A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging inEscherichia coli cells

Choe, Leila H.; Aggarwal, Kunal; Franck, Zsofia; Lee, Kelvin H.

doi:10.1002/elps.200410336

Cited by 109 publications

(88 citation statements)

References 26 publications

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“…In the simplest situation, iTRAQ is used in a pairwise comparison (10,18,21,38,39). A protein is deemed to be differentially abundant if measured ratios exceed a certain thresh- …”

Section: Selecting Significance Thresholdmentioning

confidence: 99%

“…Peptide abundance ratios are calculated by combining data from multiple fractions across MS runs and then averaging across peptides to give an abundance ratio for each parent protein. GPS Explorer, a software package developed for iTRAQ, assumes normality in the peptide ratio for a protein once an outlier filter is applied (18). The iTRAQ package ProQuant assumes that peptide ratio data for a protein follow a log-normal distribution (19).…”

mentioning

confidence: 99%

“…Some of the current approaches include outlier removal (18,25), weighted means (21,22), inclusion filters (16,22), logarithmic transformation (19), and weighted correlation analysis (23). Outlier removal methods, for example using Dixon's test, assume a normal distribution for which there is little empirical basis.…”

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confidence: 99%

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Addressing Accuracy and Precision Issues in iTRAQ Quantitation

Karp

Huber

Sadowski

et al. 2010

Molecular & Cellular Proteomics

469

459

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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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“…In the simplest situation, iTRAQ is used in a pairwise comparison (10,18,21,38,39). A protein is deemed to be differentially abundant if measured ratios exceed a certain thresh- …”

Section: Selecting Significance Thresholdmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Addressing Accuracy and Precision Issues in iTRAQ Quantitation

Karp

Huber

Sadowski

et al. 2010

Molecular & Cellular Proteomics

469

459

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“…According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13].…”

mentioning

confidence: 99%

“…R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13].…”

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confidence: 99%

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Armenta

Hoeschele

Lazar

2009

J. Am. Soc. Mass Spectrom.

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An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17␤-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of Ͻ4%. The reproducibility of protein identifications was ϳ60%-67% between duplicate, and ϳ50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P Ͻ 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ϳ2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13]. Isotope labeling approaches rely on the covalent attachment of stable isotope tags to specific amino acid residues of proteins or peptides during metabolic, enzymatic, or chemical processes. Label-free quantitation methods rely on measuring peak areas, intensities, or spectral counts, and benefit from not having to chemically alter the sample. Throughput, however, is lower, and quantitation errors are higher, as the samples are processed independently. Among chemical labeling techniques, isotope tags for relative and absolute quantitation (iTRAQ) has received much attention [14 -30]. In this approach, peptides are labeled with isobaric tags at the N-terminus and the lysine side chains. MS/MS fragmentation produces signature ions that can be used to obtain quantitative information. Perhaps the most attractive advantage of this approach is that it can be used for the simultaneous quantification (relative or absolute) of up to four/eight different samples. Simplicity, of course, is an added benefit.A...

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