Info

Marshall, T. and Williams, K.M., Recovery of protein by Coomassie Brilliant Blue precipitation prior to electrophoresis, Electrophoresis, 13, 887-888, 1992. Marshall, T. and Williams, K., Two-dimensional electrophoresis of human urinary proteins following concentration by dye precipitation, Electrophoresis, 7, 1265-1272, 1996. Marshall, T., Centriprep ultrafiltration for fractionation of serum and urinary proteins before electrophoresis, Clin. Chem., 39, 1558, 1993. Edwards, J.J., Tollaksen,...

Classical Twodimensional

ELECTROPHORESIS (2DE) BASED METHODS OF MEMBRANE PROTEIN ISOLATION AND CHARACTERIZATION There are numerous advantages to a gel-based approach to membrane proteomics. Significant technical advances mean that the techniques are relatively robust and easily accessible to many groups. The expression of one gene can result in many different protein isoforms by posttranslational modification of the expression product. 2DE offers at least a preliminary means of monitoring the subtle changes in...

Glycoprotein Detection On 2d Gels

Identification of glycoproteins on gels or blots is based on one of two general methods. One method utilizes lectin binding as immunoblots to antibodies and has limited specificity. The other scheme is based on periodate oxidation of sugar cis-hydroxyl groups and converting them to aldehydes. The aldehydes are then conjugated to a variety of reagents including digoxigenin, fluorescence, biotin, or acidic fuchsin dye. These procedures have varying sensitivities and ease of use. An...

Glycoprotein Detection Using ProQ Emerald 300 Glycoprotein Gel Stain

The buffers and solutions needed for glycoprotein detection included in the Multiplexed Proteomics glycoprotein gel stain kit are as follows 1. Pro-Q Emerald 300 reagent, component A, enough for 1l of total staining solution, which can be used to stain up to three large 2D gels (20 x 20 cm). 2. Pro-Q Emerald 300 staining buffer (component B), 1l. 3. Periodic acid, 10 g (component C). 4. SYPRO Ruby protein gel stain, 1l. In addition, the following solutions should be made up fresh before use 1....

Polystyrene Monolithic Capillary Columns In Online 2d Lcesims Proteomic Analysis

Proteomic samples can contain several thousands of proteins resulting in millions of peptides after enzymatic digestion. Bottom-up shotgun LC MS proteomics strategy requires high resolving power of liquid separation at the peptide level. Various separation methods utilized in proteomics prior to MS include one-dimensional (1D) and 2D polyacryl-amide gel electrophoresis (PAGE), 1D nano-LC, multidimensional protein identification technology (MudPIT), isoelectric focusing (IEF), and SPE, among...

Stage 3 Depletion of HSA and Human IgG from Human Plasma

The affinity matrix was tested using an Akta explorer 100 for efficiency of the depletion and nonspecific binding of the affinity matrix. Onto a small affinity column (400 l) a sample of human plasma was loaded, and the same chromatographic procedure as described previously was used. The flow-through fractions and elution fractions (Figure 3.4) were collected and used for SDS-PAGE analyses. SDS-PAGE was performed on NOVEX Tris glycine 4-20 gels according to the supplier's protocol. Figure 3.5...

Recent Advances In Destreaking Techniques

As mysterious and persistent as these streaks are and despite the numerous partially effective solutions that have been proposed, a simple but efficient solution is highly desirable. Recently our laboratory experimented on sample reduction and alkylation using tris(2-carboxyethyl)-phosphine hydrochloride (TCEP) and vinyl-pyridine (VP) before IEF and greatly improved the basic end resolution in 2D gels. Trialkylphosphines such as TBP and TCEP are powerful reducing agents, which can readily and...

Staining Proteins With Tannins

Interestingly, Syed and Sayeed93 stained proteins in polyacrylamide gels with henna, an extract from the leaves of Lawsonia inermis used for dying hair and on the Indian subcontinent for body ornamentation. Staining was reportedly more sensitive than CBB for some proteins and, unlike the sulfonated dyes, was effective over the entire pH 2-12 range. Staining occurs via the interaction of plant tannins with both proteins and carbohydrates. Arginine, histidine, lysine, and tryptophan residues of...

Proteomics Conclusion

The application of proteomic approaches to the analysis of clinical samples for the analysis of human tissues and disease offers the opportunity to identify novel proteins that may serve as biomarkers or therapeutic targets. However, the identification of minor proteins will require high-capacity methods for the depletion of abundant proteins. Combinations of approaches based on selected affinity or high-resolution separations such as protein A G and MCE offer the potential for a proteomic...

Glycoprotein Analysis

For sensitivity and specificity, mass spectrometry (MS) is unsurpassed in its ability to unravel a protein sequence. Overall the techniques for sequencing have continuously improved, but the more significant contribution to sequence analysis has been the adaptability of MS to meet newer challenges of sensitivity and specificity. Comparable progress in the characterization of molecular glycosylation has been lacking. Glycan structures are largely reported devoid of interresidue linkage and...

Simplified Immunoprobing With Compressed Gels

The problem with direct immunoprobing is that gels are thick (nominally 1.5 mm), and because of dissociability of antigen and antibody, complexes require specialized washout procedures, best achieved by precise suctioning buffer through the gels. That imposes a burden, and except where necessary for fibrinogen and von Willebrand factor7,9 the approach has been ignored. To remove that obstacle, we devised a method for constructing gels that could be compressed from 1.8 to 0.3 mm, which enabled...

Conclusions

Electrophoretic NMR proteomics is potentially a powerful tool for studying protein interactions in system biology and may be combined with the current in-cell NMR techniques.32-34 to map biological signaling events across cell membranes mediated by interactions of cell protein receptors and other proteins, ligands, or drugs.35 Without physical separation of the proteins in a free solution (or in gel matrix), ENMR can map unique proteomic profiles of coexisting proteins and protein...

Figure

1.1 (A) Barocycler NEP2017 (B) Barocycler NEP3229. FIGURE 1.2 An illustration of a PULSE Tube. FIGURE 1.2 An illustration of a PULSE Tube. loaded into a tube holder. The holder is then placed into the pressure chamber, which contains sample chamber fluid and has been equilibrated to a desired temperature. In operation (Figure 1.3B), pressure is applied in the chamber, and as the process moves toward equilibrium, the pressure difference between the fluid in the pressure chamber and the interior...

Sample Preparation By The Pct Sps Compared To Traditional Methods

Currently many laboratories use manual grinding with a mortar and pestle, sometimes at liquid nitrogen temperature, to release cellular components.5 Other current mechanical or chemical methods include sonication, bead beating, French press processing, freezer milling, rotor-stator homogenization, enzymatic digestion, and chemical dissolution all of which have inherent limitations. These limitations include safety issues, potential for contamination, extended process time, lack of automation,...

Structural Characterization Of Coexisting Proteins In Solution

A 3D ENMR experiment is necessary to give the two-dimensional correlation spectra of different protein components in solution that can be used for simultaneous sequential and stereospecific assignments of small proteins (< 20 kD) coexisting in solution. To study large proteins, multinuclear multidimensional NMR sequences can be employed and modified accordingly for the ENMR experiments.28 The first 3D ENMR experiment was demonstrated using the 3D electrophoretic COSY pulse sequence (Figure...

Conclusion

Polymeric monolithic capillary columns have become popular as alternatives to granulate packed columns due to the simplicity of their preparation and handling, advantageous flow transfer properties and separation performance at acceptable backpressure, diversity of their chemistries and functionalities of their chromato-graphic surfaces, and the elimination of need for retaining frits. In contrast to packed columns, polymeric monoliths demonstrate outstanding chemical and mechanical stability...

Liquid Chromatography Coupled With Mass Spectrometry Approaches

Although 2DE offers many advantages for the characterization and quantification of proteins, it has a number of inherent disadvantages that limit its use for the separation of membrane proteins the resolution of hydrophobic, alkaline proteins is often poor, and low-abundance proteins can be difficult to detect on gels.13,36 Studies by Pederson et al. suggest that low-abundance proteins, in the region of 10,000 copies per cell, make up 80 of the predicted yeast proteome and that therefore a 2 mg...

Introduction

Human serum is frequently used in proteomics analysis for the discovery of new disease markers, drug targets, or studying protein expression patterns. This body fluid represents the most complex sample of the human proteome, composed of homeo-static blood proteins as well as tissue leakage proteins.1 Difficulties arise in the pro-teomic analysis of serum due to the extreme concentration range of target proteins over 10 to 12 orders of magnitude. High-abundant proteins such as albumin, IgG,...

Composite Gels

Agarose gels, by themselves, do not adequately sieve proteins of moderate molecular size. To enhance sieving we constructed composites with degradable (removable) polyacrylamide fillers, as described.4,7 These fillers are constructed using dihydroxy-ethylene-bis-acrylamide (DHEBA) as a cross-linker because it is easily hydrolyzed at alkaline pH. We also add 1-allyloxy-2,3-proparnediol to the polymerization mixture because it helps produce small polyacrylamide fragments that are easily washed...

Contents

2.2 IEX Chromatography 2.2.2 IEX Chromatography Media Available 2.2.3 Illustration of IEX Chromatography Separation Correlated with 2DE 2.3 Applications of IEX Chromatography Prior to 2DE Analysis 22 2.3.1 Fractionation of Human 2.3.2 Impact on IEX Prefractionation on Resolution in 2DE 23

Highthroughput Approach For Global Proteomic Measurements

The level of proteome coverage achievable depends on both the separation quality and the MS platform being utilized. For proteomic analyses from complex peptide mixtures, the better the separations prior to tandem MS (MS MS) analysis, the more complex a mixture that can be addressed.3 The first implementation of a combined LC-MS approach for global proteomics was by Washburn et al.,4,5 who utilized 2D LC separations that involved sequential step elutions of tryptically digested peptides from a...

Qmj

FIGURE 27.6 (A) A control 2D COSY spectrum from the mixture of L-aspartic acid and 4, 9-dioxa-1, 12-dodecanediamine using conventional 2D COSY method (B) & (C) the two 2D COSY planes from the 3D Electrophoretic COSY matrix displaying the COSY spectrum of each component molecule.28 (with permission) of high salt concentrations. When electric conductivity (k) is high, the electric field (Edc IJkA) may become too low to detect the cosinusoidal electrophoretic signal modulations. Generation of...

Recent Developments In Colloidal Coomassie Chemistry

The colloidal properties of CBB, or other ionic dyes for that matter, can be manipulated by altering the type of counterion and its concentration relative to the dye concentration. The addition of ammonium salts drives the formation of larger, but decreasingly stable dye colloids. This is analogous to counterion staining except that an ammonium salt, usually ammonium sulfate, substitutes for the secondary dye as counterion for stabilization of the primary colloid. Some stabilization occurs when...

Discussion

Optic nerve contains significant lipid-rich myelin membranes and constitutes one of the more difficult tissues from which to extract soluble protein. Detergents and thiourea are essential for solubilization of many membrane proteins,28-30 and here we found that ASB-14 and dodecylmaltoside are relatively effective in extracting optic nerve proteins. Our results are consistent with other efforts to solubilize central nervous system myelin.28,31,32 However in our hands, even after multiple...

References

Fazekas, D.S., Groth, S., Webster, R.G., and Datyner, A., Biochim. Biophys. Acta, 71, 377-385, 1963. 2. Meyer, T.S. and Lambert, B.L., Biochim. Biophys. Acta, 107, 144, 1965. 3. Diezel, W., Koppenschlager, G., and Hofmann, E., Anal. Biochem., 48, 617-620, 1972. 4. Neuhoff, V., Stamm, R., and Eibl, H., Electrophoresis, 6, 427-448, 1985. 5. Shainoff, J.R., Ratnoff, O.D., Smejkal, G.B., DiBello, P.M., Welches, W.R., Lill, H., Mitkevich, O.V., and Periman, P., Thrombosis Res., 101, 91-99, 2001. 6....

Or Not 2d

That was the question posed in Shakespearian satire by Fey and Larsen in a Current Opinions in Chemical Biology review article published in 2001.13 This marked a period that seemingly began a movement away from 2DE, or at least an attempt by some researchers to avoid using it. However, as one of the major contributors to the field of protein separation, Pier Giorgio Righetti, has recently illustrated in his review of the history of electrophoresis,14 this technology is a prolific area of...

Instrumentation for Imaging Fluorescent Stains and Data Analysis

In order to image all members of the Multiplexed Proteomics technology family, both a UV light based charge-coupling device (CCD) camera system and a laser-based gel scanner would be ideal. The following instruments can be used for imaging the fluorescent stains. 1. Pro-Q Emerald 300 dye The Lumilmager (Roche Biochemicals, Mannheim, Germany) or any standard UV light based imaging system with a 300 nm excitation is suitable. As an emission filter the 520 nm bandpass in the Lumilmager or a...

Ligand Docking Using NMR Constraints T Relaxation or NOE Data

While CF STD NMR can be used to identify which ligands bind to a protein, further functional insights can be obtained if there is structural information about the protein-ligand complex. Although NMR is a powerful tool for determining protein structures (see section 26.3.2) and is playing a prominent role in structural proteomics efforts,25 it can also provide the structural data needed to guide the docking of a ligand into a protein structure. Thus, it serves the chemical proteomic role of...

Sample Preparation Basics

Due to the great diversity of protein sample types and origins, no universal sample preparation method applicable for all proteins exists.4 Unfortunately, an optimal procedure must be determined empirically in most cases and tailored for each sample type. This can be a daunting task. However, some general sample preparation guidelines should be kept in mind to avoid a number of pitfalls during sample preparation for proteomic studies. Sample preparation is not only a prerequisite for a...

The Developing Field Of Glycoproteomics

When genomes are sequenced, the information is immediately usable for bioinfor-matics, microarrays, and many other applications. Genomes can be compared and genes manipulated knowing only the DNA sequence. The function of DNA and the one-way translation of data were rarely in question. From this simple understanding the significance of the proteome rose to astonishing prominence and became the research objective of many with goals and dreams, .capable of identifying up to 1 million proteins a...

The Emerging Role of NMR Spectroscopy in Chemical Proteomics

The field of chemical proteomics is about studying protein-ligand interactions on a large scale, and NMR is emerging as a powerful method for such studies. NMR plays a role not just in designing chemical proteomic probes, as just discussed, but also in the biophysical characterization of protein-ligand interactions. An important asset of NMR spectroscopy in chemical proteomics is its ability to detect ligand binding for tight binding ligands as well as compounds with lower affinities. This...

Theory

IEX is the reversible adsorption of charged molecules to an immobilized group on a suitable solid phase that bears the opposite charge.9 In practice, the process employs the following steps (a) equilibration of the IEX surface with a suitable counterion (anion or cation) and pH so the molecule of interest will be strongly adsorbed onto the surface (b) binding of the sample and displacement of the bound counterion (molecules with the same charge as the surface will be unretained by the solid...

Three Color Staining of Gels

In some cases it might be desirable to stain a sample for glycol as well as phospho-protein content followed by total protein staining. In those cases, the gel first has to be stained with the Pro-Q Diamond dye as described in section 24.3.1 followed by the complete protocol described in 24.3.2 including the fixation step, which is very important to be repeated. This ensures better specificity. The SYPRO Ruby dye staining can be performed straight after the Pro-Q Emerald 300 dye without another...

Usage Guide

This is a detailed how-to guide for performing DIGE experiments using the GE Healthcare IPGPhor II with the IPGPhor Manifold for the first dimension and SE660 for the second. All references to H2O should be read as double distilled H2O unless stated otherwise. In recipes, the information given in each line corresponds to final concentration, the name of ingredient, and the amount used, in that order. 15.2.2 Recipes, Apparatus, and Chemicals for Sample Solubilization and Labeling 40 Methylamine...

V

IPG rehydration buffer 1 ml, 2 mg ml In this experiment, increased solubility provided by a combination of thiourea and urea and CHAPS allowed for high recovery of proteins from FFE across the entire pH range and a high loading capacity of 6 mg hr without significant precipitation. In total, 10 ml of each fraction was collected. Every other fraction from a collection 96-well plate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with Silver...

Zoom 2DE of individual FFE fractions

FIGURE 11.4 A diagram outlining a rat liver proteome fractionation with IEF-FFE and subsequent zoom 2DE of individual FFE fractions (see text). appropriate FFE separation buffer, 30 mg of protein was subjected to the FFE to ensure sufficient amounts of fractionated proteins for subsequent 2DE analysis. Continuous nature of FFE separation allowed for large amounts of protein in relatively large volume (10 ml) to be separated. TABLE 11.1 Experimental Conditions for a Rat Liver Proteome...

Silver Staining

The first published silver stain by Kerenyi and Gallyas18 was an emulation of earlier histochemical stains and enabled the visualization of proteins in agarose gels. Later, Merril et al.19 would revolutionize staining with the development of an acidic silver nitrate stain for polyacrylamide gels that extended sensitivity into the picogram range. Soon thereafter, Oakley et al.20 described the first ammonical silver stain based on the formation of silver diamine complexes. From these initially...

Pct Mp Pct Mp Pct Mp Mw

FIGURE 1.6 Proteins released from chicken muscle by PCT SPS (PCT) compared to mortar and pestle grinding (MP) using various buffers (B1-B3) shown in a 12 Tris-glycine reducing SDS-PAGE. FIGURE 1.6 Proteins released from chicken muscle by PCT SPS (PCT) compared to mortar and pestle grinding (MP) using various buffers (B1-B3) shown in a 12 Tris-glycine reducing SDS-PAGE. a variety of cell and tissue types was compared to conventional methods such as mortar and pestle grinding, sonication, and...

Methods

Ribosomal proteins were purified and analyzed using procedures described in our earlier work.74 Log-phase Saccharomyces cerevisiae were harvested, lysed, and subjected to differential centrifugation to enrich ribosomal protein particles. Acid extraction of those particles generated a protein sample (-50 g) that was resolved in the first dimension of a 2D LC system (described later in the text and in Figure 21.3A) using a Shodex SP 420N 4.6 X 35 mm nonporous cation exchange column (obtained from...

Methods And Materials

13.3.1 Clinical Samples and Cell Culture All samples were obtained with informed consent from RA patients according to protocols approved by the local Ethics Review Board. Synovial fluids samples were cleared of cellular and particulate material and frozen at -70oC until required. Synovial tissues were obtained from RA patients undergoing total knee arthroplasty. The cells were released from the dissected synovial tissue by digestion with collagenase (1 mg ml) and hyaluronidase (0.05 mg ml)...

Destreaking Techniques

Various attempts have been made to reduce the basic pH range streaks, yet achieving an optimal IEF in the alkaline region remains a challenge. For example, decreasing the protein sample concentration, anodic cup-loading, shortening IEF duration, addition of a DTT reservoir at the cathode to replenish DTT, or using an alternative reducing agent such as hydroxyethyldisulphide (HED) to form mixed disulfides with cys-teinyl thiols have all been attempted.7-9 Strategies such as decreasing protein...

Urine

14.5.1 Patterns of Healthy Individuals Urine is the filtration product of the kidneys, and its protein content in healthy individuals is minimal (protein concentrations exceeding 0.1-0.15 mg ml have been defined as pathological).70 Its composition in regard to salts and proteins may vary in time considerably, being largely influenced by factors such as liquid intake, exercise, and time. Several classes of proteins can be found in urine (a) normal serum and plasma proteins and their fragments...

Fluorescent Stains

Numerous very sensitive methods for the fluorescent labeling of proteins prior to electrophoresis have been described. Both fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) enable sensitivity in the low nanogram range,36,37 and because these compounds and their hydrolysis products do not fluoresce, the detection of low levels of protein is not impaired by background fluorescence. The nonfluorescent reagent MDPF reacts with lysine amino groups to form covalent fluorescent adducts...

Stage 1 Immunization of Llamas and Creation of the VHH Library

Two llamas were immunized using standard procedures.8 One llama was immunized with HSA, and the other llama was immunized with human Fc. Immune response of the llamas to these antigens was checked at regular intervals, using a small serum sample from the immunized animal. When the immune response reached a plateau, a larger blood sample was taken from the llama. From the peripheral blood lymphocytes, the mRNA was isolated. Using polymerase chain reaction (PCR) techniques, the VHH encoding...

Generalpurpose Cleanup For Improved Resolution And Reproducibility

Success or failure of any protein analysis depends on sample purity. Contaminants, such as salts, detergents, and ionic compounds, as well as high-abundance proteins, can compromise a 2D experiment by interfering with the protein separation or masking proteins of interest. It is crucial to eliminate these contaminants prior to analysis. 8.3.1 Removal of Interfering Contaminants Interfering substances are any contaminants that negatively impact IEF, SDS-PAGE, or both. Contaminants of this nature...

Sequencing Released Glycans

Proteomics is an endeavor to understand gene function and characterize the structural relationships of the living cell. In proteomics, MS has become a powerful analytical technology to identify proteins by the analysis of peptides (PMF) with database relationships. In considerable contrast to proteomics, where global comprehensive studies are conducted in a systematic fashion, glycomics is still lost in multiple procedures looking mostly for qualitative answers and not quantitative results. A...

Results

Albumin Depletion Pierce

4.3.1 Cibacron Blue Depletion of Rat Albumin Removed Many Nontarget Proteins Rat serum was depleted with the Millipore Montage kit to remove albumin, according to manufacturer protocol. Two-dimensional gel analyses were performed with the predepleted, postdepleted, and albumin-associated proteins. Based on the results shown in Figure 4.1, the postdepleted sample showed some removal of the albumin and some enhancement of lower abundant proteins. However, it was clear that many proteins were...

Principles Of Ipgs

As briefly discussed in the introduction, pH gradients formed with carrier ampholytes are subject to instability (cathodic drift) during extended focusing runs.8 This phenomenon is not fully understood but is characterized by a drift of the gradient toward the cathode and is accompanied by acidification at the anode. This results in flattening of the gradient in the neutral pH region and ultimately a loss of alkaline proteins. IPGs were developed to overcome these limitations of carrier...

Impact on IEX Prefractionation on Resolution in 2DE

Prefractionation of complex protein mixtures with subsequent separation on 2DE gels is the preferred approach for many proteomics applications.18,19 The objective of many differential display studies in proteomics is the comparison between the levels of proteins in experimental and control samples and the determination of quantitative and qualitative differences in individual protein expression. In many cases this is carried out by 2DE resolution linked to mass spectrometry analysis. Reducing...

Overcoming Difficulties With Basic Protein Separations

The theory and methodology of working with IPGs are well understood.19 Applying the theoretical knowledge led Righetti and coworkers to recognize that extreme pH ranges pose challenges both in preparing and running IPG strips. As already discussed in section 16.2.1, the synthesis of very basic acrylamido buffers with pK values of 10.4 and greater than 12 is a requirement for the production of very alkaline IPG gels (e.g., pH 10-12). However, early attempts to run basic IPG gels polymerized with...

B

FIGURE 22.4 Gradient nano-LC-ESI-MS of a tryptic digest of bovine catalase on the monolithic column 10 amol of digest injected on the column . A Extracted ion chromatograms for selected peptides B MS spectra at the peak maximum of the same peptides. From Ivanov et al., 2003.39 To demonstrate the potential of the ultra low i.d. monoliths in the high-sensitivity proteomic analysis, a tryptic digest of protein extract of a breast ductal carcinoma tissue section 103 cell equivalent was also...

Contributors

Cleveland Clinic Foundation Cleveland, Ohio John Buhler Research Centre Winnipeg, Manitoba, Canada Indiana University School of Medicine Indianapolis, Indiana Agilent Technologies, Inc. Wilmington, Delaware Waters Corporation Milford, Massachusetts Cole Eye Institute i31 Cleveland Clinic Foundation Cleveland, Ohio Cleveland Clinic Foundation Cleveland, Ohio Agilent Technologies, Inc. Wilmington, Delaware Bio-Rad Laboratories Hercules, California Millipore Corporation Danvers, Massachusetts...

Materials And Methods

4.2.1 Coupling of Affinity-Purified IgY to UltraLink Hydrazide Gel Coupling of affinity purified IgY antibodies to UltraLink Hydrazide beads was performed essentially as described in the manufacturer's protocol Pierce Biotechnology Inc., Rockford, Ill. . The antibody conjugation scheme employed was based on hydrazide chemistry resulting in the IgY antibodies oriented with the F ab' 2 antigen-binding regions facing outward and the Fc regions of the antibodies covalently coupled via their...