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ABRF sPRG 2018 Study: A multipathway phosphopeptide standard for rapid phosphoproteomics assay development

ABSTRACT: Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomicists. There are now a wide variety of robust and inexpensive enrichment strategies to generate phosphoproteomes, while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities (ABRF) umbrella, the Proteomics Standards Research Group (sPRG) has worked to develop a multipathway phosphopeptide standard based on a pool of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This standard contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/IGF-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here we describe a characterization of the standard spiked into a HeLa tryptic digest stimulated with both EGF and IGF1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this standard with data independent acquisition.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Brian C. Searle

PROVIDER: MSV000090564 | MassIVE | Fri Oct 21 10:03:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Mosca2012 - Central Carbon Metabolism Regulated by AKT The role of the PI3K/Akt/PKB signalling pathway in oncogenesis has been extensively investigated and altered expression or mutations of many components of this pathway have been implicated in human cancers. Indeed, expression of constitutively active forms of Akt/PKB can prevent cell death upon growth factor withdrawal. PI3K/Akt/mTOR-mediated survival relies on a profound metabolic adaptation, including aerobic glycolysis. Here, the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production and nucleotide biosynthesis has been modelled, considering two states - high and low PI3K/Akt/mTOR activity. The high PI3K/Akt/mTOR activity represents cancer cell line where PI3K/Akt/mTOR promotes a high rate of glucose metabolism (condition H) and the low PI3K/Akt/mTOR activity is characterised by a lower glycolytic rate due to a reduced PI3K/Akt/mTOR signal (condition L). This model corresponds to the high PI3K/Akt/mTOR signal (condition H). This model is described in the article: Computational Modelling of the Metabolic States Regulated by the Kinase Akt. Mosca E, Alfieri R, Maj C, Bevilacqua A, Canti G, Milanesi L. Frontiers in Systems Biology. 2012 Oct 13 Abstract: Signal transduction pathways and gene regulation determine a major reorganization of metabolic activities in order to support cell proliferation. Protein Kinase B (PKB), also known as Akt, participates in the PI3K/Akt/mTOR pathway, a master regulator of aerobic glycolysis and cellular biosynthesis, two activities shown by both normal and cancer proliferating cells. Not surprisingly considering its relevance for cellular metabolism, Akt/PKB is often found hyperactive in cancer cells. In the last decade, many efforts have been made to improve the understanding of the control of glucose metabolism and the identification of a therapeutic window between proliferating cancer cells and proliferating normal cells. In this context, we have modelled the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production and nucleotide biosynthesis. We used a computational model in order to compare two metabolic states generated by the specific variation of the metabolic fluxes regulated by the activity of the PI3K/Akt/mTOR pathway. One of the two states represented the metabolism of a growing cancer cell characterised by aerobic glycolysis and cellular biosynthesis, while the other state represented the same metabolic network with a reduced glycolytic rate and a higher mitochondrial pyruvate metabolism, as reported in literature in relation to the activity of the PI3K/Akt/mTOR. Some steps that link glycolysis and pentose phosphate pathway revealed their importance for controlling the dynamics of cancer glucose metabolism. This model is hosted on BioModels Database and identified by: MODEL1210150000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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ABRF sPRG 2018 Study: A multipathway phosphopeptide standard for rapid phosphoproteomics assay development

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