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Automated protein turnover calculations from 15N partial metabolic labeling LC/MS shotgun proteomics data.

ABSTRACT: Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS 15N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (15N) to the intensity sum of all light (14N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover.

SUBMITTER: Lyon D

PROVIDER: S-EPMC3988089 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Automated protein turnover calculations from 15N partial metabolic labeling LC/MS shotgun proteomics data.

Lyon David D Castillejo Maria Angeles MA Staudinger Christiana C Weckwerth Wolfram W Wienkoop Stefanie S Egelhofer Volker V

PloS one 20140415 4

Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent prot ...[more]

PMID: 24736476

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Project description:Highly specialized cells are fundamental for proper functioning of complex organs. Variations in cell-type specific gene expression and protein composition have been linked to a variety of diseases. Although single cell technologies have emerged as valuable tools to address this cellular heterogeneity, a majority of these workflows lack sufficient in situ resolution for functional classification of cells and are associated with extremely long analysis time, especially when it comes to in situ proteomics. In addition, lack of understanding of single cell dynamics within their native environment limits our ability to explore the altered physiology in disease development. This limitation is particularly relevant in the mammalian brain, where different cell types perform unique functions and exhibit varying sensitivities to insults. The hippocampus, a brain region crucial for learning and memory, is of particular interest due to its obvious involvement in various neurological disorders. Here, we present a combination of experimental and data integration approaches for investigation of cellular heterogeneity and functional disposition within the mouse brain hippocampus using MALDI Imaging mass spectrometry (MALDI-IMS) and shotgun proteomics (LC-MS/MS) coupled with laser-capture microdissection (LCM) along with spatial transcriptomics. Within the dentate gyrus granule cells we identified two proteomically distinct cellular subpopulations that are characterized by a substantial number of discriminative proteins. These cellular clusters contribute to the overall functionality of the dentate gyrus by regulating redox homeostasis, mitochondrial organization, RNA processing, and microtubule organization. Importantly, most of the identified proteins matched their transcripts, verifying the in situ protein identification and supporting their functional analyses. By combining high-throughput spatial proteomics with transcriptomics, our approach enables reliable near-single-cell scale identification of proteins and profiling of inter-cellular heterogeneity within similar cell-types in tissues. This methodology has the potential to be applied to different biological conditions and tissues, providing a deeper understanding of cellular subpopulations in situ.

Dataset Information

Automated protein turnover calculations from 15N partial metabolic labeling LC/MS shotgun proteomics data.

Publications

Automated protein turnover calculations from 15N partial metabolic labeling LC/MS shotgun proteomics data.

Similar Datasets

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