Isoelectric Focusing In Immobilized pH Gradients Research Articles

Isoelectric focusing array with immobilized pH gradient and dynamic scanning imaging for diabetes diagnosis

A traditional immobilized pH gradient (IPG) has a high stability for isoelectric focusing (IEF) but suffers from time-consuming rehydration, focusing and staining-imaging as well as complex performance. To address these issues, an IEF system with an array of 24 IPG columns (10 mm × 600 μm × 50 μm) and dynamic scanning imaging (DSI) was firstly designed for protein focusing. Moreover, two IPG columns (pH 4–9 and pH 6.7–7.7 of 10 mm in length) were firstly synthesized for IEF. A series of experiments were carried out based on the IEF array. In contrast to a traditional IPG IEF with more than 10 h rehydration, 5–14 h IEF and ca 10 h stain-imaging, the IEF array had the following merits: 25 min rehydration for sample loading, 4 min IEF, and 2 min dynamics scanning of 24 columns, well addressing the issues of traditional IEF. Furthermore, the IEF array had fair sensitivity (LOD of 60 ng), good recovery (95%), and high stability (1.02% RSD for intra-day and 2.16% for inter-day). Finally, the developed array was successfully used for separation and determination of HbA1c (a key biomarker for diabetes diagnosis) in blood samples. All these results indicated the applicability of the developed IEF array to diabetes diagnosis.

Deep coverage of the beer proteome

We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. SignificanceThe study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality.

Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

Proteomics profiling identify CAPS as a potential predictive marker of tamoxifen resistance in estrogen receptor positive breast cancer.

BackgroundDespite the success of tamoxifen since its introduction, about one-third of patients with estrogen (ER) and/or progesterone receptor (PgR) - positive breast cancer (BC) do not benefit from therapy. Here, we aim to identify molecular mechanisms and protein biomarkers involved in tamoxifen resistance.ResultsUsing iTRAQ and Immobilized pH gradient-isoelectric focusing (IPG-IEF) mass spectrometry based proteomics we compared tumors from 12 patients with early relapses (<2 years) and 12 responsive to therapy (relapse-free > 7 years). A panel of 13 proteins (TCEAL4, AZGP1, S100A10, ALDH6A1, AHNAK, FBP1, S100A4, HSP90AB1, PDXK, GFPT1, RAB21, MX1, CAPS) from the 3101 identified proteins, potentially separate relapse from non-relapse BC patients. The proteins in the panel are involved in processes such as calcium (Ca2+) signaling, metabolism, epithelial mesenchymal transition (EMT), metastasis and invasion. Validation of the highest expressed proteins in the relapse group identify high tumor levels of CAPS as predictive of tamoxifen response in a patient cohort receiving tamoxifen as only adjuvant therapy.ConclusionsThis data implicate CAPS in tamoxifen resistance and as a potential predictive marker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9080-y) contains supplementary material, which is available to authorized users.

A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics

Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins.

Analysis of Rat Liver Proteins by Peptide Immobilized pH Gradient Isoelectric Focusing Combined with Liquid Chromatography-Tandem Mass Spectrometry

Abstract Due to the complexity of the biological sample, it is important to develop an effective pre-separation method for the liquid chromatography-mass spectrometer (LC-MS) analysis of biological samples. In this experiment, enzymatic hydrolysate of rat liver protein was pre-separated by peptide immobilized pH gradient isoelectric focusing (IPG-IEF) method, and the pre-separated components were further separated by reversed physe liquid chromatography (RPLC) and then identified by LTQ-Orbitrap MS. A total of 2039 kinds of proteins were identified, including 18 kinds of acetylated proteins, 4 kinds of proteins which were never reported before. Next, bioinformatic analysis was carried out for these proteins. The results showed that peptide IPG-IEF coupled with LC-MS/MS is an effective technique for proteomics analysis and this method is suitable for large-scale protein identification.

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.

Defining, Comparing, and Improving iTRAQ Quantification in Mass Spectrometry Proteomics Data

The purpose of this study was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. To investigate this we have used thousands of peptide measurements to estimate variance and bias for quantification by iTRAQ (isobaric tags for relative and absolute quantification) mass spectrometry in complex human samples. A549 cell lysate was mixed in the proportions 2:2:1:1:2:2:1:1, fractionated by high resolution isoelectric focusing and liquid chromatography and analyzed by three mass spectrometry platforms; LTQ Orbitrap Velos, 4800 MALDI-TOF/TOF and 6530 Q-TOF. We have investigated how variance and bias in the iTRAQ reporter ions data are affected by common experimental variables such as sample amount, sample fractionation, fragmentation energy, and instrument platform. Based on this, we have suggested a concept for experimental design and a methodology for protein quantification. By using duplicate samples in each run, each experiment is validated based on its internal experimental variation. The duplicates are used for calculating peptide weights, unique to the experiment, which is used in the protein quantification. By weighting the peptides depending on reporter ion intensity, we can decrease the relative error in quantification at the protein level and assign a total weight to each protein that reflects the protein quantitation confidence. We also demonstrate the usability of this methodology in a cancer cell line experiment as well as in a clinical data set of lung cancer tissue samples. In conclusion, we have in this study developed a methodology for improved protein quantification in shotgun proteomics and introduced a way to assess quantification for proteins with few peptides. The experimental design and developed algorithms decreased the relative protein quantification error in the analysis of complex biological samples.

Overexpression of αvβ6 Integrin Alters the Colorectal Cancer Cell Proteome in Favor of Elevated Proliferation and a Switching in Cellular Adhesion That Increases Invasion

Many proteins enhance cancer progression toward life-threatening metastases. These include linking proteins called integrins that mediate cell adhesion to the extracellular matrix (ECM), consequently altering both function and phenotype. Specific neoexpression of the β6 integrin subunit correlates with the epithelial-to-mesenchymal transition, metastasis, and poor overall patient survival. While β6 is implicated in these processes, exactly how it affects signaling and/or proteolytic pathways in metastasis remains unclear. A membrane-enriched peptide immobilized pH gradient isoelectric focusing (IPG-IEF) shotgun proteomics study was undertaken in which subclones of the SW480 colorectal cancer cell line transfected with a vector inducing unregulated β6 integrin overexpression were compared with the "empty" mock vector control cell line. β6 overexpression induced a significant change in 708 proteins and was found to be localized across most intracellular locations, some involving cellular processes and pathways underpinning cancer progression. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000230. β6 expression increased cell proliferation 4-fold while decreasing cell adhesion to many integrin ECM substrates. β6 expression also enhanced cell invasion and promoted the expression/repression of many established cancer-related pathways.

Study on stability mechanism of immobilized pH gradient in isoelectric focusing via the Svensson–Tiselius differential equation and moving reaction boundary

An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson–Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG–IEF and non-IPG–IEF (opposite to IPG–IEF) were developed to unveil stability mechanism of IPG–IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG–IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG–IEF system, (ii) there existed high diffusional current in non-IPG–IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG–IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG–IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG–IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique.

Enhanced N-glycosylation site exploitation of sialoglycopeptides by peptide IPG-IEF assisted TiO2 chromatography

Playing an important role in a broad range of biological and pathological processes, sialylation has been drawing wide interest. The efficient sialoglycopeptides enrichment methods are therefore attracting considerable attention. In this paper, we first compared two conventional enrichment methods, lectin and TiO(2), and analyzed their characteristics. Furthermore, considering the highly negatively charged nature of sialic acids, we developed a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assisted TiO(2) chromatography (PIAT), for the highly efficient enrichment of sialoglycopeptides. In this method, peptides were first separated into 24 fractions using peptide IPG-IEF. Sialoglycopeptides were relatively concentrated in low-pH fractions of the immobilized pH strips and were captured using TiO(2) chromatography. As a result, 614N-glycosylation sites were identified in 582 sialoglycopeptides within 322 sialoglycoproteins from rat liver using PIAT. To our knowledge, this work represents one of the most comprehensive sialoglycoproteomic analyses in general and exhibits the largest database of sialoglycoproteome in rat liver currently. So the new strategy introduced here exhibits high efficiency and universality in the sialoglycopeptide enrichment, and is a powerful tool for sialoglycoproteome exploration.

Enhancing the stability of 18O-labeled peptides through removal of immobilized trypsin by ZipTips

Trypsin-catalyzed 18O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of 18O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of 18O-labeled peptides under various pH conditions. The 18O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient–isoelectric focusing (IPG–IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of 18O labeling and molecular weight or isoelectric point (p I). The approach presented here is especially applicable to microscale samples. Its ability to generate stably 18O-labeled samples without back-exchange should expand the application scope of the 18O-labeling technique.

Observed peptide pI and retention time shifts as a result of post-translational modifications in multidimensional separations using narrow-range IPG-IEF

Modified peptides constitute a sub-population among the tryptic peptides analyzed in LC-MS based shotgun proteomics experiments. For larger proteomes including the human proteome, the tryptic peptide pool is very large, which necessitates some form of sample fractionation. By carefully choosing the sample fractionation and separation methods applied as shown here for the combination of narrow-range immobilized pH gradient isoelectric focusing (IPG-IEF) and nanoUPLC-MS, significantly increased information content can be achieved. Relatively low standard deviations were obtained for such multidimensional separations in terms of peptide pI (<0.05 pI units) and retention time (<0.3 min for a 350 min gradient) for a selection of highly complex proteomics samples. Using narrow-range IPG-IEF, experimental and predicted pI were in relative good agreement. However, based on our data, retention time prediction algorithms need further improvements in accuracy to match state-of-the-art reversed-phase chromatography performance. General trends of peptide pI shifts induced by common modifications including deamidations and N-terminal modifications are described. Deamidations of glutamine and asparagines shift peptide pI by approximately 1.5 pI units, making the peptides more acidic. Additionally, a novel pI shift (+~0.4 pI units) was found associated with dethiomethyl Met modifications. Further, the effects of these modifications as well as methionine oxidation were investigated in terms of experimentally observed retention time shifts in the chromatographic separation step. Clearly, post-translational modification-induced influences on peptide pI and retention time can be accurately and reproducibly measured using narrow-range IPG-IEF and high-performance nanoLC-MS. Even at modest mass accuracy (±50 ppm), the inclusion of peptide pI (±0.2 pI units) and/or retention time (±20 min) criteria are highly informative for human proteome analyses. The applications of using this information to identify post-translationally modified peptides and improve data analysis workflows are discussed.

Identification and Functional Characterization of Cytoplasmic Determinants of Plasmid DNA Nuclear Import

Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.

CSF proteins in neurological disorders analyzed by immobilized PH gradient isoelectric focusing using narrow PH gradients

We analyzed 352 paired CSF and serum samples with isoelectric focusing and immobilized pH gradients (IPG-IEF) using three different gradients. All MS patients (n = 35) had several (from 5 to 30) oligoclonal bands (OBs) in the CSF and most of them had few (from 1-8) OBs in serum. 54.5% patients with CNS infections (n = 12) had oligoclocal bands. OBs in these patients were fewer in number and located at the more acidic pH region compared with the MS samples. Also, corresponding serum bands were more often recognized. We suggest that IPG-IEF improves the diagnostic evaluation of the oligoclonal band pattern. The possibility of the use of extremely narrow gradients makes it a valuable tool for the analysis of selective proteins in various disorders.

A Combination of Immobilised pH Gradients Improves Membrane Proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.

Imaging mass spectrometry using peptide isoelectric focusing

Imaging Mass Spectrometry (IMS) has emerged as a powerful technique in the field of proteomics. The use of Immobilized pH Gradient-IsoElectric Focusing (IPG-IEF) is also a new trend, as the first dimension of separation, in shotgun proteomics. We report a combination of these two outstanding technologies. This approach is based on the separation of shotgun-produced peptides by IPG-IEF. The peptides are then transferred by capillarity to a capture membrane, which is then scanned by the mass spectrometer to generate MS images. This high-throughput methodology allows a preview of the sample to be obtained in a single day. We report the application of this new pipeline for differential comparison of the membrane proteome of two different strains of Staphylococcus aureus bacteria in a proof-of-principle experiment.

Characterization of the Rat Liver Membrane Proteome Using Peptide Immobilized pH Gradient Isoelectric Focusing

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.

Two‐dimensional electrophoresis with cationic detergents: A powerful tool for the proteomic analysis of myelin proteins. Part 2: Analytical aspects

The ability to analyze proteins in developing and damaged myelin will be crucial to improve our understanding of the mechanisms of myelinogenesis, dysmyelination, and demyelination. Comparative two-dimensional electrophoresis (2-DE) is a powerful approach to analyze these proteins. In part 1 of this study (see accompanying paper), a method for the 2-DE analysis of myelin proteins using the cationic detergents benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was described. We obtained improved separation and found that 16-BAC is the most effective agent for separation in 2-DE of myelin proteins and that CTAB is the most effective agent for solubilization of myelin proteins. Here in part 2, major myelin proteins as well as membrane proteins with multitransmembrane domains were identified by mass spectrometry after 16-BAC/SDS-PAGE and CTAB/SDS-PAGE. In addition, a high-molecular-weight protein enriched in myelin fraction was identified as unconventional myosin ID using 16-BAC/SDS-PAGE, which had previously not been detected using immobilized pH gradient isoelectric focusing (IPG)/SDS-PAGE. From these results, we concluded that combinational analysis using IPG/SDS-PAGE, 16-BAC/SDS-PAGE, and CTAB/SDS-PAGE provides a powerful technique facilitating the proteomic analysis of myelin proteins in either developmental or pathological changes.

ITRAQ compatibility of peptide immobilized pH gradient isoelectric focusing

Immobilized pH gradient isoelectric focusing (IPG-IEF) has emerged as a highly promising alternative to strong-cation exchange fractionation as the first dimension in shot-gun proteomics. Herein is shown the compatibility of this method with iTRAQ isotope labeling for relative quantitation and validation of sequence matches from database searching.