Dataset Information

Comparative analysis of label-free and 8-plex iTRAQ approach for quantitative tissue proteomic analysis

ABSTRACT: MS-based approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics the open question still remains, which quantification method is more suitable for identification of proteins with the altered abundance, particularly for the highly complex clinical samples. In this study, a thorough comparison of label-free method (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor samples from non-muscle invasive and muscle-invasive bladder cancer. In the case of the label-based approach, the results obtained for the unfractionated and fractionated sample were included. To obtain representative results from each approach, both experiments were conducted according to routinely applicable protocols. Additionally, the sampling procedure was standardized to reduce the effect of biological and analytical variability on the quantification results.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urinary Bladder

DISEASE(S): Bladder Transitional Cell Carcinoma

SUBMITTER: Agnieszka Latosinska

LAB HEAD: Dr Antonia Vlahou

PROVIDER: PXD002170 | Pride | 2015-09-09

REPOSITORIES: Pride

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Publications

Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis.

Latosinska Agnieszka A Vougas Konstantinos K Makridakis Manousos M Klein Julie J Mullen William W Abbas Mahmoud M Stravodimos Konstantinos K Katafigiotis Ioannis I Merseburger Axel S AS Zoidakis Jerome J Mischak Harald H Vlahou Antonia A Jankowski Vera V

PloS one 20150902 9

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue sam ...[more]

PMID: 26331617

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Project description:Tissue samples in biobanks preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature (OCT)-embedded and subsequently freezing are highly valuable in experimental studies where the results can be related to clinical parameters. Today, mass spectrometry (MS)-based proteomics is the method of choice for unbiased and relative comparison of the abundances of thousands of proteins present in biological samples. MS analysis of FFPE- and OCT-preserved tissue has been limited, but it is now approachable using newly developed protocols. However, it has not been clarified how the results from different preservation methods agrees and differ. In this study, we use a unique sample set of urinary bladder cancer tissues of two different tumor stages (Ta/T1 – non-muscle invasive and T2/T3 muscle invasive); upon sampling the tumors were divided and preserved by the two methods. Thereafter a protocol for parallel processing of the samples were applied after which the samples were analyzed with label free quantitative MS analysis. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Principal component analysis showed that the largest differences between samples are based on the preservation method, but this analysis could also classify the tumors into different stages in each preservation. The overlap of quantifiable proteins between the preservation methods were 84% when all proteins and full data set were taken into consideration, but only 41% on average when proteins from a specific tumor were analyzed. Proteins involved in mitochondrial function were overrepresented among proteins present in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Moreover, univariate analysis showed that HMGCS2 protein is uniquely quantified in Ta/T1 tumors in both FFPE and OCT data, and that LGALS1, ANXA5 and plastin proteins are upregulated in T2/T3 tumors compared to Ta/T1 tumors in both FFPE and OCT data, confirming the consistency between the data sets and suggesting these proteins as biomarkers. Finally, LGALS1 data were verified by immunohistochemistry staining of an independent data set.

Dataset Information

Comparative analysis of label-free and 8-plex iTRAQ approach for quantitative tissue proteomic analysis

Publications

Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis.

Similar Datasets

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