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.     

Quantitative analysis of O-GlcNAcylation in combination with isobaric tag labeling and chemoenzymatic enrichment

Protein O-GlcNAcylation regulates various biological processes, and is associated with several diseases. Therefore, the development of quantitative proteomics is important for understanding the mechanisms of O-GlcNAc-related diseases. We previously reported selective enrichment of O-GlcNAcylated peptides, which provided high-selectivity and effective release by a novel thiol-alkyne and thiol-disulfide exchange. Here, we describe a new approach using initial isobaric tag labeling for relative quantification followed by enrichment and β-elimination/Michael addition with dithiothreitol for identification of both proteins and modification sites. The approach was validated using model proteins and peptides. This novel strategy could be used for quantitative O-GlcNAcome of biological samples.     

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.     

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases.     

Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times and high-resolution spectra to quantify the single-cell signal; however, the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time-intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS enhanced quant of single cell spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at an MS2 injection time of 750 ms using a 2 h gradient.     

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.     

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.     

Determination of Variation Parameters as a Crucial Step in Designing TMT-Based Clinical Proteomics Experiments

In quantitative shotgun proteomic analyses by liquid chromatography and mass spectrometry, a rigid study design is necessary in order to obtain statistically relevant results. Hypothesis testing, sample size calculation and power estimation are fundamental concepts that require consideration upon designing an experiment. For this reason, the reproducibility and variability of the proteomic platform needs to be assessed. In this study, we evaluate the technical (sample preparation), labeling (isobaric labels), and total (biological + technical + labeling + experimental) variability and reproducibility of a workflow that employs a shotgun LC-MS/MS approach in combination with TMT peptide labeling for the quantification of peripheral blood mononuclear cell (PBMC) proteome. We illustrate that the variability induced by TMT labeling is small when compared to the technical variation. The latter is also responsible for a substantial part of the total variation. Prior knowledge about the experimental variability allows for a correct design, a prerequisite for the detection of biologically significant disease-specific differential proteins in clinical proteomics experiments.     

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.     

Implementation and evaluation of relative and absolute quantification in shotgun proteomics with label-free methods

Tandem mass spectrometry allows for fast protein identification in a complex sample. As mass spectrometers get faster, more sensitive and more accurate, methods were devised by many academic research groups and commercial suppliers that allow protein research also in quantitative respect. Since label-free methods are an attractive alternative to labeling approaches for proteomics researchers seeking for accurate quantitative results we evaluated several open-source analysis tools in terms of performance on two reference data sets, explicitly generated for this purpose.In this paper we present an implementation, T3PQ (Top 3 Protein Quantification), of the method suggested by Silva and colleagues for LC-MS^E applications and we demonstrate its applicability to data generated on FT-ICR instruments acquiring in data dependent acquisition (DDA) mode. In order to validate this method and to show its usefulness also for absolute protein quantification, we generated a reference data set of a sample containing four different proteins with known concentrations. Furthermore, we compare three other label-free quantification methods using a complex biological sample spiked with a standard protein in defined concentrations. We evaluate the applicability of these methods and the quality of the results in terms of robustness and dynamic range of the spiked-in protein as well as other proteins also detected in the mixture. We discuss drawbacks of each method individually and consider crucial points for experimental designs. The source code of our implementation is available under the terms of the GNU GPLv3 and can be downloaded from sourceforge (http://fqms.svn.sourceforge.net/svnroot/fqms). A tarball containing the data used for the evaluation is available on the FGCZ web server (http://fgcz-data.uzh.ch/public/T3PQ.tgz).     

Advances in quantitative proteomics

Large-scale protein quantification has become a major proteomics application in many areas of biological and medical research. During the past years, different techniques have been developed, including gel-based such as differential in-gel electrophoresis (DIGE) and liquid chromatography-based such as isotope labeling and label-free quantification. These quantitative proteomics tools hold significant promise for biomarker discovery, diagnostic and therapeutic applications. They are also important for research in functional genomics and systems biology towards basic understanding of molecular networks and pathway interactions. In this review, we summarize current technologies in quantitative proteomics and discuss recent applications of the technologies.     

Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines

Within the past decade numerous methods for quantitative proteome analysis have been developed of which all exhibit particular advantages and disadvantages. Here, we present the results of a study aiming for a comprehensive comparison of ion-intensity based label-free proteomics and two label-based approaches using isobaric tags incorporated at the peptide and protein levels, respectively. As model system for our quantitative analysis we used the three hepatoma cell lines HepG2, Hep3B and SK-Hep-1. Four biological replicates of each cell line were quantitatively analyzed using an RPLC–MS/MS setup. Each quantification experiment was performed twice to determine technical variances of the different quantification techniques. We were able to show that the label-free approach by far outperforms both TMT methods regarding proteome coverage, as up to threefold more proteins were reproducibly identified in replicate measurements. Furthermore, we could demonstrate that all three methods show comparable reproducibility concerning protein quantification, but slightly differ in terms of accuracy. Here, label-free was found to be less accurate than both TMT approaches. It was also observed that the introduction of TMT labels at the protein level reduces the effect of underestimation of protein ratios, which is commonly monitored in case of TMT peptide labeling. Previously reported differences in protein expression between the particular cell lines were furthermore reproduced, which confirms the applicability of each investigated quantification method to study proteomic differences in such biological systems. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Temporal Differential Proteomes of Clostridium difficile in the Pig Ileal-Ligated Loop Model

The impact of Clostridium difficile infection (CDI) on healthcare is becoming increasingly recognized as it represents a major cause of nosocomial diarrhea. A rising number of CDI cases and outbreaks have been reported worldwide. Here, we developed the pig ileal-ligated loop model for semi-quantitative analysis comparing temporal differential proteomes in C. difficile following in vivo incubation with in vitro growth using isobaric tags for relative and absolute quantification (iTRAQ). Proteins retrieved from the in vitro cultures and the loop contents after 4, 8, and 12 h in vivo incubation were subjected to in-solution digestion, iTRAQ labeling, two-dimensional liquid chromatography/tandem mass spectrometry and statistical analyses. From a total of 1152 distinct proteins identified in this study, 705 proteins were available for quantitative measures at all time points in both biological and technical replicates; 109 proteins were found to be differentially expressed. With analysis of clusters of orthologous group and protein-protein network interactions, we identified the proteins that might play roles in adaptive responses to the host environment, hence enhancing pathogenicity during CDI. This report represents the quantitative proteomic analysis of C. difficile that demonstrates time-dependent protein expression changes under conditions that mimic in vivo infection and identifies potential candidates for diagnostic or therapeutic measures.     

Validation of analytical method for (E)-2-decenedioic acid quantification in honey samples

Natural honey is a highly sought-after product owing to its biological uniqueness. We developed a suitable analytical method for the quantitative analysis of (E)-2-decenedioic acid in Korean natural and sugar-fed honey samples. A total of 258 honey samples were screened using ultra-performance liquid chromatography (UPLC) for the detection and quantification of (E)-2-decenedioic acid. (E)-2-decenedioic acid was present in high concentrations (122–127 mg/kg) in sugar-fed honey but only in trace amounts (10.9–14.8 mg/kg) in natural honey. Findings indicate that the UPLC method is suitable and reliable for the quantitative evaluation of (E)-2-decenedioic acid in honey. We expect that this method will be applicable for detecting honey adulteration.     

Quantitative phosphoproteomics: New technologies and applications in the DNA damage response

Cells are highly responsive to their environment. One of the main strategies used by cells in signal transduction is protein phosphorylation, a reversible modification that regulates numerous biological processes. Misregulation of phosphorylation-mediated processes is often implicated in many human diseases and cancers. A global and quantitative analysis of protein phosphorylation provides a powerful new approach and has the potential to reveal new insights in signaling pathways. Recent technological advances in high resolution mass spectrometers and multidimensional liquid chromatography, combined with the use of stable isotope labeling of proteins, have led to the application of quantitative phosphoproteomics to study in vivo signal transduction events on a proteome-wide scale. Here we review recent advancements in quantitative phosphoproteomic technologies, discuss their potentials and identify areas for future development. A key objective of proteomic technology is its application to addressing biological questions. We will therefore describe how current quantitative phosphoproteomic technology can be used to study the molecular basis of phosphorylation events in the DNA damage response.Key words: proteomics, mass spectrometry, DNA damage response, phosphorylation, HILIC, SILAC     

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.     

Quantitative determination of isoflurane enantiomers in blood samples during and after surgery via headspace gas chromatography–mass spectrometry

The quantitative analysis of the chiral volatile anesthetic isoflurane (1) for biomedical applications by means of enantioselective gas chromatography (mass sensitive detector, selected ion monitoring) was studied. Two methods for the quantification of the enantiomers in blood samples drawn during and after narcosis were compared. Either the isomeric enflurane (2) was selected as an internal standard or a single enantiomer of 1 was used for the standard addition method, an approach referred to as ‘enantiomer labeling’. Concentrations up to 0.3 μmol/l of the single enantiomers could be differentiated two days after anesthesia. The presented data imply that the body clearance for (+)-(S)-1 and (−)-(R)-1 proceeds to a measurable degree of enantioselectivity.