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1.
Stable isotopes are now widely used to quantify concentration changes in proteomics. This paper focuses on the resolution of isotopically coded peptides and how isotope effects occurring during chromatographic separations can be minimized. Heavy isotope derivatizing agents used in this work were the commercially available 2H8-ICAT reagent and 13C4-succinic anhydride. The ICAT reagent derivatizes cysteine-containing peptides, whereas the succinic anhydride reacts with primary amine groups in peptides. It was observed during reversed-phase chromatography of peptides from a BSA tryptic digest differentially labeled with the 2Hr and 2H8-ICAT reagents that resolution of the isoforms exceeded 0.5 with 20% of the peptides in the digest. Three-fourths of the peptides in this group contained two cysteine residues and were doubly labeled. Only 23% of the peptides labeled with a single ICAT residue had a resolution greater than 0.4. The resolution of peptides differentially labeled with 13C- and 12C-succinate never exceeded +/- 0.01, even in the case of peptides from the BSA digest labeled with 2 mol of succinate. Because this value is within the limits of the method used to determine resolution, it was concluded the 13C- and 12C-coded isoforms of labeled peptides did not resolve. The isotope ratio in the case of 13C/12C coding could be determined from a single mass spectrum taken at any point in the elution profile. This enabled isotope ratio analysis to be completed early in the elution of a peptide from chromatography columns. 相似文献
2.
Loss of ICAT gene function leads to arrest of ureteric bud branching and renal agenesis 总被引:1,自引:0,他引:1
Hasegawa Y Satoh K Iizuka-Kogo A Shimomura A Nomura R Akiyama T Senda T 《Biochemical and biophysical research communications》2007,362(4):988-994
ICAT, inhibitor of beta-catenin and T cell factor, or Ctnnbip1, is a negative regulator of the Wnt signaling pathway that interferes with the interaction between beta-catenin and T cell factor. Some ICAT-deficient (ICAT-/-) embryos exhibit unilateral or bilateral renal agenesis. In this study, we investigated developmental processes in the ICAT-/- kidney. ICAT was highly expressed in both the ureteric bud (UB) and the surrounding metanephric mesenchymal (MM) cells in the metanephros of embryonic day E11.5-E13.5 wild-type (ICAT+/+) mouse. In the E12.5-ICAT-/- metanephros, UB branching was delayed, and a T-shaped, bifurcated UB was frequently seen; this was never seen in the E12.5-ICAT+/+ metanephros. More apoptotic MM cells were detected in the ICAT-/- metanephros than in the ICAT+/+ metanephros. These results suggest that the loss of ICAT gene function causes the arrest of UB branching and the apoptotic death of MM cells, resulting in renal agenesis. 相似文献
3.
Yi EC Li XJ Cooke K Lee H Raught B Page A Aneliunas V Hieter P Goodlett DR Aebersold R 《Proteomics》2005,5(2):380-387
Quantitative protein profiling using the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes (13)C substitution for (12)C in the heavy-ICAT reagent rather than (2)H (for (1)H) as in the original reagent, was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collision induced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses (13)C as the "heavy" element rather than (2)H, thus, eliminating the slight delay in retention time of ICAT-labeled "light" peptides on a C18-based HPLC separation that occurs with (2)H and (1)H. Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification. 相似文献
4.
Synthesis of acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in proteomic expression profiling analysis 总被引:1,自引:0,他引:1
A convenient synthesis of some homologous light isotope-coded affinity tags (ICAT-L) containing an acid-labile moiety between the affinity component biotin and an electrophilic polar linker is described. These light ICAT reagents give smooth mass spectral signals in tandem mass spectrometry (MS/MS) analyses of some commercially available cysteine-containing peptides. However, these ICAT molecules are designed for use in identification and relative quantification of whole or partially purified cellular and tissue proteomes. Since the biotin moiety can be readily cleaved off the reagent after mass tagging, undesired residual fragmentation patterns caused by biotin of derived peptides, as normally observed using biotin-containing ICAT reagents, are effectively eliminated. This strategy should enhance peptide sequence coverage significantly which, in turn, should result in improving the quality of data obtained during data-dependent peptide mass and tandem mass spectral analysis of whole proteomes. 相似文献
5.
Mario Cannataro Giovanni Cuda Marco Gaspari Sergio Greco Giuseppe Tradigo Pierangelo Veltri 《BMC bioinformatics》2007,8(1):255
Background
Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. 相似文献6.
Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates. 相似文献
7.
In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Following protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identified peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol.Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state ( 2, 3) peptides are likely to be identified, and the hydropathicity of the peptides might not be related to whether it is more likely to be identified or not. The number of identified peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identified peptides present three types of redundancy, namely identification, charge, and sequence redundancy, which may repress low abundance protein identification. 相似文献
8.
Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis 总被引:18,自引:0,他引:18
The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate. 相似文献
9.
Wåhlander A Arrigoni G Wårell K Levander F Palmgren R Maloisel JL Busson P James P 《Journal of proteome research》2007,6(3):1101-1113
We present a generic approach for quantitative differential proteomics that reduces data complexity in proteome analysis by automated selection of peptides for MS/MS analysis according to their isotope-labeling ratio. Isotopic reagents were developed that give products which fragment easily to generate a unique pair of signature ions. Using the ion-pair ratio, we show that it is possible to select only BSA peptides (with a 3:1 light heavy isotope ratio) for MS/MS when spiked in a whole yeast extract using Parent (precursor) Ion Quantitation Scanning (PIQS) for MS/MS. 相似文献
10.
Paba J Ricart CA Fontes W Santana JM Teixeira AR Marchese J Williamson B Hunt T Karger BL Sousa MV 《Journal of proteome research》2004,3(3):517-524
Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes. 相似文献
11.
A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology. 相似文献
12.
Rejtar T Hu P Juhasz P Campbell JM Vestal ML Preisler J Karger BL 《Journal of proteome research》2002,1(2):171-179
High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests. 相似文献
13.
Chiang MC Juo CG Chang HH Chen HM Yi EC Chern Y 《Molecular & cellular proteomics : MCP》2007,6(5):781-797
Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the progression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to quantitatively determine changes in protein expressions in the striatum of a transgenic HD mouse model (R6/2). The cysteine residues of striatal proteins from HD and wild-type mice were labeled, respectively, with the heavy and light forms of the ICAT reagents. Samples were trypsinized, uncovered by avidin affinity chromatography, and analyzed by nano-LC-MS/MS. Western blot analyses were used to confirm and to calibrate the ICAT ratios. Linear regression was used to uncover a group of proteins that exhibited consistent changes. In two independent ICAT experiments, we identified 427 cysteine-containing striatal proteins among which approximately 66% (203 proteins) were detected in both ICAT experiments. Approximately two-thirds of proteins identified in each ICAT experiment were detected in both ICAT experiments. In total, 68 proteins with altered expressions in HD mice were identified. Elevated expressions of two down-regulated proteins (14-3-3sigma and FKBP12) effectively reduced Htt aggregates in a striatal cell line, supporting the functional relevance of the above findings. Collectively by using a well defined protocol for data analysis, large scale comparisons of protein expressions by ICAT can be reliable and can provide valuable clues for identifying proteins critical for pathophysiological functions. 相似文献
14.
Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid chromatography coupled to tandem mass spectrometry. The novel (13)C-labeled ICAT reagents have identical elution profiles for labeled peptide pairs and broadly spread the distribution of labeled peptides during reversed-phase chromatography. A total of 560 proteins were identified and quantified, with 51 displaying more than 2-fold expression differences. In addition to some known proteins involved in salt stress, four RNA-binding proteins were found to be up-regulated by high salinity, suggesting that selective RNA export from the nucleus is important for the salt-stress response. Some proteins involved in amino acid synthesis, which have been observed to be up-regulated by amino acid starvation, were also found to increase their abundance on salt stress. These results indicate that salt stress and amino acid starvation cause overlapping cellular responses and are likely to be physiologically linked. 相似文献
15.
Jing Wu Hai Pu Xiaoqing Ren Mingrui An Shicheng Wei Jianguo Ji 《Analytical biochemistry》2010,398(1):34-44
Serum low-molecular-weight proteins (LMWPs, molecular weight <30 kDa) are closely related to the body physiological and pathological situations, whereas many difficulties are encountered when enriching and fractionating them. Using C18 absorbent (100 Å) enrichment and fractionation under urea/dithiothreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs could be enriched more than 100-fold and were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag (ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) levels, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and LTQ-Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). By virtue of its easy operation and high reproducibility to process large quantity complex serum samples, this method has potential uses in enriching LMWPs either in serum or in cell and tissue samples. 相似文献
16.
Moulder R Filén JJ Salmi J Katajamaa M Nevalainen OS Oresic M Aittokallio T Lahesmaa R Nyman TA 《Proteomics》2005,5(11):2748-2760
The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed. 相似文献
17.
Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research 总被引:3,自引:0,他引:3
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. 相似文献
18.
A pair of ICAT reagents, N-(13-iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)biotinamide (8d, ICAT-d(8)) and N-(13-iodoacetamido-4,7,10-trioxa-tridecanyl)biotinamide (8c, ICAT-d(0)), and an alternative pair of ICAT reagents, N-(10-iodoacetamido-2,5,5,6,6,9-hexadeutero-4,7-dioxa-decanyl)biotinamide (8b, s-ICAT-d(6)) and N-(10-iodoacetamido-4,7-dioxa-decanyl)biotinamide (8a, s-ICAT-d(0)), were successfully synthesized. A mixture of sodium borohydride and cobalt(II) chloride reduced the intermediate dinitrile to the diamine without loss of the deuterium labels, which occurred when Raney nickel was the reducing agent. The problem caused by unsymmetrical biotinylation of the intermediate diamine was solved by using the solid-phase method in which one end of the diamine was attached to a chlorotrityl chloride resin, followed by biotinylation of the resin-bound amine. The self-alkylation of ICAT reagents that accounted for their instability and their limitations in the applications was also studied. 相似文献
19.
Bienvenut WV Déon C Pasquarello C Campbell JM Sanchez JC Vestal ML Hochstrasser DF 《Proteomics》2002,2(7):868-876
Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses. 相似文献
20.
In this study we developed a quantitative proteomic method named ICAT switch by introducing isotope-coded affinity tag (ICAT) reagents into the biotin-switch method, and used it to investigate S-nitrosation in the liver of normal control C57BL/6J mice and type 2 diabetic KK-Ay mice. We got fifty-eight S-nitrosated peptides with quantitative information in our research, among which thirty-seven had changed S-nitrosation levels in diabetic mouse liver. The S-nitrosated peptides belonged to forty-eightproteins(twenty-eightwerenewS-nitrosated proteins), some of which were new targets of S-nitrosation and known to be related with diabetes. S-nitrosation patterns were different between diabetic and normal mice. Gene ontology enrichment results suggested that S-nitrosated proteins are more abundant in amino acid metabolic processes. The network constructed for S-nitrosated proteins by text-mining technology provided clues about the relationship between S-nitrosation and type 2 diabetes. Our work provides a new approach for quantifying S-nitrosated proteins and suggests that the integrative functions of S-nitrosation may take part in pathophysiological processes of type 2 diabetes. 相似文献