Quantitative Proteomics Using Isobaric Labeling: A Practical Guide

In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification; isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; and (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. On the basis of the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.     

High‐performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis: Observations and implications

We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label‐free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3‐based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label‐free approach offers a more linear response with a wider dynamic range than MS/MS‐based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS‐based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.     

Isobaric protein and peptide quantification: perspectives and issues

An important challenge for proteomics is the ability to compare protein levels across biological samples. Since their introduction, isotopic and isobaric peptide labeling have played an important role in relative quantitative comparisons of proteomes. One important drawback of most of the isotopic-labeling techniques is an increase in sample complexity. This problem was successfully addressed with the construction of isobaric labeling strategies, such as isobaric tag for relative and absolute quantification (iTRAQ), tandem mass tagging, the cleavable isobaric affinity tag, dimethylated leucines and isobaric peptide termini labeling. Furthermore, numerous applications for multiplexing using iTRAQ and tandem mass tagging have been reported.     

Isobaric protein and peptide quantification: perspectives and issues

An important challenge for proteomics is the ability to compare protein levels across biological samples. Since their introduction, isotopic and isobaric peptide labeling have played an important role in relative quantitative comparisons of proteomes. One important drawback of most of the isotopic-labeling techniques is an increase in sample complexity. This problem was successfully addressed with the construction of isobaric labeling strategies, such as isobaric tag for relative and absolute quantification (iTRAQ), tandem mass tagging, the cleavable isobaric affinity tag, dimethylated leucines and isobaric peptide termini labeling. Furthermore, numerous applications for multiplexing using iTRAQ and tandem mass tagging have been reported.     

Ultra-high intra-spectrum mass accuracy enables unambiguous identification of fragment reporter ions in isobaric multiplexed quantitative proteomics

Isobaric tagging using reagents such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) have become popular tools for mass spectrometry based quantitative proteomics. Because the peptide quantification information is collected in tandem mass spectra, the accuracy and precision of this method largely depend on the resolution with which precursor ions can be selected for the fragmentation and the specificity of the generated reporter ion. The latter can constitute an issue if near isobaric ion signals are present in such spectra because they may distort quantification results. We propose a simple remedy for this problem by identifying reporter ions via the accurate mass differences within a single tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. Our results show that this leads to unambiguous reporter ion identification and complete removal of interfering signals. This mode of data processing is easily implemented in software and offers advantages for protein quantification based on few peptides.     

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer.     

A novel quantitative proteomics workflow by isobaric terminal labeling

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.     

Amino acid-coded tagging approaches in quantitative proteomics

To improve the efficiency, accuracy, reproducibility, throughput and proteome coverage of mass spectrometry-based quantitative approaches, both in vitro and in vivo tagging of particular amino acid residues of cellular proteins have been introduced to assist mass spectrometry for global-scale comparative studies of differentially expressed proteins/modifications between different biologically relevant cell states or cells at different pathological states. The basic features of these methods introduce pair-wise isotope signals of each individual peptide containing a particular type of tagged amino acid (amino acid-coded mass tagging) that originated from different cell states. In this review, the applications of major amino acid-coded mass tagging-based quantitative proteomics approaches, including isotope-coded affinity tag, isobaric tags for relative and absolute quantification (iTRAQ) and stable isotope labeling by amino acids in cell culture are summarized in the context of their respective strengths/weakness in identifying those differentially expressed or post-translational modified proteins regulated by particular cellular stress on a genomic scale in a high-throughput manner. Importantly, these gel-free, in-spectra quantitative mechanisms have been further explored to identify/characterize large-scale protein-protein interactions involving various functional pathways. Taken together, the information about quantitative proteome changes, including multiple regulated proteins and their interconnected relationships, will provide an important insight into the molecular mechanisms, where novel targets for diagnosis and therapeutic intervention will be identified.     

Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it.     

MilQuant: a free, generic software tool for isobaric tagging-based quantitation

Isobaric tagging techniques such as iTRAQ and TMT are widely used in quantitative proteomics and especially useful for samples that demand in vitro labeling. Due to diversity in choices of MS acquisition approaches, identification algorithms, and relative abundance deduction strategies, researchers are faced with a plethora of possibilities when it comes to data analysis. However, the lack of generic and flexible software tool often makes it cumbersome for researchers to perform the analysis entirely as desired. In this paper, we present MilQuant, mzXML-based isobaric labeling quantitator, a pipeline of freely available programs that supports native acquisition files produced by all mass spectrometer types and collection approaches currently used in isobaric tagging based MS data collection. Moreover, aside from effective normalization and abundance ratio deduction algorithms, MilQuant exports various intermediate results along each step of the pipeline, making it easy for researchers to customize the analysis. The functionality of MilQuant was demonstrated by four distinct datasets from different laboratories. The compatibility and extendibility of MilQuant makes it a generic and flexible tool that can serve as a full solution to data analysis of isobaric tagging-based quantitation.     

Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.     

Optimization of Plasma Sample Pretreatment for Quantitative Analysis Using iTRAQ Labeling and LC-MALDI-TOF/TOF

Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome''s complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.     

Amino acid-coded tagging approaches in quantitative proteomics

To improve the efficiency, accuracy, reproducibility, throughput and proteome coverage of mass spectrometry-based quantitative approaches, both in vitro and in vivo tagging of particular amino acid residues of cellular proteins have been introduced to assist mass spectrometry for global-scale comparative studies of differentially expressed proteins/modifications between different biologically relevant cell states or cells at different pathological states. The basic features of these methods introduce pair-wise isotope signals of each individual peptide containing a particular type of tagged amino acid (amino acid-coded mass tagging) that originated from different cell states. In this review, the applications of major amino acid-coded mass tagging-based quantitative proteomics approaches, including isotope-coded affinity tag, isobaric tags for relative and absolute quantification (iTRAQ?) and stable isotope labeling by amino acids in cell culture are summarized in the context of their respective strengths/weakness in identifying those differentially expressed or post-translational modified proteins regulated by particular cellular stress on a genomic scale in a high-throughput manner. Importantly, these gel-free, in-spectra quantitative mechanisms have been further explored to identify/characterize large-scale protein–protein interactions involving various functional pathways. Taken together, the information about quantitative proteome changes, including multiple regulated proteins and their interconnected relationships, will provide an important insight into the molecular mechanisms, where novel targets for diagnosis and therapeutic intervention will be identified.     

A paired ions scoring algorithm based on Morpheus for simultaneous identification and quantification of proteome samples prepared by isobaric peptide termini labeling strategies

The isobaric peptide termini labeling (IPTL) method is a promising strategy in quantitative proteomics for its high accuracy, while the increased complexity of MS2 spectra originated from the paired b, y ions has adverse effect on the identification and the coverage of quantification. Here, a paired ions scoring algorithm (PISA) based on Morpheus, a database searching algorithm specifically designed for high‐resolution MS2 spectra, was proposed to address this issue. PISA was first tested on two 1:1 mixed IPTL datasets, and increases in peptide to spectrum matchings, distinct peptides and protein groups compared to Morpheus itself and MASCOT were shown. Furthermore, the quantification is simultaneously performed and 100% quantification coverage is achieved by PISA since each of the identified peptide to spectrum matchings has several pairs of fragment ions which could be used for quantification. Then the PISA was applied to the relative quantification of human hepatocellular carcinoma cell lines with high and low metastatic potentials prepared by an IPTL strategy.     

Index-ion triggered MS2 ion quantification: a novel proteomics approach for reproducible detection and quantification of targeted proteins in complex mixtures

Biomedical research requires protein detection technology that is not only sensitive and quantitative, but that can reproducibly measure any set of proteins in a biological system in a high throughput manner. Here we report the development and application of a targeted proteomics platform termed index-ion triggered MS2 ion quantification (iMSTIQ) that allows reproducible and accurate peptide quantification in complex mixtures. The key feature of iMSTIQ is an approach called index-ion triggered analysis (ITA) that permits the reproducible acquisition of full MS2 spectra of targeted peptides independent of their ion intensities. Accurate quantification is achieved by comparing the relative intensities of multiple pairs of fragment ions derived from isobaric targeted peptides during MS2 analysis. Importantly, the method takes advantage of the favorable performance characteristics of the LTQ-Orbitrap, which include high mass accuracy, resolution, and throughput. As such it provides an attractive targeted proteomics tool to meet the demands of systems biology research and biomarker studies.     

Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.     

MSnbase-an R/Bioconductor package for isobaric tagged mass spectrometry data visualization, processing and quantitation

MSnbase is an R/Bioconductor package for the analysis of quantitative proteomics experiments that use isobaric tagging. It provides an exploratory data analysis framework for reproducible research, allowing raw data import, quality control, visualization, data processing and quantitation. MSnbase allows direct integration of quantitative proteomics data with additional facilities for statistical analysis provided by the Bioconductor project. AVAILABILITY: MSnbase is implemented in R (version ≥ 2.13.0) and available at the Bioconductor web site (http://www.bioconductor.org/). Vignettes outlining typical workflows, input/output capabilities and detailing underlying infrastructure are included in the package.