Tandem Mass Tags:  A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS/MS

Thompson, Alan J.; Schäfer, Jürgen; Kuhn, Karsten; Kienle, Stefan; Schwarz, Josef; Schmidt, Günter; Neumann, Thomas; Hamon, Christian

doi:10.1021/ac030267r

Cited by 109 publications

(104 citation statements)

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“…Recent advances in protein quantification using tandem mass tags (TMTs) and isobaric tags for relative and absolute quantification (iTRAQ) have improved measurement precision, accuracy and reproducibility (Thompson et al, 2003;Ross et al, 2004), surpassing label-free quantification methods such as spectral counting (Li et al, 2012). TMT/ iTRAQ-based quantitative proteomics can be used with complex samples, including biological systems that are not amenable to efficient metabolic labeling with stable isotopes.…”

Section: Introductionmentioning

confidence: 99%

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

et al. 2014

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Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 1C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.

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Section: Introductionmentioning

confidence: 99%

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

et al. 2014

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“…There is significant interest in stable isotope labeling strategies of proteins or peptides as with every measurement there is the potential to use an internal reference allowing relative quantitation comparison, which significantly increases sensitivity of detection of change in abundance. Isobaric labeling techniques such as tandem mass tags (11,12) or isobaric tags for relative or absolute quantitation (iTRAQ) 1 (13,14) allow multiplexing of four, six and eight separately labeled samples within one experiment. In contrast to most other quantitative proteomics methods where precursor ion intensities are measured, here the measurement and ensuing quantitation of iTRAQ reporter ions occurs after fragmentation of the precursor ion.…”

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Addressing Accuracy and Precision Issues in iTRAQ Quantitation

Karp

Huber

Sadowski

et al. 2010

Molecular & Cellular Proteomics

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iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variancestabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variancestabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry. Molecular & Cellular Proteomics 9:1885-1897, 2010.Different techniques are being used and developed in the field of proteomics to allow quantitative comparison of samples between one state and another. These can be divided into gel-(1-4) or mass spectrometry-based (5-8) techniques. Comparative studies have found that each technique has strengths and weaknesses and plays a complementary role in proteomics (9, 10). There is significant interest in stable isotope labeling strategies of proteins or peptides as with every measurement there is the potential to use an internal reference allowing relative quantitation comparison, which significantly increases sensitivity of detection of change in abundance. Isobaric labeling techniques such as tandem mass tags (11, 12) or isobaric tags for relative or absolute quantitation (iTRAQ) 1 (13, 14) allow multiplexing of four, six and eight separately labeled samples within one experiment. In contrast to mos...

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“…Relative quantitation of proteins using a differential bottom-up proff ff teomics platform can be performed using in-vivo metabolic labeling with stable isotope-labeled amino acids (SILAC) [145], chemical labeling (e.g., isotope-code affi nity tag (ICAT) [146], isobaric tags for relative and absoffi lute quantitation (iTRAQ) [147], and tandem mass tags (TMT) [148]), or label-free methods. Label-free comparative proteomics uses MS1 ion current or MS2 spectral counting to identify differentially abundant peptides ff ff [149][150][151].…”

Section: Mass Spectrometry (Ms) Methodsmentioning

confidence: 99%

Detection of Allergen Markers in Food: Analytical Methods

et al. 2016

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Tandem Mass Tags: A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS/MS

Abstract: The PQQ solution was bubbled with argon for 30 min, followed by the addition of 7 µL of pyrrole.

Cited by 109 publications

References 0 publications

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

Addressing Accuracy and Precision Issues in iTRAQ Quantitation

Detection of Allergen Markers in Food: Analytical Methods

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