⿿Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples

Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona; Gaihede, Michael; Birkelund, Svend; Andersen, Vibeke; Stensballe, Allan

doi:10.1016/j.euprot.2015.10.001

Cited by 45 publications

(48 citation statements)

References 47 publications

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“…While RNAlater is ineffective in the preservation of fine anatomical and structural features [1], it has been shown to be effective in the preservation of nucleic acids [1,3,4]. RNAlater has been used to successfully preserve high quality DNA for up to 7 years from the date of sample collection and fixation in chimpanzee fecal material collected from remote habitats [1].…”

Section: Introductionmentioning

confidence: 99%

Transcriptome and proteome responses in RNAlater preserved tissue of Arabidopsis thaliana

et al. 2017

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Tissue preservation is a minimal requirement for the success of plant RNA and protein expression studies. The standard of snap-freezing in liquid nitrogen is not always practical or possible. RNAlater, a concentrated solution of ammonium and cesium sulfates, has become a standard preservative in the absence of liquid nitrogen. Here, we demonstrate the effectiveness of RNAlater in preserving both RNA and proteins in Arabidopsis thaliana tissues for use in RNAseq and LC-MS/MS analysis of proteins. While successful in preserving plant material, a transcriptomic and proteomic response is evident. Specifically, 5770 gene transcripts, 84 soluble proteins, and 120 membrane-bound proteins were found to be differentially expressed at a log-fold change of ±1 (P ≤ 0.05). This response is mirrored in the abundance of post-translational modifications, with 23 of the 108 (21.3%) phosphorylated proteins showing altered abundance at a log-fold change of ±1 (P ≤ 0.05). While RNAlater is effective in preserving biological information, our findings warrant caution in its use for transcriptomic and proteomic experiments.

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Section: Introductionmentioning

confidence: 99%

Transcriptome and proteome responses in RNAlater preserved tissue of Arabidopsis thaliana

et al. 2017

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“…Tissue specimens need to be properly handled to ensure not only the integrity of the morphological structure but also to avoid protein degradation. [2] Several protocols have been developed for this purpose, such as chemical fixation followed by paraffin wax embedding and snap freezing. [4] Heat-fixation of the tissue is also used, but changes in the fine structures of the cells has been observed.…”

Section: Resultsmentioning

confidence: 99%

“…In clinical application and molecular pathology, MALDI–MS imaging (MALDI–MSI) is an emerging technology that enables the spatial distribution of biomolecules within tissue to be combined with the traditional morphological information . Hence, for diagnostic or prognostic purposes, along with predicting response to therapeutic treatment, biological specimens, such as tissues and biopsies, must be properly collected and handled in order to assure the preservation of the morphological structure and the proteomic profile, avoiding degradation or alteration phenomena, contamination, and artifacts formation . So far, several attempts have been aimed at preventing sample degradation .…”

Section: Introductionmentioning

confidence: 99%

“…[1] Hence, for diagnostic or prognostic purposes, along with predicting response to therapeutic treatment, biological specimens, such as tissues and biopsies, must be properly collected and handled in order to assure the preservation of the morphological structure and the proteomic profile, avoiding degradation or alteration phenomena, contamination, and artifacts formation. [2,3] So far, several attempts have been aimed at preventing sample degradation. [4] Two of the most commonly used approaches for sample preservation and stabilization in pathology and proteomics are chemical fixation (e.g., formalin followed by paraffin wax embedding) [5,6] and snap freezing.…”

Section: Introductionmentioning

confidence: 99%

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Feasibility Study for the MALDI‐MSI Analysis of Thyroid Fine Needle Aspiration Biopsies: Evaluating the Morphological and Proteomic Stability Over Time

Piga

Capitoli

Tettamanti

et al. 2018

Proteomics Clinical Apps

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Purpose MALDI–MS imaging (MALDI–MSI) is an emerging technology that enables the spatial distribution of biomolecules within tissue to be combined with the traditional morphological information familiar to clinicians. Thus, for diagnostic or prognostic purposes, along with predicting response to therapeutic treatment, it is important to properly collect and handle biological specimens in order to avoid degradation or the formation of artifacts in the morphological structure and proteomic profile. Experimental design In this work, the morphological and proteomic stability of thyroid fine needle aspiration biopsies in PreservCyt (up to 14 days) and CytoLyt (up to 7 days) solutions at 4 °C has been verified, by MALDI–MSI analysis. Moreover, a new measure has been introduced in order to assess the similarity of the obtained MALDI–MSI spectra, by equally taking into account the number of signals (fit and retrofit), and their intensities (Spearman's correlation and spectra overlap). Results Results show no degradation of the cellular morphology and a good stability of the samples up to 14 days in PreservCyt solution. Conclusions and clinical relevance Moreover, this protocol can be easily implemented in pathological units, allowing simple sample collection and shipment to be used not only for the proteomic MALDI–MSI analysis of thyroid FNABs but also for other biological liquid based specimens.

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“…2 FFPE tissues are immediately fixed in 10% neutral buffered formalin solution (4% formaldehyde in phosphate-buffered saline (PBS), pH 7.2) after surgical resection to inhibit the enzyme and cell activity, and maintain the native state of tissues and cells, followed by dehydration and embedment of the sample in paraffin blocks. 3 Formaldehyde was reported to produce protein-protein and proteinnucleic acid crosslinking, 4 which resulted in hidden epitopes. 5 Three types of formaldehyde-induced chemical modifications were previously identified in proteins: (a) methylol (hydroxymethyl) adducts, (b) Schiff's bases, and (c) stable methylene bridges (cross-links).…”

Section: Introductionmentioning

confidence: 99%

LysargiNase enhances protein identification on the basis of trypsin on formalin‐fixed paraffin‐embedded samples

Liu

Yin

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Formalin‐Fixed Paraffin‐Embedded (FFPE) samples are valuable for proteomic studies of disease. However, the crosslink among proteins, protein vs nucleic acid, and other covalent chemical modifications like methylation introduced by formaldehyde can interfere with trypsin digestion in proteomics studies. LysargiNase was reported to have a better full‐cleavage rate at methylation and b ion coverage than trypsin. The contribution of LysargiNase in the proteomic study of FFPE samples was assessed and compared with trypsin in this study for the first time to facilitate proteomic research on FFPE samples. Methods The FFPE proteins were extracted with an “antigen retrieval” method. Digestion parameters were optimized by visualization of the digests on the tricine gel by silver staining. Then the FFPE proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and cut into 16 gel bands and in‐gel digested by trypsin and LysargiNase, respectively. Peptides were desalted with Stage‐Tips and separated via liquid chromatography. Electrospray ionization was conducted and peptide mass was measured in the LTQ Orbitrap Velos in the data‐dependent mode. Results High concentrations of enzyme facilitate the digestion efficiency of FFPE samples. A total of 32,294 peptides and 3445 proteins were identified with LysargiNase and trypsin combined in two replicates. LysargiNase increased peptide identification by 18.9% and protein identification by 13.4% on the basis of trypsin. Consistently, LysargiNase increased C‐terminal peptide identification by 47.7%. Moreover, LysargiNase showed better full‐cleavage rate (49.3%) at methylated sites than trypsin (23.9%). LysargiNase and trypsin combined can improve the b‐ion coverage by 50% on FFPE samples. Conclusions FFPE samples can be more efficiently digested at high concentrations of LysargiNase and trypsin. LysargiNase can better digest methylated peptides and improve the proteome identification by 13.4% and the b‐ion coverage by 50% on the basis of trypsin in FFPE samples.

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⿿Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples

Abstract: Graphical abstract

Cited by 45 publications

References 47 publications

Transcriptome and proteome responses in RNAlater preserved tissue of Arabidopsis thaliana

Transcriptome and proteome responses in RNAlater preserved tissue of Arabidopsis thaliana

Feasibility Study for the MALDI‐MSI Analysis of Thyroid Fine Needle Aspiration Biopsies: Evaluating the Morphological and Proteomic Stability Over Time

LysargiNase enhances protein identification on the basis of trypsin on formalin‐fixed paraffin‐embedded samples

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