Dataset Information

Evaluation of two methods for LS-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues

ABSTRACT: The proteomics of archival samples has advanced significantly during the last two decades, making possible the use of vast FFPE tissue archives in biomarker studies. However, due to the limited data regarding comparison of the performance of different protocols, there is currently no consensus on the most effective protocol for shotgun proteomic analysis of FFPE tissue. In-solution digestion method using Rapigest as detergent and FASP method, both reported to deliver superior results on FFPE tissues were compared using two label-free data-independent LC-MS/MS acquisition modes. Archival FFPE prostate tissues stored 7 years and mirroring fresh frozen samples were processed with both protocols in 3 technical replicates. Data were comparatively evaluated in terms of protein and peptide identifications in fresh frozen and FFPE samples, analysis of the fresh and FFPE tissues representative proteome (molecular weight distribution, distribution according to protein’s pI, cellular component analysis of proteins and quantitation accuracy) and technical reproducibility of the Rapigest and FASP protocols.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Prostate Gland

SUBMITTER: Katarina Davalieva

LAB HEAD: Katarina Davalieva

PROVIDER: PXD021074 | Pride | 2021-01-15

REPOSITORIES: Pride

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Publications

Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.

Davalieva Katarina K Kiprijanovska Sanja S Dimovski Aleksandar A Rosoklija Gorazd G Dwork Andrew J AJ

Journal of proteomics 20210114

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acqu ...[more]

PMID: 33453434

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Project description:Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not known. Here, we study how different noise levels are encoded by the promoter sequence using massively parallel noise measurements of thousands of synthetically designed promoters. We find that the noise levels of promoters with similar mean expression levels can vary over more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We devised a computational model that can accurately predict the mean-independent component of the noise from DNA sequence alone. Our model suggests that the effect of promoters on noise is partly mediated by the combination of non-specific DNA binding and one-dimensional sliding along the DNA that occurs when transcription factors search for their target sites. Overall, our results demonstrate that small changes in the DNA sequence of promoters can allow tuning of noise levels in a manner that is largely predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels. Expression measurements of a collection of synthetic promoters collection that was published in Sharon et al. Nature Biotechnology 2012(doi: 10.1038/nbt.2205). Two replicates of the promoter library integrated into a plasmid in yeast were measured in SC-Glu-URA medium. The promoter library was measured as described in Sharon et. al.(Sharon et al. 2012), except for the differences below. Briefly, a large collection of synthetic promoter reporter gene strains was generated by a pooled ligation of 6500 fully designed DNA oligos (obtained by synthesis on a microarray(LeProust et al. 2010) by Agilent Technologies, Santa Clara, California). The oligos were ligated upstream to a yellow fluorescent protein (YFP) gene with a short (100 bp) core promoter sequence taken from HIS3 gene promoter and into a low copy plasmid which also contains a TEF2 promoter deriving red fluorescent protein (mCherry). The resulting plasmids were then transformation into yeast (S. cerevisiae). Next, the pool of cells was grown in amino acid starvation condition (SCD without amino acid except Histidine), and sorted according to their YFP expression level into 32 expression bins (mCherry was used for gating one plasmid copy cells and for normalization). The DNA of the promoters in each bin were then amplified and sent to multiplexed parallel sequencing. Each sequencing result was mapped to a specific promoter and expression bin, resulting in a distribution of cells that contain each promoter across all expression bins. The following differences were applied relative to the description in Sharon et. al.(Sharon et al. 2012). The medium used both for growing the cells and for their sorting was SC-Glu-URA (synthetic complete media with 2% glucose and without uracil) medium without amino acids, except for Histidine. In order to achieve expression distributions with high resolution that would allow good assessment of expression noise, the library cells were sorted into 32 bins according to their ratio of YFP and mCherry expression level, thereby normalizing for extrinsic noise effects. Each of the two extreme expression bins contained 2% of the library cells and each of the remaining 30 bins contained 3.2%. We collected a total of 10,000,000 cells. As previously described, the mapping of cells to bins involves parallel sequencing of the amplified promoter regions. For this purpose, Illumina Hi-Seq 2000 was used to obtain >30,000,000 mapped reads. The two replicates were separately generated from the ssDNA oligo library and separately measured as described above.

Dataset Information

Evaluation of two methods for LS-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues

Publications

Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.

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