Isobaric tagging approaches in quantitative proteomics: the ups and downs.
ABSTRACT Isobaric tagging has proven to be a popular quantitative proteomics tool and has been rapidly adopted to study a wide range of biological questions in the few years since its commercialization. While the flexibility and multiplexing capacity afforded by this technology are clear attractions, it is not without its shortcomings. As the speed and sensitivity of mass spectrometers have improved and the application of isobaric tags to all manner of biological systems has increased, significant issues with quantitative accuracy and precision have come to light. Here we review the issues associated with the use of isobaric tagging methods and discuss the possible solutions which have been proposed to improve their precision and accuracy to approach the levels required within quantitative proteomics.
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ABSTRACT: Pulsed Q dissociation enables combining LTQ ion trap instruments with isobaric peptide tagging. Unfortunately, this combination lacks a technique which accurately reports protein abundance ratios and is implemented in a freely available, flexible software pipeline. We developed and implemented a technique assigning collective reporter ion intensity-based weights to each peptide abundance ratio and calculating a protein's weighted average abundance ratio and p-value. Using an iTRAQ-labeled standard mixture, we compared our technique's performance to the commercial software MASCOT, finding that it performed better than MASCOT's nonweighted averaging and median peptide ratio techniques, and equal to its weighted averaging technique. We also compared performance of the LTQ-Orbitrap plus our technique to 4800 MALDI TOF/TOF plus Protein Pilot, by analyzing an iTRAQ-labeled stem cell lysate. We found highly correlated protein abundance ratios, indicating that the LTQ-Orbitrap plus our technique yields results comparable to the current standard. We implemented our technique in a freely available, automated software pipeline, called LTQ-iQuant, which is mzXML-compatible; supports iTRAQ 4-plex and 8-plex LTQ data; and can be modified for and have weights trained to a user's LTQ and other isobaric peptide tagging methods. LTQ-iQuant should make LTQ instruments and isobaric peptide tagging accessible to more proteomic researchers.Proteomics 10/2010; 10(19):3533-8. · 4.43 Impact Factor
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ABSTRACT: Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.Molecular & Cellular Proteomics 10/2005; 4(9):1240-50. · 7.25 Impact Factor
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ABSTRACT: Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.Molecular & Cellular Proteomics 06/2002; 1(5):376-86. · 7.25 Impact Factor