and extraction buffers corrected for ionic strength differences were determined to be the key elements for efficient protein extraction [39][40][41][42]. ...
Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen-retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behavior in FFPE human brain, with structural validation. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.
... However, chlorinated solvents are extremely toxic and harmful to the environment. More recently, alternative eco-friendly solvent microextraction methods using long carbon chain alcohols, ionic liquids, and supramolecular solvents have been preferred [30,31]. Interest in microextraction methods using deep eutectic solvents (DESs) has increased because they are more environmentally friendly, economic and more useful than conventional solvents [32][33][34][35]. ...
The parabens, which are harmful to our bodies, are primarily utilized as preservatives in medicine, personal care products and cosmetics. A novel, more efficient, fast and cheap vortex-assisted liquid phase microextraction method based on deep eutectic solvents (DESs) was developed for the determination of parabens. The microextraction conditions were optimized using these solvents and the analytical parameters of the method were determined under optimal microextraction conditions. After extraction, the chromatographic separation of parabens was undertaken using high-performance liquid chromatography-UV detection. Experimental parameters, such as DES type, DES volume, dilution solvent volume and vortex extraction time were optimized. DES6 [ChCl-Ethylene glycol (1/2)] was the most suitable DES to work in this study. Detection limits for this method of 0.053 µg mL⁻¹ for methylparaben, 0.061 µg mL⁻¹ for ethylparaben, 0.049 µg mL⁻¹ for propylparaben and 0.052 µg mL⁻¹ for butylparaben were obtained. Correlation coefficients (R²) for a concentration range of 0.1–100 µg mL⁻¹ were higher than 0.9992 and relative standard deviation (RSD) values below 2.91% at parabens concentration of 2.5 µg mL⁻¹ were obtained. The results of spike/recovery values of real samples were greater than 84%. When compared with other methods, the main advantages include lower LOD, short extraction time, rapidity, repeatability and simplicity.
... Data was collected by MS and MS/MS spectral acquisition for all fractions using a MALDI TOF/TOF mass spectrometer (Bruker Daltonics), fitted with a LIFT cell and a smart beam laser run at 2 kHz. Bruker flexAnalysis was used for spectral processing with protein identification performed with Proteinscape 3.0 via a MASCOT database search (SwissProt) for human tryptic peptides (modifications allowed for oxidation and phosphorylation of the peptides) having a peptide tolerance of 50 ppm (a ppm of 50 was used based on results from previous FFPE tissue studies [26,27]) and 2 missed cleavages. A minimum of b 1% false discovery rate was used for protein identification. ...
Biological significance:
This study is the first to perform an imaging mass spectrometry investigation in the human brainstem and also within sudden infant death syndrome (SIDS). LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue; with abnormal expression patterns present for 41/285 and 9/55 proteins in SIDS using IMS. The abnormal proteins are critical for neurological development; with the impairment supporting the hypothesis that SIDS may be due to delayed neurological maturation. The brainstem regions mostly affected included the raphe nuclei, hypoglossal and pyramids. This study highlights that basic cyto-architectural proteins are affected in SIDS and that abnormal expression of these proteins in other CNS disorders should be examined.
Key sentences:
LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue. Abnormal expression patterns were present for 41/285 and 9/55 proteins in SIDS using IMS. Brainstem regions mostly affected included the raphe nuclei, hypoglossal and pyramids.
Compared with conventional optical microscopy techniques, mass spectrometry imaging (MSI) or imaging mass spectrometry (IMS) is a powerful, label-free analytical technique, which can sensitively and simultaneously detect, quantify, and map hundreds of biomolecules, such as peptides, proteins, lipid, and other organic compounds in cells and tissues. So far, although several soft ionization techniques, such as desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS) have been used for imaging biomolecules, matrix-assisted laser desorption/ionization (MALDI) is still the most widespread MSI scanning method. Here, we aim to provide a comprehensive review of MALDI-MSI with an emphasis on its advances of the instrumentation, methods, application, and future directions in single cell and biological tissues.
In this review, quality by design (QbD) initiatives are described as applications for
many types of processes, such as in the pharmaceutical industry, biotechnology
field, and for analytical methods development. Design space (DS) and
design of experiments (DoE) provide useful results for analytical methods
development and simulation of advanced processes in traditional manufacturing
relationships. The three topics, QbD, DS and DoE are the best way to achieve
strong and efficient industrial production.
Aim
Report our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis.
Background
Nasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis.
Methods
FFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients’ received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan–Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated.
Results
28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein–Barr (EBV) and Herpes simplex (HSV) viruses’ related proteins significantly present in early-stage primary NPC (T1 and T2, p < 0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients (p < 0.01). Median survival from this M1 group was <1 year (p < 0.001).
Conclusions
FFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed.
A simple electrochemical sensor for dopamine was developed based on activated carbon (AC) as a modifier for carbon paste electrode (CPE). The proposed electrode (AC-CPE) was used to study the electrocatalytic oxidation of dopamine by cyclic voltammetry (CV), chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS). Firstly, the electrochemical behaviour of the modified electrode demonstrated that the substantial of the cathodic and anodic peak potentials were positively and negatively shifted, respectively; accompanied by an enhancement of current peak of DA. Then, the diffusion coefficient (D = 1.01 × 10⁻⁵ cm² s⁻¹) and the kinetic parameters such as the electron transfer coefficient (α = 0.48), apparent electron transfer rate constant (ks = 0.80 s⁻¹) and catalytic reaction rate constant (kcat = 40.90 × 10⁴ mol L⁻¹ s⁻¹) for dopamine at the surface of the AC-CPE were determined using electrochemical approaches. The AC−CPE showed favourable electron transfer kinetics and electro-catalytic activity towards oxidation/reduction of the dopamine. Differential pulse voltammetry (DPV) was used successfully for the determination of DA in the linear response range from 1.0 × 10⁻⁷ to 1.0 × 10⁻³ mol L⁻¹ with a detection limit of 3.09 × 10⁻⁸ mol L⁻¹. The AC−CPE offered high electro-catalytic activity in dopamine sensing individually and simultaneously in the presence of hydroquinone, catechol, paracetamol and phenol. These results demonstrated that the AC−CPE exhibited an excellent anti-interference property by peaks separation. The practical analytical utility of the AC−CPE is illustrated by dopamine determination in blood samples.
An effective three step proteomic workflow is proposed to overcome the pitfalls caused by polymers present in OCT-embedded tissue during preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water, in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min ultrasonic duty time). As proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCC) and renal onco-cytomas (RO). A successful cluster of proteomic profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recov-ery from OCT samples.
The drugs are emerging pollutants with great evidence in several scientific studies directed to the removal and separation of these contaminants from aquatic environments. The present study provides an efficient methodology for the determination of the topiramate in aqueous solutions by salting-out assisted liquid-liquid extraction-gas chromatography coupled to mass spectrometry, obtaining optimum recovery efficiency of topiramate, 99.0%. The methodology validation showed linearity with detection limits of 10.23 mg/L and quantification of 30.99 mg/L. The application of the method was founded in monitoring of adsorption assay of drug in perlita expandida, that has been demonstrated efficient (34.0% adsorption removal), since topiramate is a potential micropollutants of aqueous environment.
In livestock and poultry, broad-spectrum antibiotics such as tetracyclines and fluoroquinolones are widely used; thus, controlling food productions made from these animals is a necessary task. Meat may contain residues of antibiotics, even in low concentrations, which can cause a selection pressure for antibiotic resistance. Therefore, measurement of amounts of antibiotics in meat is of major importance. A total of 41 beef and 41 chicken meat samples were collected for 1 year. Tetracycline and ciprofloxacin were extracted from samples and tested by an indirect competitive enzyme-linked immunosorbent assay. Overall, 100% of the beef and more than 95% of the chicken meat samples were positive for ciprofloxacin. Only one of the chicken meat samples had concentrations of ciprofloxacin higher than maximum residue limit (MRL). For ciprofloxacin, none of the beef meat samples exceeded the MRL. For tetracycline, 75% of the beef and 58% of the chicken meat samples were positive. All of the samples had concentrations of tetracycline lower than MRL. It was revealed that the chicken meat samples had higher levels of both antibiotics than those of beef samples. The amounts of tested antibiotics were not high in the meat samples, consequently using of beef and chicken meat by consumers in Iran is not resulted in entrance of high amounts of the antibiotics into human body.
Burn injury inevitably leads to changes in the endogenous production of cytokines, as well as adrenal and gonadal steroids. Previous studies have reported gender-related differences in outcome following burn injury, which suggests that gonadal steroids may play a role. The aim of this study was to assess alterations in concentration of endogenous steroids in patients with burn injury.
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.
As a review for the 20th anniversary of publishing the antigen retrieval (AR) technique in this journal, the authors intend briefly to summarize developments in AR-immunohistochemistry (IHC)-based research and diagnostics, with particular emphasis on current challenges and future research directions. Over the past 20 years, the efforts of many different investigators have coalesced in extending the AR approach to all areas of anatomic pathology diagnosis and research and further have led to AR-based protein extraction techniques and tissue-based proteomics. As a result, formalin-fixed paraffin-embedded (FFPE) archival tissue collections are now seen as a literal treasure of materials for clinical and translational research to an extent unimaginable just two decades ago. Further research in AR-IHC is likely to focus on tissue proteomics, developing a more efficient protocol for protein extraction from FFPE tissue based on the AR principle, and combining the proteomics approach with AR-IHC to establish a practical, sophisticated platform for identifying and using biomarkers in personalized medicine.
Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.
In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.
These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for 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 microg of protein yielded approximately 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 approximately 17% after one year of storage to approximately 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.
To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies.
Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry.
MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues.
Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.
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.
A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.
Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.
High-throughput proteomic studies of archival formalin-fixed paraffin-embedded (FFPE) tissues have the potential to be a powerful tool for examining the clinical course of disease. However, advances in FFPE tissue-based proteomics have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications in proteins. To help address these problems, we have developed a procedure for the formation of 'tissue surrogates' to model FFPE tissues. Cytoplasmic proteins, such as lysozyme or ribonuclease A, at concentrations approaching the protein content in whole cells, are fixed with 10% formalin to form gelatin-like plugs. These plugs have sufficient physical integrity to be processed through graded alcohols, xylene, and embedded in paraffin according to standard histological procedures. In this study, we used tissue surrogates formed from one or two proteins to evaluate extraction protocols for their ability to quantitatively extract proteins from the surrogates. Optimal protein extraction was obtained using a combination of heat, a detergent, and a protein denaturant. The addition of a reducing agent did not improve protein recovery; however, recovery varied significantly with pH. Protein extraction of >80% was observed for pH 4 buffers containing 2% (w/v) sodium dodecyl sulfate (SDS) when heated at 100 degrees C for 20 min, followed by incubation at 60 degrees C for 2 h. SDS-polyacrylamide gel electrophoresis of the extracted proteins revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins, regardless of the extraction protocol employed. Additionally, protein extracts from surrogates containing carbonic anhydrase:lysozyme (1:2 mol/mol) had disproportionate percentages of lysozyme, indicating that selective protein extraction in complex multiprotein systems may be a concern in proteomic studies of FFPE tissues.
Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable informational resource of histologically characterized specimens for proteomic studies. In this article, the authors review the advancement performed in the field of FFPE proteomics focusing on formaldehyde treatment and on strategies addressed to obtain the best recovery in the protein/peptide extraction. A variety of approaches have been used to characterize protein tissue extracts, and many efforts have been performed demonstrating the comparability between fresh/frozen and FFPE proteomes. Finally, the authors report and discuss the large numbers of works aimed at developing new strategies and sophisticated platforms in the analysis of FFPE samples to validate known potential biomarkers and to discover new ones.
A simple and rapid method is described for the quantitative extraction and determination of Cd and Pb in mussel tissue (Mytilus edulis). The method is based on the quantitative ultrasound-assisted extraction (i.e. sample mass at mg level) of the two metals
using diluted nitric acid as extractant. The extraction procedure is carried out in autosampler cups of the graphite furnace
(typically, less than 20 mg). A two-level full factorial design (24) was applied to optimize the variables influencing the ultrasound extraction process. These variables were: extraction time,
ultrasound amplitude, nitric acid concentration and particle size. Optimization results showed that acid concentration and
particle size were the more significant variables. Determination of Cd and Pb in extracts obtained after ultrasound treatment
was carried out by Electrothermal Atomic Absorption Spectrometry. The method was validated by statistically comparing the
metal contents found with the certified ones corresponding to the BCR 278 mussel tissue. No significant differences were observed
for P = 0.05. LODs for Cd and Pb in mussel tissue were 0.019 and 0.37 μg g–1. RSDs values (corresponding to between-batch precision for n = 5) were 2.2 and 6.7% for Cd and Pb, respectively. The method was applied to measure the contents of Cd and Pb in mussels
used as pollution bioindicators from the Galician coast (Ria de Vigo, Spain).
Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.
Formalin fixation, followed by paraffin embedding, is long established as the standard procedure for the stabilization and preservation of tissue architecture, essential for enabling microscopic examination and long-term storage of samples. During the years, this has led to the generation of a worldwide repository of patient tissues with associated complete clinical records. As such, this represents a golden mine for all those attempting to identify proteomic signatures of disease, aimed to the understanding of pathological processes and to the identification of new biomarkers. However, access to this resource has been hampered by the stable cross-linked network generated on tissue molecules during formalin fixation. Recently, researchers have been actively working to overcome this limitation, reaching unexpected achievements. This review aims to discuss and compare the various strategies devised for extracting full-length proteins or peptides from fixed tissues, and to provide a general perspective on studies comparing matched fixed and fresh-frozen tissue proteomes, applying proteomic techniques for biomarker discovery from archival tissues, and attempting to exploit gel-based approaches. In addition, the concomitant progresses in understanding the impact of tissue processing variables and the extent and nature of formaldehyde-induced modifications are presented. In conclusion, the future perspectives and open challenges in this field are discussed.
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.
Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time-the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications.
Formalin-fixed, paraffin-embedded (FFPE) tissues have been used to discover disease-associated protein changes using mass spectrometry. Protein post-translational modifications such as glycosylation are known to associate with disease development. In this study, we investigated whether FFPE tissues preserve such modifications and therefore can be used as specimen of choice to identify the disease-associated modifications. We isolated the glycopeptides from the tryptic digest of frozen and FFPE lung tissues using solid-phase extraction of glycopeptides and analyzed them using mass spectrometry. The glycopeptides identified from FFPE lung tissue were compared to the ones from frozen lung tissue regarding their relative abundance, unique glycosylation sites, and subcellular locations. The results from our study confirmed that glycosylation in FFPE tissues are preserved and FFPE tissues can be used for discovery of new disease associated changes in protein modifications. Furthermore, we demonstrated the feasibility of applying the strategy of glycopeptide isolation from tryptic peptides of FFPE tissue to other tissues such as liver and heart.
The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of cross-link reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, full-length, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study.
Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients with PBC. Only one of 22 normals (5%), one of 20 with collagen-vascular diseases (5%), and one of seven with nonimmunologic liver disease (14%) were positive for this hepatocyte membrane antibody. Bouin's fixation prior to IF is a simple technique which appears to alter the hepatocyte membrane so that HMA become detectable. The strong association of HMA with CAH and PBC suggests that this test might be of value and may contribute towards a further understanding of the pathogenesis of these conditions.
Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.
Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.
- S R Shi
- Y Shi
- C R Taylor
S.R. Shi, Y. Shi, C.R. Taylor, J. Histochem. Cytochem. 59 (2011) 13–32.
- L Giusti
- A Lucacchuni
L. Giusti, A. Lucacchuni, Expert Rev. Proteomics 10 (2013) 165–177.
Estadística para investigadores, Reverté
- Gep Box
- Wg Hunter
- Hunter
GEP Box, JS Hunter, WG. Hunter, Estadística para investigadores, Reverté, 1st
ed., Barcelona, 1989, pp. 299–384.
- N J Nirmalan
- C Hughes
- J Peng
- T Mckenna
- J Lanridge
- D A Cairns
- P Harnden
- P J Selby
- R E Banks
N.J. Nirmalan, C. Hughes, J. Peng, T. McKenna, J. Lanridge, D.A. Cairns,
P. Harnden, P.J. Selby, R.E. Banks, J. Proteome Res. 10 (2011) 896–906.
- Y Tian
- K Gurley
- D L Meany
- C J Kemp
- H Zhang
Y. Tian, K. Gurley, D.L. Meany, C.J. Kemp, H. Zhang, J. Proteome Res. 8 (2009)
1657–1662.
- C B Fowler
- R E Cunningham
- T O Leary
- J T Mason
C.B. Fowler, R.E. Cunningham, T. O'Leary, J.T. Mason, Lab Invest. 87 (2007)
836–846.
- C B Fowler
- T J Wauvright
- T D Veenstra
- T J O 'leary
- J T Mason
C.B. Fowler, T.J. Wauvright, T.D. Veenstra, T.J. O'Leary, J.T. Mason, J. Proteome
Res. 11 (2012) 2602–2608.
- S Magdeldin
- T Yamamoto
S. Magdeldin, T. Yamamoto, Proteomics 12 (2012) 1045–1058.
- H Xu
- L Yang
- W Wang
- S.-R Shi
- C Liu
- Y Liu
- X Fang
- C R Taylor
- C S Lee
- B M Balgley
H. Xu, L. Yang, W. Wang, S.-R. Shi, C. Liu, Y. Liu, X. Fang, C.R. Taylor, C.S. Lee,
B.M. Balgley, J. Proteome Res. 7 (2008) 1098–1108.
- P Ostasiewicz
- D F Zielinska
- M Mann
- J R Wisniewski
P. Ostasiewicz, D.F. Zielinska, M. Mann, J.R. Wisniewski, J. Proteome Res. 9
(2010) 3688–3700.
On the right are represented the number of proteins identified for each condition. Samples were separated by 1D SDS-PAGE and the gel-lanes were excised as shown in Fig. 5, and submitted to in-gel digestion. Then samples were analysed by
Fig. 6. Venn diagram comparing the proteins identified in each of the four conditions tested (#13–#16). On the right are represented the number of proteins identified for
each condition. Samples were separated by 1D SDS-PAGE and the gel-lanes were excised as shown in Fig. 5, and submitted to in-gel digestion. Then samples were analysed
by MALDI-TOF/TOF MS.
[17] T. Guo, W. Wang, P.A. Rudnick, T. Song, Z. Zhuang, R.J. Weil, D.L. DeVoe, C.S. Lee,
B.M. Balgley, J. Histochem. Cytochem. 55 (2007) 763–772.
- C B Fowler
- I E Chesnick
- C D Moore
- T O Leary
- J T Mason
- S R Shi
- C Liu
- B M Balgley
- C Lee
- C R Taylor
C.B. Fowler, I.E. Chesnick, C.D. Moore, T. O'Leary, J.T. Mason, PLoS One 5 (2010)
e14253.
[20] S.R. Shi, C. Liu, B.M. Balgley, C. Lee, C.R. Taylor, J. Histochem. Cytochem. 54
(2006) 739–743.
- A Tanca
- D Pagnozzi
- M F Addis
A. Tanca, D. Pagnozzi, M.F. Addis, Proteomics Clin. Appl. 6 (2012) 7–21.
- R W Sprung
- J W C Brock
- J P Tanksley
- M Li
- M K Washington
- R J C Slebos
- D C Liebler
R.W. Sprung, J.W.C. Brock, J.P. Tanksley, M. Li, M.K. Washington, R.J.C. Slebos,
D.C. Liebler, Mol. Cell Proteomics 8 (2009) 1988–1998.