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Comprehensive proteome and phosphoproteome profiling shows negligible influence of RNAlater on protein abundance and phosphorylation.

ABSTRACT: Certain tumors such as pancreatic ductal adenocarcinoma (PDAC) are known to contain a variety of hydrolytic enzymes including RNases and proteases that may lead to degradation of RNA and proteins during sample processing. For such tumor tissues with RNA instability, RNAlater containing a high concentration of quaternary ammonium sulfates that denature RNA-hydrolyzing enzymes is often used to protect RNAs from hydrolysis. Although a few studies have been carried out to determine the effect of RNAlater on DNA and RNA, whether RNAlater influences the proteome and phosphoproteome is largely unknown. In this study we carried out a systematic and comprehensive analysis of the effect of RNAlater on the proteome and phosphoproteome using high-resolution mass spectrometry. PDAC tissues from three patients were individually pulverized and the tissue powders of each patient were divided into two portions, one of which was incubated in RNAlater at 4 °C for 24 h (RNAlater tissue) while the other was kept at - 80 °C (frozen tissue). Comprehensive quantitative profiling experiments on the RNAlater tissues and the frozen tissues resulted in the identification of 99,136 distinct peptides of 8803 protein groups and 17,345 phosphopeptides of 16,436 phosphosites. The data exhibited no significant quantitative changes in both proteins and phosphorylation between the RNAlater tissues and the frozen tissue. In addition, the phosphoproteome data showed heterogeneously activated pathways among the three patients that were not altered by RNAlater. These results indicate that the tissue preservation method using RNAlater can be effectively used on PDAC tissues for proteogenomic studies where preservation of intact DNA, RNA and proteins is prerequisite. Data from this study are available via ProteomeXchange with the identifier PXD010710.

SUBMITTER: Bae J

PROVIDER: S-EPMC6482574 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Comprehensive proteome and phosphoproteome profiling shows negligible influence of RNAlater on protein abundance and phosphorylation.

Bae Jingi J Kim Su-Jin SJ Lee Seung-Eun SE Kwon Wooil W Kim Hongbeom H Han Youngmin Y Jang Jin-Young JY Kim Min-Sik MS Lee Sang-Won SW

Clinical proteomics 20190425

Certain tumors such as pancreatic ductal adenocarcinoma (PDAC) are known to contain a variety of hydrolytic enzymes including RNases and proteases that may lead to degradation of RNA and proteins during sample processing. For such tumor tissues with RNA instability, RNAlater containing a high concentration of quaternary ammonium sulfates that denature RNA-hydrolyzing enzymes is often used to protect RNAs from hydrolysis. Although a few studies have been carried out to determine the effect of RNA ...[more]

PMID: 31049047

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Project description:Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ?99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process.

Dataset Information

Comprehensive proteome and phosphoproteome profiling shows negligible influence of RNAlater on protein abundance and phosphorylation.

Publications

Comprehensive proteome and phosphoproteome profiling shows negligible influence of RNAlater on protein abundance and phosphorylation.

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