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.     

Amine-reactive isobaric tagging reagents: requirements for absolute quantification of proteins and peptides

Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented.     

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.     

同位素标记相对和绝对定量蛋白组技术研究进展

近年来定量蛋白组学技术迅速发展,目前常用的有双向荧光差异凝胶电泳、同位素亲和标记、15N同位素标记、同位素标记相对和绝对定量和细胞培养条件下稳定同位素标记技术等。同位素标记相对和绝对定量技术以其高通量、高灵敏度、高重复性、高动态检测限和能对各种复杂样品进行相对和绝对定量研究等优势而备受研究者青睐,目前已发展到在同一实验中分析8组样品,增加了实验设计的灵活性。我们就同位素标记相对和绝对定量技术在定量蛋白组史中的地位作用、研究策略,以及在病毒致病机制研究和医药临床相关问题中的应用做简要综述。     

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.     

Higher-energy collision-activated dissociation without a dedicated collision cell

Beam-type collisional activation dissociation (HCD) offers many advantages over resonant excitation collision-activated dissociation, including improved identification of phosphorylated peptides and compatibility with isobaric tag-based quantitation (e.g. tandem mass tag (TMT) and iTRAQ). However, HCD typically requires specially designed and dedicated collision cells. Here we demonstrate that HCD can be performed in the ion injection pathway of a mass spectrometer with a standard atmospheric inlet (iHCD). Testing this method on complex peptide mixtures revealed similar identification rates to collision-activated dissociation (2883 versus 2730 IDs for iHCD/CAD, respectively) and precursor-product-conversion efficiency comparable to that achieved within a dedicated collision cell. Compared with pulsed-q dissociation, a quadrupole ion trap-based method that retains low-mass isobaric tag reporter ions, iHCD yielded isobaric tag for relative and absolute quantification reporter ions 10-fold more intense. This method involves no additional hardware and can theoretically be implemented on any mass spectrometer with an atmospheric inlet.     

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.     

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.     

Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS3 acquisition

Recently, we reported a novel proteomics quantitation scheme termed “combined precursor isotopic labeling and isobaric tagging (cPILOT)” that allows for the identification and quantitation of nitrated peptides in as many as 12–16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co‐isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple‐stage mass spectrometry (MS³) data acquisition, a selective MS³ method that focuses on product selection of the y₁ fragment of lysine‐terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS³ acquisition methods.     

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).     

Performance of isobaric and isotopic labeling in quantitative plant proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.     

Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells.     

IsobariQ: software for isobaric quantitative proteomics using IPTL, iTRAQ, and TMT

Isobaric peptide labeling plays an important role in relative quantitative comparisons of proteomes. Isobaric labeling techniques utilize MS/MS spectra for relative quantification, which can be either based on the relative intensities of reporter ions in the low mass region (iTRAQ and TMT) or on the relative intensities of quantification signatures throughout the spectrum due to isobaric peptide termini labeling (IPTL). Due to the increased quantitative information found in MS/MS fragment spectra generated by the recently developed IPTL approach, new software was required to extract the quantitative information. IsobariQ was specifically developed for this purpose; however, support for the reporter ion techniques iTRAQ and TMT is also included. In addition, to address recently emphasized issues about heterogeneity of variance in proteomics data sets, IsobariQ employs the statistical software package R and variance stabilizing normalization (VSN) algorithms available therein. Finally, the functionality of IsobariQ is validated with data sets of experiments using 6-plex TMT and IPTL. Notably, protein substrates resulting from cleavage by proteases can be identified as shown for caspase targets in apoptosis.     

iTRAQ标记技术与差异蛋白质组学的生物标志物研究

结合多维液相色谱和串联质谱分析,iTRAQ技术已成为差异蛋白质组学定量研究的主要工具之一。而寻找和发现区别于正常生理状态下的疾病特异表达蛋白质,有利于阐明疾病的发病机理,对疾病的预防、诊断、预后和疗效监测具有重要作用,并有助于用作新靶点来开发临床治疗药物。本文重点就该技术在医学领域中进行差异蛋白质组分析并寻找标记蛋白质的研究进行综述。     

LTQ‐iQuant: A freely available software pipeline for automated and accurate protein quantification of isobaric tagged peptide data from LTQ instruments

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.     

Electron transfer dissociation of iTRAQ labeled peptide ions

Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.     

Preprocessing Significantly Improves the Peptide/Protein Identification Sensitivity of High-resolution Isobarically Labeled Tandem Mass Spectrometry Data

Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994.Mass spectrometry-based proteomics has been widely applied to investigate protein mixtures derived from tissue, cell lysates, or from body fluids (1, 2). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)¹ is the most popular strategy for protein/peptide mixtures analysis in shotgun proteomics (3). Large-scale protein/peptide mixtures are separated by liquid chromatography followed by online detection by tandem mass spectrometry. The capabilities of proteomics rely greatly on the performance of the mass spectrometer. With the improvement of MS technology, proteomics has benefited significantly from the high-resolution and excellent mass accuracy (4). In recent years, based on the higher efficiency of higher energy collision dissociation (HCD), a new “high–high” strategy (high-resolution MS as well as MS/MS(tandem MS)) has been applied instead of the “high–low” strategy (high-resolution MS, i.e. in Orbitrap, and low-resolution MS/MS, i.e. in ion trap) to obtain high quality tandem MS/MS data as well as full MS in shotgun proteomics. Both full MS scans and MS/MS scans can be performed, and the whole cycle time of MS detection is very compatible with the chromatographic time scale (5).High-resolution measurement is one of the most important features in mass spectrometric application. In this high–high strategy, high-resolution and accurate spectra will be achieved in tandem MS/MS scans as well as full MS scans, which makes isotopic peaks distinguishable from one another, thus enabling the easy calculation of precise charge states and monoisotopic mass. During an LC-MS/MS experiment, a multiply charged precursor ion (peptide) is usually isolated and fragmented, and then the multiple charge states of the fragment ions are generated and collected. After full extraction of peak lists from original tandem mass spectra, the commonly used search engines (i.e. Mascot (6), Sequest (7)) have no capability to distinguish isotopic peaks and recognize charge states, so all of the product ions are considered as all charge state hypotheses during the database search for protein identification. These multiple charge states of fragment ions and their isotopic cluster peaks can be incorrectly assigned by the search engine, which can cause false peptide identification. To overcome this issue, data preprocessing of the high-resolution MS/MS spectra is required before submitting them for identification. There are usually two major preprocessing steps used for high-resolution MS/MS data: deisotoping and deconvolution (8, 9). Deisotoping of spectra removes all isotopic peaks except monoisotopic peaks from multi-isotopic peaks. Deconvolution of spectra translates multiply charged ions to singly charged ions and also accumulates the intensity of fragment ions by summing up all the intensities from their multiply charged states. After performing these two data-preprocessing steps, the resulting spectra is simpler and cleaner and allows more precise database searching and accurate bioinformatics analysis.With the capacity to analyze multiple samples simultaneously, stable isotope labeling approaches have been widely used in quantitative proteomics. Stable isotope labeling approaches are categorized as metabolic labeling (SILAC, stable isotope labeling by amino acids in cell culture) and chemical labeling (10, 11). The peptides labeled by the SILAC approach are quantified by precursor ions in full MS spectra, whereas peptides that have been isobarically labeled using chemical means are quantified by reporter ions in MS/MS spectra. There are two similar isobaric chemical labeling methods: (1) isobaric tag for relative and absolute quantification (iTRAQ), and (2) tandem mass tag (TMT) (12, 13). These reagents contain an amino-reactive group that specifically reacts with N-terminal amino groups and epilson-amino groups of lysine residues to label digested peptides in a typical shotgun proteomics experiment. There are four different channels of isobaric tags: TMT two-plex, iTRAQ four-plex, TMT six-plex, and iTRAQ eight-plex (12–16). The number before “plex” denotes the number of samples that can be analyzed by the mass spectrum simultaneously. Peptides labeled with different isotopic variants of the tag show identical or similar mass and appear as a single peak in full scans. This single peak may be selected for subsequent MS/MS analysis. In an MS/MS scan, the mass of reporter ions (114 to 117 for iTRAQ four-plex, 113 to 121 for iTRAQ eight-plex, and 126 to 131for TMT six-plex upon CID or HCD activation) are associated with corresponding samples, and the intensities represent the relative abundances of the labeled peptides. Meanwhile, the other ions from the MS/MS spectra can be used for peptide identification. Because of the multiplexing capability, isobaric labeling methods combined with bottom-up proteomics have been widely applied for accurate quantification of proteins on a global scale (14, 17–19). Although mostly associated with peptide labeling, these isobaric labeling methods have also been applied at protein level (20–23).For the proteomic analysis of isobarically labeled peptides/proteins in “high–high” MS strategy, the common consensus is that accurate reporter ions can contribute to more accurate quantification. However, there is no evidence to show how the ions related to isobaric labeling affect the peptide/protein identification and what preprocessing steps should be taken for high-resolution isobarically labeled MS/MS. To demonstrate the effectiveness and importance of preprocessing, we examined how the combination of preprocessing steps improved peptide/protein sensitivity in database searching. Several combinatorial ways of data-preprocessing were applied for high-throughput data analysis including deisotoping to keep simple monoisotopic mass peaks, deconvolution of ions with multiple charge states, and preservation of top 10 peaks in every 100 Dalton mass range. After systematic analysis of high-resolution isobarically labeled spectra, we further processed the spectra and removed interferential ions that were not related to the peptide. Our results suggested that the preprocessing of isobarically labeled high-resolution tandem mass spectra significantly improved the peptide/protein identification sensitivity.     

Mass spectrometry in plant proteomic analysis

Abstract

The current revolution in proteomics has been generated by the combination of very sensitive mass spectrometers coupled to microcapillary liquid chromatography, specific proteolysis of protein mixtures and software that is capable of searching vast numbers of mass measurements against predicted peptides from sequenced genomes. The challenges of post‐genomic plant biology include characterization of protein function, post‐translational modifications and composition of protein complexes as well as deciphering protein complements in intracellular compartments – proteomes of cell organelles. In this review we summarize the current mass spectrometry methods currently being used in plant proteomics and discuss the various tagging strategies that are being used for purification and proteomic analysis of plant protein complexes.

Abbreviations: BCCD, biotin carboxyl carrier protein domain; CBP, calmodulin‐binding protein; CID, collision‐induced dissociation; ESI, electrospray ionization; EST, expressed sequence tag; FT‐ICR, Fourier transform ion cyclotron resonance; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin; HILEP, hydroponic isotope labelling of entire plants; His, histidine; HPB, HA–PreScission–Biotin; HPLC, high‐performance liquid chromatography; ICAT, isotope‐coded affinity tags; ICPL, isotope‐coded protein label; iTRAQ, isobaric tag for relative and absolute quantification; LC, liquid chromatography; MALDI, matrix‐assisted laser desorption ionization; MBP, maltose‐binding protein; MS, mass spectrometry; SDS‐PAGE, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis; SILAC, stable isotope labelling with amino acids in cell culture; SILIP, stable isotope labelling in planta; Strep, streptavidin; TAP, tandem affinity purification; TBP, TATA‐box‐binding protein; TOF, time‐of‐flight; UPLC, ultraperformance liquid chromatography     

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.