A large-scale targeted proteomics assay resource based on an in vitro human proteome

Abstract

Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.

PHGDH as a Key Enzyme for Serine Biosynthesis in HIF2α-Targeting Therapy for Renal Cell Carcinoma

“…mRNA profiles were generated by single-read deep sequencing using Illumina HiSeq 2500/2000. In vitro proteome-assisted multiple reaction monitoring for protein absolute quantification (iMPAQT) analysis was performed at Kyusyu University in Japan (12). …”

“…To identify key molecules within the cells which could still proliferate, we performed quantitative proteomics (12) and RNA sequencing. From this analysis, we identified phosphoglycerate dehydrogenase (PHGDH), a key enzyme involved in serine biosynthesis that functions to convert the glycolytic intermediate 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP).…”

Extension of the yeast metabolic model to include iron metabolism and its use to estimate global levels of iron‐recruiting enzyme abundance from cofactor requirements

“…The measurement of intracellular metabolite pools and protein abundances are useful approaches to understand these homeostatic mechanisms, and have been applied to many different systems ranging from bacteria such as Escherichia coli (Bennett et al, ) to tumour cell lines (Madhukar, Warmoes, & Locasale, ; Matsumoto et al, ). Although total protein content and absolute quantification of individual proteins have been reported for a number of biological systems (Bennett et al, ; Carroll et al, ; Madhukar et al, ; Matsumoto et al, ; Picotti, Bodenmiller, Mueller, Domon, & Aebersold, ), the properties of metabolic systems cannot be defined by studying enzyme proteins in isolation. Enzymes are often present in lower copy numbers compared to other components of the proteome, such as ribosomal proteins.…”

“…Many of these reactions were involved with the transport or exchange of lipid metabolism intermediates across organelles, as well as of amino acids including glycine, L-alanine, L-leucine, serine and valine and of small inorganic molecules including ammonia, bicarbonate, carbon dioxide, hydronium ion, oxygen, phosphate and water (Supporting Information 3).The rewiring of lipid metabolism and the changes in the fluxes in the energy pathways in response to a perturbation induced to mimic the reduction of ARH1 function in the cell was in line with earlier findings on its role in biological systems. Apart from acting as an essential component of the ISC machinery, the Arh1 protein is an ortholog of the human adrenodoxin reductase(Manzella, Barros, & Nobrega, 1998), which was reported to function in the mitochondrial electron transfer chain that catalyses the conversion of cholesterol into pregnenolone. Expression of human ARH1 on a retroviral vector was shown to restore the LDL receptor function in cells from patients suffering from familial hypercholesterolemia(Eden et al, 2002), demonstrating the enzyme's role in neutral lipid metabolism, as also captured by our analysis in the model yeast system.Because iron cofactors, specifically Fe-S clusters, were used extensively as cofactors by enzymes catalysing mitochondrial reactions (particularly those that affect energy generation routes via aerobic respiration), constraining the flux of such a reaction was observed to lead to a reduction in the oxygen uptake flux, also by 50%.…”

Prediction of Hopeless Peptides Unlikely to be Selected for Targeted Proteome Analysis

“…After establishing comprehensive SRM assay methods, such as the SRMAtlas of human and yeast proteins, 9–13) these methods could be reused owing to their basic compatibility among triple quadrupole mass spectrometers. 14) However, SRM assay methods for the targeted proteome analysis of non-model organisms, such as various industrially important bacteria for biomaterial productions, are under continuous development.…”