References

R., High energy ions from a Q-switched laser, Canadian Journal of Physics 42 (July, 1964), 1413-1416, 1964. 2. Fenner, N. C. and Daly, N. R., Laser used for mass analysis, The Review of Scientific Instruments 37(8), 1068-1070, 1966. 3. Vastola, F. J. and Pirone, R. H., Ionization of organic solids by laser irradiation, Advances in Mass Spectrometry 4, 107-111, 1968. 4. Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., and Yoshida, T., Protein and polymer analyses up to m z...

Data Analysis In Seldi Clinical Proteomics Research

Analysis of SELDI array data consists of several preprocessing and postprocessing steps. Data preprocessing involves the manipulations required to organize the data, such as TOF to mass calibration, baseline subtraction, and signal intensity normalization. Postprocessing consists of using analytical tools so that one can draw conclusions from the empirical results. These steps are similar in many ways to those used to process DNA microarray images. For example, SELDI mass spectra data must be...

Isotope Coding And Ms Detection For Relative Protein Quantification

Isotope coding of proteins is the process by which proteins (or their corresponding peptides) are labeled with a combination of stable isotopes used to differentiate control from treated samples while permitting relative quantification between proteins of two distinct proteomes to be determined. These chemical tagging or chemical labeling strategies involve the modification of functional groups of amino acid side chains and FIGURE 13.2 Mass spectrometry-based proteomics using liquid...

Ray Crystallography [1278

The majority of protein structures are determined with x-ray crystallography, with a smaller fraction determined using NMR (15-20 ). As such, most structural proteomics projects make central use of x-ray crystallography or a combination of crystallography and NMR 25,26 . The first step in determining a protein structure using x-ray crystallography is to obtain protein, usually by overexpressing in Escherichia coli and then to obtain diffraction-grade crystals. These tend to be the rate-limiting...

Future Prospects

A major challenge for the development of new proteomic tools and methods is to determine ways to identify low-abundance proteins in complex mixtures. Low-abundance proteins, such as transcription factors and other signaling molecules, play a very dynamic role in the function of the cell. Being able to better monitor their association with different protein complexes and their post-translational modification state would greatly increase the amount of information that we can gain from proteomic...

Ppm

FIGURE 12.2 ENMR spectra acquired on a Bruker 250-MHz spectrometer, for a mixture of 1 mM Ala-Gly-Gly and 1 mM ethylenediamine in D2O. (Johnson, C.S., Jr., and He, Q. In Advanced Magnetic Resonance, vol. 13, ed. W.S. Warren. San Diego Academic Press, 1989, 131-159. With permission.) Another ENMR experiment was successfully carried out using the transmembrane segment S4 peptide of the shaker K+ channel protein (2.8 kDa). The S4 peptide, which contains 23 amino acid residues...

Twodimensional Separations And Miniaturization

Beyond the previously discussed CIEF-LC-MS and CIEF-CZE-MS approaches, several other combinations of two separation techniques with independent (orthogonal) selectivity have been developed in order to handle biological samples of high complexity 55 . Different electrophoretic and chromatographic modes have been coupled to achieve good sensitivity (i.e., application of a system capable of high loadability as a first dimension) and high separation efficiency. CZE is attractive as a second...

Exenmr Characterization Of Protein Reaction Interfaces

ENMR experiments can be designed to visualize protein conformational changes during protein interactions in the presence of other proteomic molecules. By applying a DC electric field, multicomponent protein interactions can be studied by using intermolecular nuclear Overhauser effects (NOEs) to identify interface residues in polypeptide chains or, by mapping the altered chemical shifts, molecular dynamic parameters and residual dipolar coupling patterns 35 . These exchange ENMR (Ex-ENMR)...

Mxy

FIGURE 18.9 Mek1 ATP PD318088complex. The structure of Mekl bound to ATP is drawn as sticks, and the noncompetitive inhibitor PD318088 is shown as sticks highlighted with a surface (PDB code 1S9J). The chemical structure of PD318088 is shown on the right. FIGURE 18.9 Mek1 ATP PD318088complex. The structure of Mekl bound to ATP is drawn as sticks, and the noncompetitive inhibitor PD318088 is shown as sticks highlighted with a surface (PDB code 1S9J). The chemical structure of PD318088 is shown...

B

FIGURE 5.3 Schematic representation of two tandem mass spectrometers with MALDI ionization source. (A) The MALDI-TOF-TOF instrument (B) the MALDI-Q-TOF instrument. Collision-induced dissociation is accomplished by selecting an ion of interest with the first mass analyzer (e.g., Q or TOF) and introducing that ion into a collision cell. The selected ion then collides with a collision gas, resulting in fragmentation. The fragments are then analyzed by the second analyzer (e.g., TOF) to obtain a...

Twodimensional Enmr Signal Separation Of Coexisting Proteins In Solution

A decade ago, the principle of 2D ENMR was demonstrated using a spin-echo sequence on a high-resolution superconducting NMR spectrometer (fig. 12.1) 9 . A new dimension of electrophoretic flow was introduced as the second dimension the chemical shift was displayed in the first dimension 6-9,12 . Signals from different ionic species can be separated in the new flow dimension according to different electrophoretic mobilities. Two different approaches were developed to generate 2D ENMR spectra...

Selected Applications Of Metabonomics

16.4.1 Genetic Differences and Other Physiological Effects In order to determine therapeutic or toxic effects or to understand the biochemical alterations caused by disease, it is necessary first to understand any underlying physiological sources of variation. To this end, metabonomics has been used to separate classes of experimental animals such as mice and rats according to a number of inherent and external factors based on the endogenous metabolite patterns in their biofluids 42 . Such...

Table 102

Possible Applications of Antibody and Antigen Microarrays in Biomedicine Protein profiling and biomarker searching Post-translational modifications and signal transduction Immunoassays and diagnostic tools Assessment of immune response Prognostic of therapeutic treatment Target discovery and guiding vaccines Screening biomolecules and drug discovery Cytotoxicity of leads binding remains a critical issue in high-throughput dissection of antigen-antibody interactions. Improving the affinity and...

Identifying Sources Of Variation

Consider correlation in replicate spectra to which preprocessing has been applied. A property of such spectra with interesting ramifications is high correlation of the spectral intensities at widely separated values of m z. Such correlation implies sources of variation that affect more than one point in the spectrum, and such sources can often be associated with physical mechanisms in the measurement system. The approach to reproducibility presented in this chapter is based on such...

Posttranslational Modifications Using Isotope Coding

Protein function is highly modulated by post-translational modifications (PTMs). The complexity of these modifications, which vary between organisms and during modulation of cellular function, present the most challenging aspect of proteomic analysis. Because of their diversity, there are many methods to enrich and label particular PTMs for quantitative analysis using stable isotope-coded mass spec-trometry. In this chapter, we will focus on just two of the more important PTMs phosphorylation...

Data Interpretation Using Chemometrics

In chemistry, the term chemometrics is generally applied to describe the use of PR and related multivariate statistical approaches to chemical numerical data. The general aim of PR is to classify an object or to predict the origin of an object based on identification of inherent patterns in a set of experimental measurements or descriptors. PR can also be used for reducing the dimensionality of complex data sets for example, by 2D or 3D mapping procedures to enable easy visualization of any...

Coregen

Most pharmaceutically interesting ligands can be represented in terms of the ringlinker frameworks that comprise them 31 . Recent analysis of 119 published kinase inhibitors from at least 18 different targets illustrated that a basis set of four rings and eight linkers is sufficient to describe about 90 of ring and linker occurrences, respectively. A similar result was derived from a larger set of approximately 40,000 kinase inhibitors from curated patents 30 . Tools that combine elements of de...

Interpretation Of Results

It is important to be aware that paramagnetism may be associated with denatured proteins. Occasionally, sophisticated EPR studies have been carried out on essentially dead enzyme systems the active systems are EPR silent under the conditions used. It is also important to select the appropriate host organism for expression of gene products. If it does not have the necessary machinery, the metal center or cofactor will not be inserted or the wrong one will be inserted. 19.5.1 No Insertion of...

H

FIGURE 16.4 High-resolution 400-MHz 'H MAS NMR spectra of various tissues, with sample spinning at 4.2 kHz. free solution where molecules can tumble isotropically and rapidly. NMR spectroscopy on a tissue matrix in an MAS experiment is the same as solution-state NMR, and all common pulse techniques can be employed in order to study metabolic changes and to perform molecular structure elucidation. Typical 'H NMR spectra from a range of tissue types are shown in figure 16.4. In most cases, a...

N

And maintained by electron spin-lattice relaxation. The signal decreases if the populations become equalized, which tends to happen if too much microwave power is applied, a phenomenon known as power saturation. EPR spectroscopy is analogous to the more familiar nuclear magnetic resonance (NMR) method used in chemistry of organic compounds. However, there are some significant differences from NMR. Unpaired electrons are generally much more rare than nuclear spins, so fewer species are present...

Basic Principles Of Electrophoretic

Measurement of electrophoretic flow of ionic species was first attempted by Packer et al. in 1972 17 , followed by the first successful demonstration by Holz et al. in the 1980s using a horizontal-bore electromagnetic NMR spectrometer 18-21 . Johnson et al. later pioneered the development of the high-resolution 1D and 2D ENMR techniques on a vertical-bore superconducting NMR spectrometer 7-9,13,14,22-24 . More recently, He et al. extended 2D ENMR into multidimensional ENMR in order to...

Info

Tamoxifen Nafoxidine Testosterone Dexamethaso m Corticosterone holo-Tf, pH 7.4 apo-Tf, pH 6.3 holo-Tf, pH 7.4 holo-Tf, pH 7.4 apo-Tf, pH 6.3 holo-Tf, pH 7.4 Tamoxifen Nafoxidine Testosterone Dexamethaso m Corticosterone FIGURE 14.4 Functional and structural analyses of binding pairs. (A) Kinetic screen of ligands binding to ER-a ligand-binding domain. Responses were generated from the injection of nonestrogen agonists (gray), estrogen agonists, antagonists, and control ligands (all shown in...

Future Prospects And Conclusions

Over the course of the last 12 years, SELDI protein array technology has demonstrated utility in the analysis of protein complexes, the discovery of relevant biomarkers, and the facile creation of assays for clinical research, drug discovery, or basic biological research. To do so, significant advances in the areas of surface chemistry, protein purification, bioinformatics, and laser desorption-based mass spectrometry were achieved. Future improvements are expected on all of these fronts. In...

Exploiting The Similarities In Protein Binding Sites Modular Approaches To Drug Design

The major focus of applied work on protein-ligand interactions is for purposes of drug design. The existence of similarities in binding sites has multiple implications for those endeavors. The first is that a drug is not a key that can open only one lock. Rather, all drugs show varying degrees of interaction with a number of proteins. Successful drugs, therefore, are not ones that interact exclusively with a target of interest. Rather, they are compounds that display the highest affinity for...

Technical Aspects

Classical in vivo NMR spectroscopy is a well established technique and has been used for many years for the observation of metabolites and the investigation of metabolic fluxes in systems ranging from suspensions of bacteria and other cells to entire perfused organs 26-32 . So far, investigations of macromolecules in living cells have been possible only in a few very special cases 33-36 . Recently, however, we and others have demonstrated that magnetic resonance spectroscopy can be used to...

The Scope Of Proteomic And Chemical Proteomic Studies

Proteomic studies strive to characterize groups of proteins in a systematic manner. This can involve studies of pools of proteins isolated from tissue extracts or subsets (subproteomes) of these protein mixtures (part II). Studies can also be of individual purified proteins, but carried out in a highly parallel way, as in structural proteomics. In both cases, studies are systems based (chapter 1) because they focus on groups of proteins that are related by the networks of interactions they...

Contents

17.1.1 Structural Proteomics Centers, Web Sites, and Information 35G 17.1.2 Advantages of NMR-Based Structural Proteomics 35G 17.2 Steps in NMR-Based Structural 17.2.1 Information 17.2.2 Target 17.2.3 Choice of Protein Production 17.2.4 Cloning and Construct 17.2.5 Small-Scale Screening for Protein Production Level and 17.2.6 Screening of U-15N -Protein for Suitability as an NMR Target 356 17.2.7 Production of U-13C, 17.2.8 NMR Data 17.2.9 Backbone and Side-Chain 17.2.1G NOE Assignments and...

Q

Monitor the kinetics of site-specific phosphorylation of different signaling proteins in Jurkat T lymphocytes 54 . The phosphorylation state of 62 signaling components was established in cells stimulated via CD3 and CD28 membrane receptors. In the same context, reverse phase protein arrays were widely used to study post-translational modifications of proteins at different stages of breast cancer progression in patients before and after therapy 55 . In earlier studies, detection from reverse...

E u2 E j

FIGURE 8.3 Leading canonical correlation coefficient for all pairs of intervals. Pairs with highest correlation are of greatest interest. FIGURE 8.3 Leading canonical correlation coefficient for all pairs of intervals. Pairs with highest correlation are of greatest interest. These R2 values depend on the particular interval of the pair, the particular pair, and the mass-to-charge ratio u.

I

Phase displayed array Total protein array FIGURE 10.1 (See color insert.) Fluorescence detection of molecular interactions with high-throughput protein microarrays. A variety of antibody and antigen arrays and four main types of molecules employed for binding assays are shown. Both primary detection (labeled molecules) and secondary detection (labeled secondary binders) can be used to detect bound molecules on spots. concentration, binding affinity, and post-translational modification of...

Microcolumn Purification Strategies

Several instruments in the new generation of mass spectrometers provide similar results with respect to sensitivity of protein identification. As an example, we have tested several MALDI instruments and found that these show similar performance for identification of in-gel and in-solution digested proteins by peptide mass fingerprinting 49 . Thus, the limitation in MS protein identification often lies in the sample preparation steps. Consequently, efforts have been made to develop new methods...

Wpz

12C 13C isotope coding, 262 C-glycosylation, 83 C-terminal isotope labeling, relative protein quantification with, 264-265 Cajal bodies, 215 Calmodulin, 314 interaction with phenoxybenzamine, 309 Cancer biopsies, tissue heterogeneity in, 197 Cancer diagnosis, metabonomics studies in, 338 Cancer progression, protein profiling, 193, 195 Cancer-specific antigens, 198 Cancer susceptibility, 321 Canonical correlation analysis (CCA). See Functional canonical correlation analysis (CCA) Canonical...

S

See also Yeast MCAT approach with, 264 protein chaperones in, 214-215 SILAC method with, 266 Swi Snf complex from, 218 ENMR experiment, 228-229 ENMR spectra of, 229 movement in lipid membranes, 230 SAGA histone acetyltransferase complex, 218 Sample amounts limitations with MALDI-MS, 86 low CE requirements, 58 low SELDI requirements, 112 miniaturization with CE-MS, 48, 60-62 with miniaturized NMR probes, 326 and p-values, 120 Sample detection, in SELDI MS, 108-109 Sample...

Identification Of Glycosylation Sites

Determination of glycosylation sites directly from intact glycopeptides can be problematic. This is particularly the case when the glycosylation is heterogeneous and the signal therefore is distributed over a population of different glycopeptides, which confounds the interpretation of the mass spectrum. The heterogeneity may also limit the detection of low-abundance peptides, particularly if poorly ionizable and or large

C

FIGURE 18.3 Comparison of the conformations sampled by traditional docking and CORES. The inhibitor SU-5271 was modeled in the active site of EGFR (PDB code 1M17). The resulting predicted ligand structures obtained with molecular docking (GLIDE 2.5, Schrodinger, Inc., NY) are shown in panel A. Molecular modeling of the same compound using CORES is shown in panel B. quantitate the frequency with which related ligands bind in the same orientation in the kinase active site. For 74 different...

Preface

A significant challenge in presenting an overview of Spectral Techniques in Proteomics is in defining the scope of the topic. Proteomics means different things to different people for years, the dominant technique employed was 2D gel electrophoresis, followed by mass spectrometry (MS). While many exciting MS applications are presented (e.g., matrix-assisted laser desorption ionization MALDI , electrospray ionization ESI , tandem MS, liquid chromatography LC -MS, surface-enhanced laser...

Chemical Proteomics Studies of Protein Ligand Interactions in Pools and Pathways

Chapter 13 Characterizing Proteins and Proteomes Using Isotope-Coded Mass Chapter 14 Surface Plasmon Resonance Biosensors' Contributions to Proteome Chapter 15 Application of In-Cell NMR Spectroscopy to Investigation of Protein Behavior and Ligand-Protein Interaction inside Living Chapter 16 An Overview of Metabonomics Techniques and Applications 321 John C. Lindon, Elaine Holmes, and Jeremy K. Nicholson Chapter 17 NMR-Based Structural Chapter 18 Leveraging X-Ray Structural Information in Gene...

Magnetic Resonance 3341 NMR [125

NMR (nuclear magnetic resonance) spectroscopy is widely used to study the structure and dynamics of proteins. It is based on the measurement of the spin angular momentum properties of nuclei such as protons. In the presence of a static magnetic field (Bo), the nuclei of protons and other NMR active atoms (e.g., 13C, 15N, 31P, 19F) behave as if they are spinning and possess a spin angular momentum I.1 is related to magnetic moment by i yI, where y is the gyromagnetic ratio. As such, these nuclei...

Blc

ICAT reagents, 259-260, 261 Immobilization effects on analyte ligand interaction, 290-291 in SPR biosensor technology, 289 Immobilized metal ion affinity chromatography (IMAC), 56, 268, 270 for selecting histidinyl peptides, 265 Immobilized supports, and PhIAT limitations, assessment with antigen arrays, 195-196 Immunoaffinity chromatography, 164 Immunoglobulin titers, 196 Immunological reagents, as screening tools, 172 Immunoproteomics, 9, 411 antigen identification and, 158-159 challenges to,...

Proteins Analyzed By Cems

Protein Deconvolution Example

CE is frequently used for the separation of intact proteins. The use of CE in protein and proteomic studies in recent years has been reviewed in great detail 45-47 . The reason for this widespread use of CZE is the open tubular principle, circumventing the problem that many proteins stick to surfaces and are thus difficult to handle in chromatography. The combination of CZE with ESI-MS is even more powerful due to the ability of ESI-MS to determine exact masses after charge deconvolution of the...

Assessment Of Immune Response With Antigen Arrays

Many diagnostic approaches currently use conventional immunoassays and most popular among them is the enzyme-linked immunosorbent assay (ELISA), an outstanding multiplexed approach to assess enzymatic and binding parameters of proteins, including antigens and antibodies, in individual chambers of microtiter plates. Antigen microarrays provide an alternative to immunoassays by high-throughput monitoring of target molecules in a single assay. This is crucial for the precise comparison of binding...

Mass Spectral Studies Of Proteome And Subproteome Mixtures

20.2.1 Chapter 4 Capillary Electrophoresis-Mass Spectrometry (CE-MS) for Characterization of Peptides and Proteins Efficient analysis of protein mixtures often requires in-line separation technology before the mass spectral analysis. In this regard, capillary electrophoresis-mass spectrometry (CE-MS) is complementary to liquid chromatography (LC)-MS. It offers an orthogonal separation strategy, usually coupled to electrospray ionization (ESI). It also offers advantages in situations where...

Cellular Survival During Nmr Experiments

Another parameter that strongly influences the applicability of in-cell NMR experiments is the survival rate of the cells in the NMR tube. In particular, the high cell density can lead to oxygen starvation and lack of nutrients. If the sensitivity of the selected system (mainly the overexpression level) is high enough, the NMR spectra can be measured relatively fast (less than an hour). During longer experiments or series of experiments such as relaxation studies, however, significant changes...

Protein Profiling With Antibody Arrays

The measurement of gene expression in cells is of great interest to elucidate networks of protein ligand nucleic acid relationships in diseased and normal organisms. But DNA microarray-based assessment of mRNA does not necessarily reflect the abundance of the corresponding proteins in cells. Moreover, it does not give valuable information about post-translational modifications, which govern many cellular processes 35,36 . Therefore, alternative protein array-based approaches can provide not...