Dataset Information

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

ABSTRACT: Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level) combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level). Here, we use the gastric carcinoma cell line MKN-45 treated with the c-Met inhibitor SU11274 and PKC inhibitor staurosporine to investigate the most efficient and most comprehensive strategies for both total protein and PTM analysis. Under the conditions used, total protein analysis yielded few changes in response to either compound, while analysis of phosphorylation identified thousands of sites that changed differentially between the two treatments. Both metal affinity and antibody-based enrichments were used to assess phosphopeptide changes, and the data generated by the two methods was largely complementary (non-overlapping). Label-free quantitation of peptide peak abundances was used to accurately determine fold-changes between control and treated samples. Protein interaction network analysis allowed the data to be placed in a biologically relevant context, and follow-up validation of selected findings confirmed the accuracy of the proteomic data. Together, this study provides a framework for start-to-finish proteomic analysis of any experimental system under investigation to maximize the value of the proteomic study and yield the best chance for uncovering actionable target candidates.

SUBMITTER: Stokes MP

PROVIDER: S-EPMC5217380 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

Stokes Matthew P MP Farnsworth Charles L CL Gu Hongbo H Jia Xiaoying X Worsfold Camilla R CR Yang Vicky V Ren Jian Min JM Lee Kimberly A KA Silva Jeffrey C JC

Proteomes 20150807 3

Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level) combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level). Here, we use ...[more]

PMID: 28248267

Similar Datasets

Project description:Camellia oleifera Abel. (C. oleifera), as an important woody tree species producing edible oils in China, has attracted enormous attention due to its abundant unsaturated fatty acids and their associated benefits to human health. To reveal novel insights into the characters during the maturation period of this plant as well as the molecular basis of fatty acid biosynthesis and degradation, we conducted a conjoint analysis of the transcriptome and proteome of C. oleifera seeds from Hainan Island. Using RNA sequencing (RNA-seq) technology and shotgun proteomic method, 59,391 transcripts and 40,500 unigenes were obtained by TIGR Gene Indices Clustering Tools (TGICL), while 1691 protein species were identified from Mass Spectrometry (MS). Subsequently, all genes and proteins were employed in euKaryotic Orthologous Groups (KOG) classification, Gene Ontology (GO) annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to investigate their essential functions. The results indicated that the most abundant pathways were biological metabolic processes. There were 946 unigenes associated with lipid metabolism at the transcriptome level, with 116 proteins at the proteome level; among these, 38 specific proteins were involved in protein-protein interactions, with the majority being related to fatty acid catabolic process. The expression levels of 21 candidate unigenes encoding target proteins were further detected by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, Gas Chromatography Mass Spectrometry (GC-MS) was carried out to determine the fatty acid composition of C. oleifera oil. These findings not only deepened our understanding about the molecular mechanisms of fatty acid metabolism but also offered new evidence concerning the roles of relevant proteins in oil-bearing crops. Furthermore, the lipid-associated proteins recognized in this research might be helpful in providing a reference for the synthetic regulation of C. oleifera oil quality by genetic engineering techniques, thus resulting in potential application in agriculture.

Dataset Information

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

Publications

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets