Isotope-coded Affinity Tag Research Articles

Analytical Characteristics of Cleavable Isotope-Coded Affinity Tag-LC-Tandem Mass Spectrometry for Quantitative Proteomic Studies

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.

Proteomic analyses to identify novel therapeutic targets for the treatment of advanced prostate cancer.

At present there is no cure for advanced prostate cancer once it progresses to an androgen independent stage. Hormonal therapy, radiotherapy, and chemotherapy all have limited durations of efficacy for men diagnosed with androgen independent disease and patients will succumb over a period of several months to two years. The androgen receptor (AR) has been suspected to play an important role in the mechanism of progression to androgen independence. This is because the AR is a transcription factor that 'normally' mediates the effects of androgen to regulate expression of genes involved in proliferation and survival of prostate cells. Thus identifying and characterizing the proteins that interact with the AR to facilitate an activated receptor is of critical importance. Proteomic approaches such as isotope-coded affinity tags (ICAT), isotope Tags for Relative and Absolute Quantification (iTRAQ)(TM), Stable Isotope Labeling with Amino acids in Cell culture (SILAC), Tandem Affinity Purification (TAP) of tagged proteins (TAP-tag) and Multidimensional Protein Identification Technology (MudPIT) provide large scale unbiased strategies and have not been previously applied to identify proteins that interact with the AR. Here an example of the power of these proteomic approaches to identify potential therapeutic targets for prostate cancer is provided. Application of MudPIT identified 82 peptides in endogenous complexes immunoprecipitated with the AR from prostate cancer cells. Identification of these novel proteins may ultimately lead to the development of better therapies for the treatment or prevention of advanced prostate cancer.

Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.

P4-145: Proteomics of mitochondria in Alzheimer’s disease brain

Most cellular energy in the form of ATP is produced by mitochondria. Mitochondria also produce free radicals which can damage cellular structures. There is increasing evidence that mitochondrial dysfunction plays an important role in the pathology of a number of neurodegenerative diseases, including Alzheimer's disease (AD). Dysfunction of mitochondrial energy metabolism would lead to reduced ATP production and increased production of free radicals. Our hypothesis is that the proteins of the mitochondrial energy production system are reduced or oxidatively damaged in AD brains. To test this hypothesis, we are using a quantitative proteomics approach to compare the levels of the mitochondrial proteins in AD versus normal brain during the course of the disease. We are examining this issue using genetically altered triple transgenic mice (3xTg–AD; APPSwe, PS1M146V, tauP301L) which develop age–dependent accumulation of both amyloid plaques and neurofibrillary tangles, as well as age–associated memory impairments. Quantitative proteomics will be achieved using isotope–coded affinity tags (cICAT), followed by liquid chromatography and tandem mass spectrometry analysis (LC–MS/MS). Quantitative proteomics of mitochondrial proteins will be carried out using mitochondria isolated from the cortex and hippocampus of wild–type and 3xTg–AD mice at three stages of AD progression: before (2 months old); during (6 months of age); and after (15 months old) the appearance of amyloidal and tangle pathology and cognitive impairment. The findings from these studies should help further define the role of mitochondrial energy dysfunction in the pathogenesis of AD.

Neuroproteomics: Relevance to anxiety disorders

Despite advances in the treatment of anxiety disorders, there is a need for medications with greater efficacy and fewer side effects. Advances in techniques to facilitate high throughput, mass analysis of proteins potentially allows for new drug targets, with a shift in focus from membrane receptor proteins and enzymes of neurotransmitter metabolism to molecules in intracellular signal transduction and other pathways. A computerized literature search was done to collect studies on recently developed proteomic techniques and their application in psychiatric research. Particular techniques, such as two-dimensional electrophoresis, two-dimensional differential gel electrophoresis, isotope-coded affinity tags, and isotope tags for relative and absolute quantification, are reviewed. In addition, a combination of these techniques with MALDI-TOF/TOF and ESI-Q-TOF mass spectrometry analysis is discussed in relation to possible novel signaling pathways relevant to anxiety disorders, and to the development of biomarkers for the evaluation of these conditions.

Concepts and Approaches Towards Understanding the Cellular Redox Proteome

The physiological activity of a significant subset of cell proteins is modified by the redox state of regulatory thiols. The cellular redox homeostasis depends on the balance between oxidation of thiols through oxygen and reactive oxygen species and reduction by thiol-disulfide transfer reactions. Novel and improved methodology has been designed during recent years to address the level of thiol/disulfide regulation on a genome-wide scale. The approaches are either based on gel electrophoresis or on chromatographic techniques coupled to high end mass spectrometry. The review addresses diagonal 2D-SDS-PAGE, targeted identification of specific redox-interactions, affinity chromatography with thioredoxins and glutaredoxins, gel-based and non-gel based labelling techniques with fluorophores (such as Cy3, Cy5, ICy), radioisotopes, or with isotope-coded affinity tags (ICAT), differential gel electrophoresis (DIGE) and combined fractional diagonal chromatography (COFRADIC). The extended methodological repertoire promises fast and new insight into the intricate regulation network of the redox proteome of animals, bacteria, and plants.

Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Discovery in Aging and Alzheimer’s Disease

Systems biology offers enormous potential to understand the complexity of human brain aging and neurodegenerative diseases. Proteomics has an important role in these investigations because of its unique strengths and because of the potential central pathogenic contribution of pathological protein to several of these diseases. Here we have reviewed the methods and presented some examples of liquid chromatography-electrospray ionization-tandem mass spectrometry-based proteomics, with and without quantification using isotope-coded affinity tags, in the investigation of aging and Alzheimer's disease. As protocols and methods for improved quantitative high-throughput proteomics constantly improve, this approach will likely continue to provide deeper insight into human brain aging and neurodegenerative diseases.

Proteomic analysis of opsins and thyroid hormone-induced retinal development using isotope-coded affinity tags (ICAT) and mass spectrometry.

Analyses that reveal the relative abundance of proteins are informative in elucidating mechanisms of retinal development and disease progression. However, popular high-throughput proteomic methods do not reliably detect opsin protein abundance, which serve as markers of photoreceptor differentiation. We utilized thyroid-hormone (TH) treatment of rainbow trout (Oncorhynchus mykiss) as a model of cone apoptosis and cone regeneration. We used this model to investigate if emerging proteomic technology allows effective analysis of retinal development and opsin protein abundance. We also sought to begin a characterization of proteomic changes in the retina occurring with TH treatment and address whether TH affects proliferation or photoreceptor differentiation. Retinal homogenates were prepared from control and TH-treated fish. Peptides from control and treated homogenates were differentially labeled, using isotope-code affinity tags (ICAT) and analyzed using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry (capLC-ESI-MS/MS). This method identifies proteins and quantifies their relative abundance between two samples. The relative abundance of many retinal proteins changed during TH treatment. These included proteins from every functional class. We detected 1,684 different peptides, and our quantification suggests that 94 increased and 146 decreased in abundance more than 50% during TH treatment. Cell-cycle proteins appear to be increased, consistent with TH-inducing cell proliferation, similar to its effect in Xenopus. Other proteins associated with retinal development, such as deltaA and tubulins, changed in abundance during TH treatment. Rod opsin and three cone opsins were identified and the relative abundance of each changed with TH treatment. ICAT and capLC-ESI-MS/MS are an effective complement to other molecular approaches that investigate the mechanisms of retinal development. Unlike other proteomic techniques, this approach does not require development of species- or tissue-specific methodology, such as characterizing two dimensional (2D) gels or antibodies, in order to be practical as a high-throughput approach. Importantly, this technology was able to assess the relative abundance of opsin proteins. These findings represent the first high-throughput proteomic analysis of the retina and demonstrate the technique's ability to provide useful information in retinal development.

Quantitative proteomics identifies a change in glial glutamate metabolism at the time of female puberty

Mammalian puberty requires activation of luteinizing hormone–releasing hormone (LHRH) neurons. In turn, these neurons are controlled by transsynaptic and glia-to-neuron communication pathways, which employ diverse cellular proteins for proper function. We have now used a high throughput relative quantitative proteomics technique to identify such proteins. We selected the method of two-dimensional liquid chromatography tandem mass spectrometry (2DLC–MS/MS) and cleavable isotope-coded affinity tags (cICAT), to both identify and quantify individual proteins within a complex protein mixture. The proteins used derived from the hypothalamus of juvenile (25-day-old) and peripubertal (first proestrus, LP) female rats, and their identity was established by analyzing their mass spectra via database searching. Five proteins involved in glutamate metabolism were detected and two of them appeared to be differentially expressed. They were selected for further analysis, because of their importance in controlling glutamate synthesis and degradation, and their preferential expression in astroglial cells. One, glutamate dehydrogenase (GDH) catalyzes glutamate synthesis; its hypothalamic content detected by 2DLC–MS/MS increases at first proestrus. The other, glutamine synthetase (GS), catalyzes the metabolism of glutamate to glutamine; its content decreases in proestrus. Western blot analysis verified these results. Because these changes suggested an increased glutamate production at puberty, we measured glutamate release from hypothalamic fragments from juvenile 29-day old rats, and from rats treated with PMSG to induce a premature proestrus surge of luteinizing hormone (LH). To determine the net output of glutamate in the absence of re-uptake we used the excitatory amino acid transporter (EAAT) inhibitor l- trans-pyrrolidine-2,4-dicarboxylic acid (PDC). PDC elicited significantly more glutamate- and LHRH-release from the proestrus hypothalamus. Thus, an increase excitatory drive to the LHRH neuronal network provided by glutamatergic inputs of glial origin, is an event contributing to the pubertal activation of LHRH secretion.

Isotope-Coded Affinity Tagging Technique Using Avidin Functional Affinity Electrophoresis: An Alternative to an Avidin Affinity Column

Avidinfunctionalaffinityelectrophoresis(AFAEP)issubstitutedforanavidinaffinitycolumn(AAC) to capture biotinylated peptides in the Isotope-Coded Affinity Tagging (ICAT) technique which is a valuable tool in quantitative proteomics. In this new technique, the AFAEP-captured ICAT-labeled biotinylated peptides are extracted with the biotin tag intact from the polyacrylamide gel piece with aqueous 95% formamide (pH 8.2) at 65C for 20 min, and then detected by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Bovine serum albumin (BSA) and the 12 C- and 13 C-ICAT reagents are used to test this AFAEP-ICAT technique. The results show that both AFAEP and AAC methods providequantitativeinformationoftherelativeamountsof 12 C-and 13 C-ICAT-labeledbiotinylatedtryptic peptides of BSA in a sample. Compared with AAC, the AFAEP is cheaper to perform, more stringent in capturing the biotinylated peptides, and capable of simultaneously processing multiple samples.

Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry

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.

A Proteome Reference Map and Proteomic Analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.

Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry.

Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using (12)C- (control) and (13)C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.

PROTEOMICS TECHNOLOGY AND THERAPEUTICS

1. Proteins are the workhorses of biological systems and, thus, it is not surprising that the field of proteomics has flourished in recent years. The present review briefly surveys the general proteomic approaches taken to identify drug targets and to evaluate drug efficacy and toxicity in the preclinical and clinical settings. These generally fall into three basic categories: a profiling approach, a functional approach and a structural approach. 2. The current 'tools' used and applied to the drug-discovery process will be highlighted. These include the two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, isotope-coded affinity tag and protein biochips. 3. Clearly, proteomics has begun to set a foothold in every stage of the drug-discovery process. Future developments in this area will likely make a significant impact in our quest for better, safer and cost-effective drugs.

Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

BackgroundIsotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer.MethodsPaired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed.Results353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts.ConclusionICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

Degradomics: Systems biology of the protease web. Pleiotropic roles of MMPs in cancer

The major role of matrix metalloproteinases (MMPs) is for homeostatic regulation of the extracellular environment, not simply to degrade matrix as their name suggests. We designed and printed a dedicated, focused DNA microarray, the CLIP-CHIP, that enables the analysis of every human and murine protease, protease homologue and inhibitor on a system-wide basis in cancer. We have also developed novel proteomic approaches to identify cleaved substrates of proteases in complex milieu. Isotope coded affinity tag (ICAT) and iTRAQ labeling of conditioned medium proteins secreted by MDA-MB-231 breast carcinoma cells and Mmp2 -/- murine fibroblasts transfected with protease (MT1-MMP or active MMP-2) or their inactive mutant forms enabled quantitative proteomics to be performed. Comparison of the relative abundance ratios of identical peptides from the two samples identified proteins in the conditioned medium that may have been degraded (low ratios) and those that were shed from the cell membrane (high ratios). MS/MS was used to sequence and identify the potential substrates. These analyses have revealed a plethora of new bioactive substrates and biological roles for MMPs. Biochemical confirmation of cleavage of the potential substrates was performed and the cleavage sites identified by MALDI-TOF. In these studies we discovered and confirmed that CTGF, galectin-1, death receptor-6, HSP90alpha, procollagen C-proteinase enhancer protein, the chemokine fractalkine, and cystatin C were novel MT1-MMP or MMP-2 substrates. These sophisticated cellular control functions highlight new intervention points in multiple pathways to treat early stage cancer.

Relative and absolute quantitative expression profiling of cytochromes P450 using isotope‐coded affinity tags

The development of a novel method for absolute quantification of proteins based on isotope-coded affinity tagging using ICAT reagents is described. The method exploits synthetic peptide standards to determine protein content at the femtomole level in biological samples. The approach is generally applicable to any subset of proteins, but is particularly appropriate for quantitative analysis of multiple, closely related isoforms, and for hydrophobic proteins that are poorly represented in 2-D gels. Relative and absolute quantification techniques are applied to an important group of microsomal metabolic enzymes, the cytochromes P450 (P450), which are critical in determining the disposition, safety and efficacy of drugs in man. Measurement of the P450 induction profile in response to chemicals is a fundamental aspect of drug safety evaluation and is currently achieved by low-throughput methods employing poorly discriminatory antibodies or substrates. Tagging technology is shown to supersede conventional methods for P450 profiling in terms of discriminatory power and throughput, exemplified by the simultaneous detection of distinct induction profiles for cyp2c subfamily members in response to phenobarbitone: cyp2c29 expression, but not cyp2c40 or cyp2c50, was induced threefold by treatment. This technology should abbreviate the drug development pathway, and provide a widely applicable, rapid means of quantifying proteins.

New findings on protein expression in copper deficient rat heart using proteomic approaches

Dietary Cu-deficiency not only causes a hypertrophic cardiomyopathy but also increases cancer risk in rodent models. The present studies were undertaken to determine the effect of Cu-deficiency on protein profiles in rat heart tissue. Male Sprague-Dawley rats were fed diets that were either copper adequate (6.0 μg copper/g diet n = 6) or copper deficient (0.3 μg copper/g diet n = 6) for 5 wk. An approach using liquid chromatography mass spectrometry analysis coupled with DNA gel shift and immunodection dot blotting revealed a 132 kDa protein complex in the protein extract from hearts of Cu-deficient, but not Cu-adequate rats. This complex contained a collagen alpha (I) chain precursor as well as a leucine-rich protein 130 (DNA binding protein). These data suggest that Cu-deficiency may have a potential to influence DNA stability and mRNA transcript processing. Another approach to determine the effect of Cu deficiency on protein profiles used the combination of the isotope-coded affinity tag (ICAT) method and Western blotting analysis. ICAT analysis suggested that protein profiles from heart tissue of Cu-deficient rats were different from those of Cu-adequate rats; seven major protein species differed by more than a 100% increase or a 50% decrease. Western blotting analysis confirmed an 85% increase in fibulin-5 (also known DANCE/EVEC) in Cu-deficient rat heart, and suggested a critical biological function of Cu in controlling vascular redox state, scaffolding of cells to elastic fiber, and preventing elastinopathy in the rat heart.

Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in the brains of Alzheimer’s disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3β, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is the first report of a phosphorylation site on complexin 2.

Comparative Study of Three Proteomic Quantitative Methods, DIGE, cICAT, and iTRAQ, Using 2D Gel- or LC−MALDI TOF/TOF

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.