Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A new protocol of analyzing isotope-coded affinity tag data from high-resolution LC-MS spectrometry.

ABSTRACT: Isotope-coded affinity tags (ICAT) is a labeling technique that provides insights into quantitative molecular changes. In this paper, we propose a new protocol to identify and analyze ICAT labeled peak pairs in high-resolution LC-MS data. Our major contributions are: (1) we use isotope distance constraint, ICAT distance constraint, and LC-span constraint to identify ICAT labeled peak pairs and (2) we propose to trigger tandem MS/MS scanning based on the ratio estimation value of identified ICAT peak pairs instead of the peak intensity values. Compared with current approaches that choose peaks with high intensity values for tandem MS/MS scanning, the new protocol can improve the scanning efficiency and accuracy.

SUBMITTER: Yu W

PROVIDER: S-EPMC4878675 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A new protocol of analyzing isotope-coded affinity tag data from high-resolution LC-MS spectrometry.

Yu Weichuan W Liu Junfeng J Colangelo Chris C Gulcicek Erol E Zhao Hongyu H

Computational biology and chemistry 20070320 3

Isotope-coded affinity tags (ICAT) is a labeling technique that provides insights into quantitative molecular changes. In this paper, we propose a new protocol to identify and analyze ICAT labeled peak pairs in high-resolution LC-MS data. Our major contributions are: (1) we use isotope distance constraint, ICAT distance constraint, and LC-span constraint to identify ICAT labeled peak pairs and (2) we propose to trigger tandem MS/MS scanning based on the ratio estimation value of identified ICAT ...[more]

PMID: 17499548

Similar Datasets

Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies.

Project description:Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.

| S-EPMC1867626 | biostudies-literature

Comparative glycomics using a tetraplex stable-isotope coded tag.

Project description:This study illustrates the utility of tetraplex stable isotope coded tags in mass spectrometric glycomics using three carbohydrate classes. The teteraplex tags allow for the direct comparison of glycan compositions within four samples using capillary scale hydrophilic interaction chromatography with online mass spectrometry. In addition, the ability to discern glycan structural isomers is shown based on the tandem mass spectra of each composition using nanospray ionization. Results are shown for chondroitin sulfate proteoglycans, low molecular weight heparins, full length heparins, and N-glycans from alpha-1-acid glycoproteins from four mammalian species. The data demonstrate the value of the tetraplex stable isotope tagging approach for producing high-quality glycomics compositional profiling and fine structural analysis.

| S-EPMC2854645 | biostudies-literature

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.

Project description:An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naïve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.

| S-EPMC1444949 | biostudies-literature

Affinity Purification Coupled to Stable Isotope Dilution LC-MS/MS Analysis to Discover IgG4 Glycosylation Profiles for Autoimmune Pancreatitis.

Project description:Type 1 autoimmune pancreatitis (AIP) is categorized as an IgG4-related disease (IgG4-RD), where a high concentration of plasma IgG4 is one of the common biomarkers among patients. IgG Fc-glycosylation has been reported to be potential biosignatures for diseases. However, human IgG3 and IgG4 Fc-glycopeptides from populations in Asia were found to be isobaric ions when using LC-MS/MS as an analytical tool. In this study, an analytical workflow that coupled affinity purification and stable isotope dilution LC-MS/MS was developed to dissect IgG4 glycosylation profiles for autoimmune pancreatitis. Comparing the IgG4 and glycosylation profiles among healthy controls, patients with pancreatic ductal adenocarcinoma (PDAC), and AIP, the IgG4 glycosylations from the AIP group were found to have more digalactosylation (compared to PDAC) and less monogalactosylation (compared to HC). In addition, higher fucosylation and sialylation profiles were also discovered for the AIP group. The workflow is efficient and selective for IgG4 glycopeptides, and can be used for clinical biosignature discovery.

| S-EPMC8584136 | biostudies-literature

Comparison of a label-free quantitative proteomic method based on peptide ion current area to the isotope coded affinity tag method.

Project description:Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA) and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT) method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

| S-EPMC2623286 | biostudies-literature

Isotope-Coded Labeling for Accelerated Protein Interaction Profiling Using MS.

Project description:Protein interaction surface mapping using MS is widely applied but comparatively resource-intensive. Here, a workflow adaptation for use of isotope-coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible.

| S-EPMC4535432 | biostudies-literature

Proteomic profiling of lipopolysaccharide-activated macrophages by isotope coded affinity tagging.

Project description:Lipopolysaccharide (LPS), a glycolipid component of the outer membranes of Gram-negative bacteria, initiates proinflammatory, proapoptotic, and antiapoptotic pathways upon binding to macrophage TLR4. Macrophages that are exposed to LPS become activated and exhibit altered morphology and response to infection. We performed isotope coded affinity tagging (ICAT), multidimensional liquid chromatography, and mass spectrometry to identify proteins that are differently expressed between naive and LPS-activated macrophages. We performed replicate ICAT analyses on RAW 264.7 cultured mouse macrophages as well as C57BL/6 bone marrow derived mouse macrophages. We identified and obtained relative abundances for 1064 proteins, of which we identified 36 as having significantly different expression levels upon activation by LPS. We also compared our results with a two color microarray gene expression assay performed by the Institute for Systems Biology and observed approximately 75% agreement between mRNA transcription and protein expression regarding up- or down-regulation of gene products. We used Western blot analysis to confirm the findings of ICAT and mRNA for one protein, sequestosome 1, the cellular concentration of which was observed to increase upon activation by LPS.

| S-EPMC3519239 | biostudies-literature

Chemotaxonomic profiling of Penicillium setosum using high-resolution mass spectrometry (LC-Q-ToF-MS).

Project description:In the present study, secondary metabolites produced by an endophytic fungus Penicillium setosum were extracted using colony agar plug and culture broth extraction methods. High resolution LC-MS was used to explore the chemical nature of the secondary metabolites, as well, compare the reliability of the methods. P. setosum was chemotaxonomically distinguished from other members of section Lanata-divaricata, by its ability to produce mycotoxin, patulin and also by the presence of certain phenol-derived compounds, like quercetin, dihydroflavonols (dihydroquercetin and dihydromyricetin), kaempferol, luteolin, while some Penicillium specific compounds such as, citromycetin and andrastin D reveal its similarity towards section Lanata-Divaricata members. For the first time, the presence of dihydroquercetin is remarkably and spectrometrically confirmed from a microbial source. In addition, a few polyketides, anthroquinone compounds, hydrocarbons, and fatty acids were also detected in the culture extract. Being the first report on the production of polyphenolic compounds by an endophytic fungus of Penicillium species, the current research is crucial, and moreover the starin itself is a novel species.

| S-EPMC6819834 | biostudies-literature

White spot syndrome virus proteins and differentially expressed host proteins identified in shrimp epithelium by shotgun proteomics and cleavable isotope-coded affinity tag.

Project description:Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twenty-eight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, > or =20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.

| S-EPMC2168766 | biostudies-literature

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

Project description:Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

| S-EPMC1403294 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data