Dataset Information

Quality control in quantitative proteomics: a case study for the human CSF proteome

ABSTRACT: Proteome studies using mass spectrometry (MS)-based quantification is a main approach for the discovery of new biomarkers. However, a number of analytical conditions in front and during MS data acquisition can affect the accuracy of the obtained outcome. Therefore, comprehensive quality assessment of the acquired data plays a central role in quantitative proteomics, though due to immense complexity of MS data it is often neglected. Here, we practically address the quality assessment of quantitative MS data describing key steps for the evaluation including the levels: raw data, identification and quantification. With this, four independent datasets from cerebrospinal fluid, an important biofluid for neurodegenerative diseases biomarker studies, were assessed demonstrating that already sample processing-based differences are reflected on all three levels but with a varying impact on the quality of the quantitative data.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: Svitlana Rozanova

LAB HEAD: Prof. Katrin Marcus

PROVIDER: PXD037650 | Pride | 2023-04-12

REPOSITORIES: Pride

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Publications

Quality Control-A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome.

Rozanova Svitlana S Uszkoreit Julian J Schork Karin K Serschnitzki Bettina B Eisenacher Martin M Tönges Lars L Barkovits-Boeddinghaus Katalin K Marcus Katrin K

Biomolecules 20230307 3

Proteomic studies using mass spectrometry (MS)-based quantification are a main approach to the discovery of new biomarkers. However, a number of analytical conditions in front and during MS data acquisition can affect the accuracy of the obtained outcome. Therefore, comprehensive quality assessment of the acquired data plays a central role in quantitative proteomics, though, due to the immense complexity of MS data, it is often neglected. Here, we address practically the quality assessment of qu ...[more]

PMID: 36979426

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Project description:To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately. Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).

Project description:BACKGROUND: The genetics of transcript-level variation is an exciting field that has recently given rise to many studies. Genetical genomics studies have mainly focused on cell lines, blood cells or adipose tissues, from human clinical samples or mice inbred lines. Few eQTL studies have focused on animal tissues sampled from outbred populations to reflect natural genetic variation of gene expression levels in animals. In this work, we analyzed gene expression in a whole tissue, pig skeletal muscle sampled from individuals from a half sib F2 family shortly after slaughtering. RESULTS: QTL detection on transcriptome measurements was performed on a family structured population. The analysis identified 335 eQTLs affecting the expression of 272 transcripts. The ontologic annotation of these eQTLs revealed an over-representation of genes encoding proteins involved in processes that are expected to be induced during muscle development and metabolism, cell morphology, assembly and organization and also in stress response and apoptosis. A gene functional network approach was used to evidence existing biological relationships between all the genes whose expression levels are influenced by eQTLs. eQTLs localization revealed a significant clustered organization of about half the genes located on segments of chromosome 1, 2, 10, 13, 16, and 18. Finally, the combined expression and genetic approaches pointed to putative cis-drivers of gene expression programs in skeletal muscle as COQ4 (SSC1), LOC100513192 (SSC18) where both the gene transcription unit and the eQTL affecting its expression level were shown to be localized in the same genomic region. This suggests cis-causing genetic polymorphims affecting gene expression levels, with (e.g. COQ4) or without (e.g. LOC100513192) potential pleiotropic effects that affect the expression of other genes (cluster of trans-eQTLs). CONCLUSION: Genetic analysis of transcription levels revealed dependence among molecular phenotypes as being affected by variation at the same loci. We observed the genetic variation of molecular phenotypes in a specific situation of cellular stress thus contributing to a better description of muscle physiologic response. In turn, this suggests that large amounts of genetic variation, mediated through transcriptional networks, can drive transient cell response phenotypes and contribute to organismal adaptative potential. A wide genomic project was designed to combine genetic and transcriptome analysis. This project involved about 1,000 F2 pigs from 15 half-sib families to identify QTL for meat quality traits (Cherel et al., unpublished data). In this study, we focused on one family of 57 half sib F2 pigs. Total RNA was isolated from each of the 57 muscle samples using a commercial kit, and controlled for quality and concentration using an AGILENT 2100 bioanalyzer. cDNA was synthesized and labelled from 5 Âµg total RNA by simultaneous reverse transcription of mRNA using SuperScriptTM II RNase H- Reverse Transcriptase and 33P-deoxy-CTP. Each muscle sample mRNA was hybridized at 68Â°C during 24 h to one array. The arrays were then exposed to radioisotopic-sensitive imaging plates that were scanned thereafter with a phosphor imaging system at a 25 Âµm resolution. Hybridization images were quantified using a semi-automated software through a grid process with a fixed spot diameter. The output was then subjected to the data analysis process.

Dataset Information

Quality control in quantitative proteomics: a case study for the human CSF proteome

Publications

Quality Control-A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome.

Similar Datasets

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