共查询到20条相似文献,搜索用时 15 毫秒
1.
Yusuke Kawashima Yoshio Kodera Anil Singh Masaomi Matsumoto Hiroyuki Matsumoto 《Clinical proteomics》2014,11(1):4
Background
Numerous formaldehyde-fixed and paraffin-embedded clinical tissues have been created in the past decades and stored in pathological depositories at hospitals as well as in clinical laboratories worldwide. In addition to the archived tissues, formaldehyde-fixation is also mandatory for preparing proteomics samples from diseased patients or animal models in order to inactivate contagious agents. Protein extraction from formaldehyde-fixed tissues is hampered by the Schiff base formation between the amino groups of proteins and formaldehyde. Although achievement of the highest extraction efficiency of proteins from the formaldehyde-fixed tissues is essential for obtaining maximum proteomics information, no attention has been paid to the concentration dependence of tris(hydroxymethyl)aminomethane on the extraction efficacy. We suspected that the concentration of tris(hydroxymethyl)aminomethane affects the protein extraction efficiency because of its property as a primary amine that reverses the Schiff base formation between the primary amines of proteins and formaldehyde. Thus we pursued optimization of the component and protocol of protein extraction buffer to achieve better extraction efficiency of proteins from formaldehyde-fixed and paraffin-embedded tissues.Results
In order to simulate protein extraction from diseased tissues we made formaldehyde-fixed and paraffin-embedded samples from mouse liver slices and investigated the protein extraction efficiency and speed by changing the concentration of the protein extraction buffer component tris(hydroxymethyl)aminomethane under various extraction conditions. We find, as expected, that tris(hydroxymethyl)aminomethane significantly affects the performance of protein extraction from the formaldehyde-fixed and paraffin-embedded samples both in the extraction yield and in the extraction speed.Conclusions
We recommend the concentration of tris(hydroxymethyl)aminomethane in protein extraction buffer to be higher than 300 mM when extraction is conducted for 90 min at 90°C to achieve the most efficient protein extraction in a shorter time. The information will be essential for performing the most efficient protein extraction from formaldehyde-fixed and paraffin-embedded tissue samples for proteomics analysis. 相似文献2.
A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA. 相似文献
3.
Because fresh‐frozen tissue samples associated with long‐term clinical data and of rare diseases are often unobtainable at the present time, formalin‐fixed paraffin‐embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross‐link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97–99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high‐yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic. 相似文献
4.
Alessandro Tanca Marcello Abbondio Salvatore Pisanu Daniela Pagnozzi Sergio Uzzau Maria Filippa Addis 《Clinical proteomics》2014,11(1):28
Background
The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.Experimental design
DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.Results
DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.Conclusions
These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth. 相似文献5.
John B.A. Okello Jaymi Zurek Melanie Kuch Nelson K. Sewankambo Debi Poinar 《Analytical biochemistry》2010,400(1):110-117
Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13 years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen’s WaxFree method and our in-house phenol-chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion’s RecoverAll method recovered the most amplifiable RNA. 相似文献
6.
Background
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control.Results
A total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (RSC) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc.Conclusions
Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein–protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10−45), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10−5). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis.Electronic supplementary material
The online version of this article (doi:10.1186/s12014-015-9091-8) contains supplementary material, which is available to authorized users. 相似文献7.
DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution 总被引:6,自引:1,他引:5
Shi SR Datar R Liu C Wu L Zhang Z Cote RJ Taylor CR 《Histochemistry and cell biology》2004,122(3):211-218
Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique. 相似文献
8.
Izaskun Ibarguren 《Journal of biological education》2017,51(3):284-294
All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to reinforce the following concept: what makes cells different is they make different proteins. The students performed a denaturing polyacrylamide gel electrophoresis (SDS–PAGE) to separate the proteins extracted from different animal tissues, and then obtained and compared the corresponding protein profiles. In addition, the students performed a Western blot analysis to detect the presence or absence of a tissue-specific protein in the different tissue extracts. This laboratory exercise allowed students to understand the basis of the cell differentiation better and also provided them the opportunity to learn a variety of analytical laboratory techniques. 相似文献
9.
Elena A Takano Thomas Mikeska Alexander Dobrovic David J Byrne Stephen B Fox 《BMC biotechnology》2010,10(1):1-11
Background
mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease.Results
We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7).Conclusions
Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well. 相似文献10.
Keiichi Hatakeyama Kanako Wakabayashi-Nakao Yutaka Aoki Shun-ichiro Ogura Ken Yamaguchi Takashi Nakajima Taka-Aki Sato Tohru Mochizuki Isamu Hayashi 《Proteome science》2012,10(1):1-7
We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR. 相似文献
11.
12.
Allred CC Krennmayr T Koutsari C Zhou L Ali AH Jensen MD 《Journal of lipid research》2011,52(2):408-415
CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport. Western blotting is the most widely used method to measure tissue CD36 protein content, but it is time consuming, technically demanding, and semiquantitative. To more precisely measure adipose tissue CD36 content we developed an enzyme linked immunosorbent assay (ELISA) after establishing that: 1) the anti-CD36 antibodies gave a single distinct band on traditional Western blots, and 2) the vast majority of adipocyte CD36 resides in the plasma membrane. By using serial dilutions of each sample and including a calibrator sample and quality control sample on each plate, we could achieve inter- and intra-assay variability of ~ 10%. We found that CD36 content in omental and abdominal subcutaneous adipose tissue varied over a 2-5-fold range depending upon the means of data expression (per units of tissue protein, weight, or lipid). Omental CD36 content in women decreased markedly (P = 0.01) as a function of fat cell size. For the most part, tissue CD36 content was not correlated with CD36 mRNA. This ELISA method for tissue CD36 content should enhance research into the role of this protein on tissue fatty acid uptake. 相似文献
13.
C H Sissons E M Hancock H E Perinpanayagam T W Cutress 《The Journal of applied bacteriology》1989,67(4):433-440
Staphylococcal cell protein and urease can be solubilized after growth in Todd-Hewitt broth supplemented with 0.5% yeast extract by extraction for 18-24 h in phosphate buffer, pH 7.0. In general 20% (but up to 100%) of the urease present in the original cells could be solubilized. Less protein was solubilized. Species examined included coagulase-negative staphylococci, Staphylococcus intermedius and Staph. aureus. Extracts of Staph. epidermidis prepared by this procedure gave electrophoretic urease and protein patterns similar to those prepared by sonication. The procedure was simple and minimized handling of the cells. 相似文献
14.
C.H. Sissons E.M. Hancock H.E.R. Perinpanayagam T.W. Cutress 《Journal of applied microbiology》1989,67(4):433-440
Staphylococcal cell protein and urease can be solubilized after growth in Todd-Hewitt broth supplemented with 0.5% yeast extract by extraction for 18–24 h in phosphate buffer, pH 7.0. In general 20% (but up to 100%) of the urease present in the original cells could be solubilized. Less protein was solubilized. Species examined included coagulase-negative staphylococci, Staphylococcus intermedius and Staph. aureus. Extracts of Staph, epidermidis prepared by this procedure gave electrophoretic urease and protein patterns similar to those prepared by sonication. The procedure was simple and minimized handling of the cells. 相似文献
15.
An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues 总被引:33,自引:0,他引:33
The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1–2 nM okadaic acid. In contrast, type 1 protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 μM okadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts. 相似文献
16.
Gowda S Shrivastav A Selvakumar P Dimmock JR Sharma RK 《Biochemical and biophysical research communications》2004,314(4):984-987
Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT. 相似文献
17.
The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns. 相似文献
18.
Lenne Nigul Allan Olspert Merike Meier Heiti Paves Tiit Talpsep Erkki Truve 《Plant Molecular Biology Reporter》2004,22(4):399-407
E2Tag has been used for tagging of bacterial, yeast, and mammalian proteins. A set of plant expression vectors was constructed
for tagging proteins with E2Tag. Detection of E2-tagged proteins with specific antibodies inNicotiana benthamiana andNicotiana tabacum was easily done without any nonspecific background activity. No effect on the biological activity of the enzyme GUS, used
as a marker, was detected. The subcellular localization of E2-tagged plant protein AtRLI2 with yet unknown functions was described. 相似文献
19.
Julia V. Gefter Angel L. Shaufl Mitchell P. Fink Russell L. Delude 《Cell and tissue research》2009,336(1):79-89
The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms.
Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms
expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE
mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three
distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms
with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa
(αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas
aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from
RAGE−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain
two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous
RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed
solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct
RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking
the H-300 epitope.
This work was supported by NIH grants R01 GM37631 and GM68481. 相似文献
20.
The effect of pure human insulin-like growth factor I (IGF I) on the colony formation of late stage erythroid precursor cells (CFU-e) from fetal mouse liver and adult bone marrow was studied in a serum-free culture system. We found that IGF I in physiological concentrations stimulated erythroid colony formation. The combined effect of IGF I and erythropoietin was smaller than the sum of their single effects. The number of colonies induced by IGF I was linearly dependent on the number of plated cells. Our results indicate that IGF I is the first clearly defined mitogen that stimulates the late stages of erythroid differentiation independently of erythropoietin. 相似文献