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1.
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.  相似文献   

2.
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.  相似文献   

3.
In this paper, we describe the use of iTRAQ (isobaric Tags for Relative and Absolute Quantitation) tags for comparison of protein expression levels between multiple samples. These tags label all peptides in a protein digest before labeled samples are pooled, fractionated and analyzed using mass spectrometry (MS). As the tags are isobaric, the intensity of each peak is the sum of the intensity of this peptide from all samples, providing a moderate enhancement in sensitivity. On peptide fragmentation, amino-acid sequence ions also show this summed intensity, providing a sensitivity enhancement. However, the distinct distribution of isotopes in the tags is such that, on further fragmentation, a tag-specific reporter ion is released. The relative intensities of these ions represent the relative amount of peptide in the analytes. Integration of the relative quantification data for the peptides allows relative quantification of the protein. This protocol discusses the rationale behind design, optimization and performance of experiments, comparing protein samples using iTRAQ chemistries combined with strong cation exchange chromatographic fractionation and MS.  相似文献   

4.
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.  相似文献   

5.
Pachl F  Fellenberg K  Wagner C  Kuster B 《Proteomics》2012,12(9):1328-1332
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.  相似文献   

6.
Methods for isobaric tagging of peptides, iTRAQ or TMT, are commonly used platforms in mass spectrometry based quantitative proteomics. These two methods are very often used to quantitate proteins in complex samples, e.g., serum/plasma or CSF supporting biomarker discovery studies. The success of these studies depends on multiple factors, including the accuracy of ratios of reporter ions reflecting quantitative changes of proteins. Because reporter ions are generated during peptide fragmentation, the differences of chemical structure of iTRAQ balance groups may have an effect on how efficiently these groups are fragmented and thus how differences in protein expression will be measured. Because 4-plex and 8-plex iTRAQ reagents do have different structures of balanced groups, it has been postulated that indeed differences in protein identification and quantitation exist between these two reagents. In this study we controlled the ratios of tagged samples and compared quantitation of proteins using 4-plex versus 8-plex reagents in the context of a highly complex sample of human plasma using ABSciex 4800 MALDI-TOF/TOF mass spectrometer and ProteinPilot 4.0 software. We observed that 8-plex tagging provides more consistent ratios than 4-plex without compromising protein identification, thus allowing investigation of eight experimental conditions in one analytical experiment.  相似文献   

7.
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.  相似文献   

8.
9.
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.  相似文献   

10.
Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI,1 the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 μg protein as the starting material.  相似文献   

11.
草菇子实体不同成熟阶段的比较蛋白质组学分析   总被引:1,自引:0,他引:1  
采用iTRAQ标记结合二维液相色谱串联质谱技术对草菇不同成熟阶段的差异蛋白质组进行研究。首先将提取的草菇不同成熟阶段蛋白样品进行SDS-PAGE分析,其次将经二维液相色谱串联质谱技术获取的串联质谱数据通过MASCOT软件搜库,之后对鉴定蛋白质数据进行了KEGG代谢通路分析。试验共计获得2 335个不同肽段, 鉴定到1 039个蛋白质,其中1 030个蛋白质具有定量信息。与蛋形期相比,在伸长期和成熟期阶段显著上调蛋白质85个,下调蛋白质103个。KEGG代谢通路分析结果显示,草菇不同成熟阶段中的68个差异蛋白质可定位于4种伞菌目模式真菌(灰盖鬼伞、双色蜡蘑、可可丛枝病菌和裂褶菌)的45个不同生物代谢途径,全景展示出草菇成熟阶段差异表达蛋白质定位的代谢网络。结果表明,iTRAQ标记技术结合二维液相色谱串联质谱可对不同生长发育时期的草菇蛋白样品进行有效地分离和鉴定。  相似文献   

12.
White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.  相似文献   

13.
In this experimental study, differential labeling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) proteomic approach was used to investigate differences in the proteome of rat spinal cord at 24 h following a moderate contusion injury. Spinal cord protein samples from the injury epicenter (or from sham controls) were trypsinized and differentially labeled with iTRAQ isotopic reagents. The differentially labeled samples were then combined into one sample mixture, separated by LC, and analyzed using MS/MS. Proteins were quantified by comparing the peak areas of iTRAQ reporter fragment ions in MS/MS spectra. The outcome of this analysis revealed that proteins involved in ubiquitination, endocytosis and exocytosis, energy metabolism, inflammatory response, oxidative stress, cytoskeletal disruption, and vascular damage were significantly altered at 24 h following spinal cord injury (SCI). This study demonstrates the utility of the iTRAQ method in proteomic studies and provides further insights into secondary events that occur during acute times following SCI.  相似文献   

14.
Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.Stable isotope labeling techniques have become very popular in recent years to perform quantitative mass spectrometry experiments with high precision and accuracy. In contrast to label-free approaches, multiplexed isotopically labeled samples can be simultaneously analyzed resulting in increased reproducibility and accuracy for quantification of peptides and proteins from different biological states. Isotopic labeling strategies can be grouped into two major categories: isobaric labels and nonisobaric labels. In the former category are iTRAQ1 (isobaric tags for relative and absolute quantification (1)) and TMT (tandem mass tags (2)) mass tags. In the nonisobaric labeling category are methods such as mTRAQ (mass differential tags for relative and absolute quantification), stable isotope labeling by amino acids in cell culture (SILAC (3)), and reductive dimethylation (4). Isobaric labeling techniques allow relative quantification of peptides based on ratios of low m/z reporter ions produced by fragmentation of the precursor ion, whereas nonisobaric labeling yields precursors with different masses that can be directly quantified from MS1 intensity. iTRAQ and mTRAQ reagents provide a great opportunity to directly compare capabilities of reporter and precursor ion quantification since both labels have identical chemical structures and differ only in their composition and number of 13C, 15N, and 18O atoms. In fact, iTRAQ-117 and mTRAQ-Δ4 are identical mass tags with a total mass of 145 Da (Fig. 1A). To achieve 4-plex quantification capabilities for iTRAQ labels, the composition of stable isotopes is arranged in a way to obtain the reporter ion/balancing group pairs 114/31, 115/30, 116/29, and 117/28 (1). Three nonisobaric mTRAQ labels were generated by adding or removing four neutrons to the mTRAQ-Δ4 label resulting in mTRAQ-Δ8 and mTRAQ-Δ0, respectively. Both iTRAQ and mTRAQ reagents are available as N-hydroxy-succinimide esters to facilitate primary amine labeling of peptides.Open in a separate windowFig. 1.A, Labeling strategy for comparative evaluation of iTRAQ and mTRAQ tags. Peptides were labeled with the indicated iTRAQ and mTRAQ reagents for combined phosphoproteome and proteome analysis. B, Selection of optimal instrument methods for analysis of iTRAQ- and mTRAQ-labeled peptides. Unfractionated proteome samples (1 ug) and phosphoproteome samples (enriched from 250 μg peptides) were analyzed for iTRAQ samples with a CID/HCD-Top8 method, whereas for mTRAQ we compared CID-Top16 acquisition to HCD-Top8. Note that duty cycle times were for all instrument methods ∼3.1 s.One potential advantage of an iTRAQ labeling strategy is its additive effect on precursor intensities when samples are multiplexed, resulting in increased sensitivity. However, iTRAQ ratios have been demonstrated to be prone to compression. This occurs when other nonregulated background peptides are co-isolated and cofragmented in the same isolation window of the peptide of interest and contribute fractional intensity to the reporter ions in MS2-scans (57). Because most peptides in an experiment are present at 1:1:1:1 ratios between multiplexed samples, all ratios in the experiment tend to be dampened toward unity when cofragmentation occurs. This inaccuracy led to the development of mTRAQ labels to facilitate accurate precursor-based quantification of proteins initially identified in iTRAQ discovery experiments with targeted assays, such as multiple reaction monitoring (MRM) (8). Although iTRAQ has been widely used in discovery-based proteomics studies, mTRAQ has only appeared in a small number of studies thus far (8).In this study we investigated the advantages and disadvantages of iTRAQ and mTRAQ labeling for proteome-wide analysis of protein phosphorylation and expression changes. We selected epidermal growth factor (EGF)-stimulated HeLa cells as a model system for our comparative evaluation of iTRAQ and mTRAQ labeling, as both changes in the phosphoproteome (9) as well as the proteome (10) are well described for EGF stimulation. We show that iTRAQ labeling yields superior results to mTRAQ in terms of numbers of quantified phosphopeptides, proteins and regulated components. By means of spike-in experiments with GluC generated peptides of known ratios we find that iTRAQ quantification is more precise but less accurate than mTRAQ due to ratio compression. We identify a linear relationship of observed versus expected logarithmic GluC generated peptide ratios as well as for logarithmic iTRAQ and mTRAQ ratios of the phosphoproteome and proteome analysis. This indicates a uniform degree of ratio compression over two orders of magnitude throughout iTRAQ data sets and explains why iTRAQ ratio compression does not compromise the ability to detect regulated elements in these experiments.  相似文献   

15.
S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.  相似文献   

16.
Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site‐specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong‐cation exchange, the peptides were analyzed by LC‐MS/MS on an Orbitrap mass spectrometer, which collects CID and high‐energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.  相似文献   

17.
MOTIVATION: Mass spectrometry (MS) data are impaired by noise similar to many other analytical methods. Therefore, proteomics requires statistical approaches to determine the reliability of regulatory information if protein quantification is based on ion intensities observed in MS. RESULTS: We suggest a procedure to model instrument and workflow-specific noise behaviour of iTRAQ reporter ions that can provide regulatory information during automated peptide sequencing by LC-MS/MS. The established mathematical model representatively predicts possible variations of iTRAQ reporter ions in an MS data-dependent manner. The model can be utilized to calculate the robustness of regulatory information systematically at the peptide level in so-called bottom-up proteome approaches. It allows to determine the best fitting regulation factor and in addition to calculate the probability of alternative regulations. The result can be visualized as likelihood curves summarizing both the quantity and quality of regulatory information. Likelihood curves basically can be calculated from all peptides belonging to different regions of proteins if they are detected in LC-MS/MS experiments. Therefore, this approach renders excellent opportunities to detect and statistically validate dynamic post-translational modifications usually affecting only particular regions of the whole protein. The detection of known phosphorylation events at protein kinases served as a first proof of concept in this study and underscores the potential for noise models in quantitative proteomics.  相似文献   

18.
iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150–2200 m/z) and reporter ions (low mass region, 113–121 m/z) for protein identification and relative quantification, respectively. After collision‐induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR‐Qq‐ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis‐specific parameters, their impact of quantification and identification, and their cross‐interactions.  相似文献   

19.
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.  相似文献   

20.
【目的】旨在采用iTRAQ标记结合二维液相色谱串联质谱技术对草菇不同生长发育阶段的差异蛋白质组进行研究。【方法】首先将提取的草菇不同生长阶段蛋白样品进行SDS-PAGE分析,其次将经二维液相色谱串联质谱技术获取的串联质谱数据通过MASCOT软件搜库,之后对鉴定蛋白质数据进行了主成分分析(Principal componentanalysis,PCA)、层次聚类(Hierarchy clustering)分析、K-均值(K-means)聚类和GeneOntology(GO)注释分析。【结果】试验结果显示,共计获得2 335个不同肽段,鉴定到1 039个蛋白质,其中1 030个蛋白质具有定量信息。在子实体阶段中显著上调蛋白质64个,下调蛋白质150个。生物信息学分析表明,iTRAQ标记技术结合二维液相色谱串联质谱可对不同生长发育时期的草菇蛋白样品进行有效地分离和鉴定。【结论】这一研究结果为深入研究草菇乃至其他大型担子菌子实体形成和发育的分子机制提供借鉴。  相似文献   

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