Dataset Information

Combination of bottom up 2D-LC-MS and intact protein separation enhances coverage of proteome and low molecular weight short open reading frame encoded peptides of the archeon Methanosarcina mazei

ABSTRACT: The recent discovery of an increasing number of small open reading frame (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. In the present study we evaluated analytical approaches which will allow for simultaneous analysis of widest parts of proteome together with the predicted sORF, as for biological investigations and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. We performed a full proteome analysis of the methane producing archeae Methanosarcina mazei cytosolic proteome using a high/low pH reversed phase LC-MS bottom-up approach. The second analytical approach was based on semi-top strategy, encompassing a separation at intact protein level using a GelFree system, followed by digestion and LC-MS analysis. A high overlap in identified proteins was found for both approaches yielding the most comprehensive coverage of the cytosolic proteome of this organism achieved so far. The application of the second approach and an adjustment of the search criteria for database searches further led to a significant increase of sORF peptide identifications, finally allowing to identify 30 sORF gene products.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Methanosarcina Mazei Go1

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD004325 | Pride | 2016-09-26

REPOSITORIES: Pride

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Publications

Combination of Bottom-up 2D-LC-MS and Semi-top-down GelFree-LC-MS Enhances Coverage of Proteome and Low Molecular Weight Short Open Reading Frame Encoded Peptides of the Archaeon Methanosarcina mazei.

Cassidy Liam L Prasse Daniela D Linke Dennis D Schmitz Ruth A RA Tholey Andreas A

Journal of proteome research 20160902 10

The recent discovery of an increasing number of small open reading frames (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. For biological and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. Consequently in the present study we evaluated analytical approaches that will allow for simultaneous analysis of widest parts of the proteome together with the predicted sO ...[more]

PMID: 27557128

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Project description:The microcirculation serves crucial functions in adult heart that are distinct from those carried out by epicardial vessels. Microvessels are also governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. While great progress has been made in understanding and treating coronary artery disease (CAD), there are few treatment options for patients with microvascular heart disease, primarily due to our limited understanding of underlying pathology. The advent of high-throughput analytical approaches of mRNA and protein expression in specific cells provides an opportunity to transform our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in an adult heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. Furthermore, in-vitro functional and molecular analysis requires an integrated workflow that combines primary cell culture with high-throughput proteomic or transcriptomic analysis. Our goal was an integrated process for simultaneous isolation, culture, and proteomic profiling of the three main cell types comprising the microcirculation in adult mouse heart: endothelial cells (ECs), pericytes (PCs) and vascular smooth muscle cells (VSMCs). We developed an integrated platform for simultaneous isolation and culture of ECs, PCs and VSMCs from adult mouse heart, coupled with unbiased mass spectrometry (MS)-based characterization of protein expression in these cells. We defined microvascular cell proteomes, identified novel protein markers and confirmed established cell-specific markers. Isolating and analyzing microvascular cell types separately from the same preparation of adult mouse hearts allowed for separate investigations into the unique contributions of the different cell types to health and disease, and interactions among different cell types.

Dataset Information

Combination of bottom up 2D-LC-MS and intact protein separation enhances coverage of proteome and low molecular weight short open reading frame encoded peptides of the archeon Methanosarcina mazei

Publications

Combination of Bottom-up 2D-LC-MS and Semi-top-down GelFree-LC-MS Enhances Coverage of Proteome and Low Molecular Weight Short Open Reading Frame Encoded Peptides of the Archaeon Methanosarcina mazei.

Similar Datasets

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