Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents*S

By P. Ross, Yulin Huang, J. Marchese, B. Williamson, K. Parker, S. Hattan, Nikita Khainovski, S. Pillai, S. Dey, S. Daniels and 7 more. Molecular & Cellular Proteomics, 2004

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We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Δ and xrn1Δ mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5′ to 3′ decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/7289f6ccbe433da35bd8b14fb782b665af909b46

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Citations in the corpus

Ultrasensitive proteome analysis using paramagnetic bead technology (2014, positive): “The two most frequently applied chemical labeling approaches, reductive dimethylation (Boersema et al , 2009) and isobaric mass tagging (Thompson et al , 2003; Ross et al , 2004), are based on amine-reactive chemistry and thus have specific buffer and solution composition requirements.”
Phospho.ELM: a database of phosphorylation sites—update 2008 (2007, background): “Over the past few years MS, combined with enrichment strategies for phosphorylated proteins e.g. isotope-coded aﬃnity tags (ICAT) (3), stable isotopic amino acids in cell culture (SILAC) (4) and isobaric reagent iTRAQ (5), has been increasingly employed to identify novel phosphorylation sites.”
Insights into the regulation of protein abundance from proteomic and transcriptomic analyses (2012, neutral): “— or by isotopically labelling peptides during sample preparation in a method such as isobaric tag for relative and absolute quantification (iTRAQ”
Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans (2010, methodology): “The extent of age-dependent insolubility in these strains was quantified by conducting quantitative mass spectrometry using the stable-isotope iTRAQ reagents [27], which allowed us to analyze four different samples simultaneously (Figure S2).”
Integrated Proteogenomic Characterization of Clear Cell Renal Cell Carcinoma (2019, background): “TMT Labeling of Peptides: Tandem-mass-tag (TMT) quantitation utilizes reporter ion intensities to determine protein abundance and facilitate quantitative proteomic analysis (Ross et al., 2004).”
Correlation of mRNA and protein in complex biological samples (2009, neutral): “Stable isotope labeling of peptides can be achieved either by metabolic labeling of intact proteins during cell culture (e.g. SILAC [25]) or by chemical labeling of peptides after tryptic digestion (e.g. ICAT, iTRAQ [26,27]).”