Formaldehyde fixation.

Fox, Cecil H.; Johnson, F B; Whiting, J; Roller, P P

doi:10.1177/33.8.3894502

Cited by 975 publications

(798 citation statements)

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“…The stable isotope labeling techniques developed in the last two decades for relative quantitation experiments space from in vivo metabolic incorporation of heavy isotopes, or isotopically to fix tissues and cells and it is known as a cross-linking agent involving a wide set of amino acid residues [14,15]. Metz et al reported a systematic investigation on a series of synthetic peptides in the presence of glycine or NaCNBH 3 [16,17].…”

Section: Introductionmentioning

confidence: 99%

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

et al. 2014

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Research Article pH-regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experimentsAmong the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH 3 features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde could be employed as well without a substantial increase in terms of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless, only a single paper reports about a diethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation. Keywords:Diethylation / Dimethylation / Quantitative analysis / Reductive amination / Stable isotope labeling DOI 10.1002/elps.201300484Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionOne of the most crucial issues and growing areas of modern proteomics is the relative quantification of the differential expression of proteins in two or more samples representing various conditions of biological systems. The stable isotope labeling techniques developed in the last two decades for relative quantitation experiments space from in vivo metabolic incorporation of heavy isotopes, or isotopically [3][4][5][6]. Many recent reviews give a comprehensive picture of the current tendency for relative quantitative proteomics with stable isotope labeling [7][8][9][10][11].Reductive amination with formaldehyde and NaCNBH 3 is a simple reaction that involves all the free amino groups of peptides, namely N-termini and lysine residues, replacing hydrogens with two methyl groups [12]. A differential isotope labeling can be achieved by employing d(0)-formaldehyde and d(2)-formaldehyde obtaining a mass increment of 28 and 32 Da, respectively, for each derivatized reactive site. Acetaldehyde and d(4)-acetaldehyde are still relatively inexpensive reagents and can be used as diethylating agents, by the same approach, providing a wider separation, in terms of mass units, between the differentially labeled peptides [13].The chemistry of both formaldehyde and acetaldehyde in biological systems features interesting aspects for proteomics investigations. Formaldehyd...

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Section: Introductionmentioning

confidence: 99%

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

et al. 2014

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“…A major advantage afforded by formalin-fixed paraffin-embedded (FFPE) specimens is the preservation of cellular and architectural morphologic detail in tissue sections. 1 The standard buffered formalin fixative in which biopsy specimens are processed is an aqueous solution containing 37% formaldehyde and 10-15% methyl alcohol. Formaldehyde is a highly reactive dipolar compound that results in the formation of protein-nucleic acid and proteinprotein crosslinks in vitro.…”

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confidence: 99%

Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS

Crockett

Lin

Vaughn

et al. 2005

Laboratory Investigation

142

140

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There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC g) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research. Laboratory Investigation ( Keywords: bottom-up proteomics; formalin fixed; paraffin embedded; tandem mass spectrometry Formalin fixation and tissue embedding in paraffin wax is a universal approach for tissue processing prior to light microscopic evaluation. A major advantage afforded by formalin-fixed paraffin-embedded (FFPE) specimens is the preservation of cellular and architectural morphologic detail in tissue sections. 1 The standard buffered formalin fixative in which biopsy specimens are processed is an aqueous solution containing 37% formaldehyde and 10-15% methyl alcohol. Formaldehyde is a highly reactive dipolar compound that results in the formation of protein-nucleic acid and proteinprotein crosslinks in vitro. [1][2][3][4][5] FFPE specimens have not been routinely used for global mass spectrometry-based proteomic studies. This relates to the fact that formaldehyde-induced crosslinking also renders proteins relatively insoluble and unsuitable for routine biochemical extraction and analysis. This crosslinking effect has precluded protein extraction from FFPE for routine Western blotting analysis of protein expression. 2 Thus, while recently developed enzymatic and heatinduced antigen retrieval methods have facilitated the immunohistologic detection of proteins in tissue, many proteins are still not detectable using these approaches. 6,7 In addition, proteins lacking widely available antibodies to their formalin-resistant epitopes are largely undetectable by immunohistochemistry. Although recent advances in molecular biotechnology have permitted the global analysis of proteins expressed in various cellular and tissue systems, the diminished capacity to extract intact proteins has largely precluded the proteomic analysis of FFPE samples with definitive identification of the proteins present within cells and tissues in biopsy material.The development of strategies to permit utilization of the universa...

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“…This finding is consistent with previous reports that formalin does not change the dimensions in human coronary arteries 3 or shrink most tissues. 4 Decalcification, irrespective of the amount of calcium removed, resulted in shrinkage of 5-10%. Since lumens, lesion widths, and reference distances were measured only to the nearest 0.5 mm, however, adding 10% to each of these measurements would not change the data enough to affect our interpretation.…”

Section: Discussionmentioning

confidence: 99%

Multicenter validation study of real-time ultrasonography, arteriography, and pathology: pathologic evaluation of carotid endarterectomy specimens.

Schenk

Bond

Aretz

et al. 1988

Stroke

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The morphologic description and measurements of endarterectomy specimens are usually believed to be accurate and are used as the gold standard against which the findings of diagnostic procedures are judged. Pathology data on 289 endarterectomy specimens from five participating centers and the corresponding angiography and B-mode ultrasonography data provided a basis for scrutinizing the validity of using the morphologic measurements as a standard. Discrepancies of > 1 mm between pathology and angiography measurements of minimum residual lumen occurred in 35% of the cases and between pathology and B-mode ultrasonography measurements in 64% of the cases. Discrepancies of > 1 mm between pathology-and angiography-measured lesion width occurred in 81% of the cases and between pathology and B-mode ultrasonography measurements in 64% of the cases. The cases representing mismatches of > 1 mm at one participating center were subjected to a rigorous review, with remeasurement of all morphologic features, in an attempt to explain the discrepancies. Various types of artifactual distortion of the specimens, the presence of slit-like and occluded lumens that were likely related to loss of perfusion pressure, and an inability to match planes of interrogation used in angiography and B-mode ultrasonography with pathology planes contributed significantly to the existence of mismatches. On the other hand, fixation and decalcifkation produced minimal and insignificant distortional changes. We conclude that the acquisition of quantitative data from endarterectomy specimens and the acceptance of morphologic data as a standard are limited by a number of problems that can be defined but have been difficult to resolve. (Stroke 1988; 19:289-296) T he morphologic description and measurements of endarterectomy specimens are usually believed to be accurate and are used as the gold standard against which the findings of diagnostic procedures are compared to assess their validity. Such was the approach used in a multicenter National Institutes of Health-spon- Supported by National Heart, Lung, and Blood Institute contracts N01- HV-129I6, 12904, 12912, 12913, 12914, 12915, and 12917. Address for correspondence: Eric A. Schenk, MD, Department of Pathology, University of Rochester Medical Center, Rochester, NY 14642.Received March 13, 1987; accepted October 6, 1987. sored study to evaluate B-mode ultrasonographic imaging (B-scan) of the carotid arteries. Five clinical centers and one data coordinating center collaborated in this study.The main purpose of the study was to assess the accuracy with which B-scan describes lumen diameter, plaque thickness, and percent stenosis within 1.5 cm of the carotid artery bifurcation. The B-scan findings were compared with those obtained by both conventional and digital angiography and by pathologic examination of the endarterectomy specimen. Details about the overall design, the methods for ultrasonography and angiography, and the findings have been published.'-2 This communication focuses on the patholo...

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Formaldehyde fixation.

Cited by 975 publications

References 0 publications

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS

Multicenter validation study of real-time ultrasonography, arteriography, and pathology: pathologic evaluation of carotid endarterectomy specimens.

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