Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175


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This guide describes the principles of polymer MS and best practices in polymer characterization.

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It provides a guide to developing appropriate sample preparation protocols for different polymers. Complete with examples of applications and experiments, this is an excellent reference for scientists, researchers, graduate students, and others. Permissions Request permission to reuse content from this site. Table of contents Preface. Time-of-flight MS for polymer characterization. Polymer analysis with Fourier Transform mass spectrometry.

Bain, C. Formation of monolayers by the coadsorption of thiols on gold: variation in the head group, tail group, and solvent. Journal of the American Chemical Society, , — Analytical Chemistry, 69 22 , — Latourte, L. Desorption behavior and distributions of fluorinated polymers in MALDI and electrospray ionization mass spectrometry. Analytical Chemistry, 69 , — Juhasz, P. Matrix-assisted laser desorption ionization mass spectrometry with 2- 4-hydroxyphenylazo benzoic acid matrix.

Journal of the American Society for Mass Spectrometry, 4 , — Chou, C. International Journal of Mass Spectrometry, , 15— Wei, J. Desorption-ionization mass spectrometry on porous silicon. Nature, , — Northen, T. Clathrate nanostructures for mass spectrometry. Hopfgartner, G. Weinheim: Wiley-VCH.

MALDI Mass Spectrometry for Synthetic Polymer Analysis

Google Scholar. Vogel, M. Cohen, S. Analytical Chemistry, 68 , 31— Trimpin, S. Rapid Communications in Mass Spectrometry, 15 , — Bolbach, G. Influence of target preparation and laser irradiance in matrix-assisted UV laser-desorption of proteins. Analusis, 21 , — Laser desorption ionization of proteins with molecular masses exceeding 10, Daltons. Analytical Chemistry, 60 , — Cornett, F.

Matrix-assisted laser desorption at visible wavelengths using a two-component matrix. Organic Mass Spectrometry, 27 , — Hutchens, T. New desorption strategies for the mass spectrometric analysis of macromolecules. Hillenkamp, F. Gross Eds. Dordrecht: Kluwer. Vorm, O. Analytical Chemistry, 66 , — Onnerfjord, P. Rapid Communications in Mass Spectrometry, 13 , — Perera, I. Hensel, R. Axelsson, J. Improved reproducibility and increased signal intensity in MALDI as a result of electrospray sample preparation.

Rapid Communications in Mass Spectrometry, 11 , — Nuwaysir, L. Castoro, J. Rapid Communications in Mass Spectrometry, 6 , — Kahr, M. Silver nitrate chemical ionization for analysis of hydrocarbon polymers by laser desorption Fourier transform mass spectrometry. Yao, J. Coaxial capillary and conductive capillary interfaces for collection of fractions isolated by capillary electrophoresis. Journal of American Society Mass Spectrometry, 9 , — Wessely-Szponder, J.

Analysis of antimicrobial peptides from porcine neutrophils. Journal of Microbiological Methods, 83 , 8— Smolira, A. Vacuum, 78 , — Sweeney, S. Journal of Immunology, , — Ramanathan, B. Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity. Microbiology Infections, 4 , — Panyutich, A. Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrin. Infections of Immunotherapy, 65 , — Anderson, R.

Biochemistry and Biophysical Research Communication, , — Treffers, C. Isolation and characterisation of antimicrobial peptides from deer neutrophils. International Journal of Antimicrobial Agents, 26 , — Personalised recommendations. Cite article How to cite? The information obtained from proteome analysis can aid in identification of therapeutic targets or surrogate markers in understanding the initiation and progression of a disease state.

Thus, proteomics research can be a valuable tool in drug discovery and for the first time offers the scientist an integration of genomics, mRNA analysis and protein expression Blackstock and Weir, One of the most commonly utilized techniques for protein separation has been based on gel separation. On the other hand, for complex protein mixtures i. In this approach proteins are separated by isoelectric point in the first dimension and subsequently by their mol.

However, visualization does not provide unambiguous protein identification and, therefore, scientists typically use western blotting or classical Edman sequencing for this purpose.


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Fortunately, integration of the inherent benefits of MS i. Figure 10 depicts a simplified strategy which is being widely utilized in high throughput polypeptide characterization. Typically, samples obtained from different cellular fractions are processed by 2D SDS isoelectric focusing gel electrophoresis. Each cell or tissue type may require a specific visualization approach, such as Coomassie blue or silver staining or fluorescence tagging, for protein detection Hancock et al.

Subsequently, gel images are electronically retrieved by high resolution scanners and analyzed spot finding using pattern recognition techniques against 2D gel database queries. Sophisticated computer software packages can be employed to enhance contrast, subtract background, align images, remove artifacts and perform gel comparisons. Proteome maps are compared against databases for identification of up- or down-regulation in a disease state. The resulting information could be utilized to identify biomarkers in clinical and toxicological studies. Conceptually, a similar and complementary approach but with higher accuracy i.

Sometimes, affinity chromatography techniques are necessary to enrich a specific class of proteins prior to additional sample manipulations Link et al. A number of software packages are currently available to query large databases and enhance the speed of the MS protein identification process Jaffe et al. In addition, 2D gel sample components below a mol. The following examples will clarify the above discussions.

Recently, a high throughput protein identification double parallel digestion, DPD method was reported by Sanchez and co-workers Bienvenut et al. In the DPD approach partially digested proteins were obtained using an immobilized trypsin membrane and transblotted. The resulting peptides were trapped on a polyvinylidene difluoride membrane and scrutinized by MS. The DPD approach was successfully applied to a mini-2D gel electrophoresis of Escherichia coli extract. Several critical issues, however, need to be further improved in order to realize the full potential of 2D gel analysis in conjunction with MS analysis.

These include a reduction in background chemical noise i. Keratin interference can even originate from inadequately purified trypsin, which is widely used for peptide mapping in MS experiments Zhang,Y. Thus, a clean sample preparation environment, automation, minimum sample handling procedures and higher quality gel materials compatible with MS analysis could alleviate the problem of possible contamination.

In addition, post-translational modifications, oxidation of protein during sample preparation and poor recovery of large proteins from the gel have the potential to extend the duration of the unambiguous analyte s identification process. Smith and co-workers Pasa-Tolic et al. The cells were cultured in normal as well as rare isotope i. The abundance of several intact and isotopically distinctive proteins was qualitatively monitored for up to several hours using this method. Another related area in proteome analysis is protein expression mapping, which is defined as the quantitative measurement of protein dynamics in a specimen i.

In this approach proteome analysis is typically performed in a subtractive fashion whereby alterations in individual proteins for two or more states are compared.


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An isotope-containing affinity tag ICAT , which consisted of an affinity tag biotin , a linker containing a stable isotope and a reactive moiety with a propensity to react with free sulfhydryl groups i. Two sets of cell states or tissue extracts were independently treated with isotopically light and heavy 8 Da higher in mol. The cells were combined and subjected to proteolytic cleavage. Peptide sequence information was obtained by tandem mass spectrometry experiments and identified by computer searches against protein data banks. Quantitation of proteins was performed using the ratios of the respective light and heavy ICAT-labeled peptides, which were generated by enzymatic digestion.

The stable isotope labeling procedure was a clever approach to assist in the identification of two peptides with identical sequences and mol.

Theory of MALDI-TOF Mass Spectrometry

Since all the physical characteristics of the two identical protein samples from the two cell states remain the same, the resulting peptide fragments obtained by enzymatic cleavage yield identical mass spectra. Thus, incorporation of specific stable isotopes in one cell state results in mass shifts, which in turn serve as an internal standard for all other cell states within the same experiment Mann, Since the light and heavy ICAT-tagged peptides are chemically identical, one can safely assume that they would yield analogous MS detection ionization responses and behave as mutual internal standards for quantification purposes.

In general, the above strategies for qualitative and quantitative identification of key cellular proteins could have great potential in several areas of drug development, such as pharmacogenomics Evans and Relling, In the science of pharmacogenomics genetic polymorphisms in transporters, drug metabolizing enzymes e. Currently these investigations are rather tedious and empirical. In most cases the human genetic variations resulting in different drug responses are realized in large studies at the post-marketing stages i.

Some of these idiosyncratic responses are toxic and thus it is essential to identify them prior to Phase III clinical studies Gould Rothberg et al. Therefore, it is of interest to elucidate the identity and pharmacogenomic traits i. MS technology offers a viable platform which can be utilized to assay differential protein expression following drug treatment. Considering the recent report by Sweedler and co-workers Li et al. This technology offers an exciting window of opportunity to study discrete small cells and processes such as synaptosomes, vesicular transport between the endoplasmic reticulum and the various Golgi compartments, hepatocytes and protein packaging pathways.

A growing body of literature has been devoted to the application of MS to the detection of non-covalent complexes Przybylski and Glocker, ; Loo, ; Jespersen et al. Furthermore, mass spectrometric determination of 1 H- 2 H exchange has provided complementary information to existing nuclear magnetic resonance NMR data on water soluble peptide and protein tertiary structures and conformations Gross, ; Weber-Ban et al.

For example, interaction of the HIV protein gp, on the exterior of the HIV virus, with CD4 glycoprotein, on the surface of T helper cells, is of interest in understanding the tissue specificity of HIV virus infection. CD4 is a surface glycoprotein which is involved in the T helper cell reaction with other cells cell—cell adhesion.

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As glycoproteins, gp and CD4 both show a high degree of oligosaccharide heterogeneity. This approach yielded higher resolution and accuracy in comparison with sugar gradient sedimentation experiments. However, to determine the stoichiometry of a macromolecular non-covalent assembly by MALDI-MS, several key issues need to be considered. In MALDI-MS of intact non-covalent complexes the nature of the matrix, the laser power and the laser penetration depth in the target sample are among the most critical parameters. In the presence of organic solvents and acidic MALDI matrices denaturation of protein complexes is likely to occur Glocker et al.

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In addition, repeated laser pulses on a given spot may dissociate the non-covalent complex. The analysis of specific interactions of nucleic acid sequences with repressors and other regulatory proteins has commonly been performed using electrophoretic gel mobility shift assay. However, this technique requires radiolabeled DNA and does not provide accurate i. In recent years MS techniques have provided a complementary approach to the existing methods for deciphering the mol. Specific sequences of DNA strands were immobilized i.

The specific binding of aminoglycoside antibiotics to rRNA subdomains was examined by Griffey et al. Aminoglycoside antibiotics i. Elegant MS experiments demonstrated specific binding of several antibiotics to rRNA subdomains, provided estimated binding affinities and determined their respective binding sites. Robinson and co-workers Last and Robinson, ; Rostom and Robinson, presented a collection of elegant work on protein folding and multi-protein complexes using ESI-MS.

Signals corresponding to the transthyretin tetramer 4T and non-covalent complexes with one 4TR and two 4TRR molecules of retinol-binding protein were reflective of previously known solution chemistry observations. Although the majority of published MS work has been on previously characterized non-covalent complexes, it is clear that MS offers enormous possibilities in conjunction with NMR and X-ray crystallography data for the investigation of less well defined macromolecular complexes.

Nonetheless, it is imperative to perform proper control experiments to rule out the possibility of false positive observations during the MS analysis of non-covalent complexes. Variations in solution pH, instrument parameters ionization interface temperature and voltages and chemical modification of the analyte under investigation are some of the measures that can be taken to distinguish between specific interactions and non-specific aggregations Loo, Chip and other miniaturized technologies are rapidly being adapted to analyze changes in gene expression Epstein and Butow, DNA microarray technology e.

DNA microarrays consisting of thousands of individual gene sequences can be printed in a high density format on a glass microscope slide or deposited on a miniature matrix by a photolithographic process Knapp et al. Many diseases stem from gene mutations, which may take place via various routes such as replacement, deletion, insertion or duplication of a single or multiple base units that are the building blocks of the gene. In the area of gene expression, where many human genes have already been characterized, the sensitivity of detection of mRNA at the level of one in thousands is of interest.

Driven by chemistry, the process of drug research and discovery is increasingly led in the post-genomic era by advances in biotechnology and bioinformatics Drews, ; Sander, The trend towards sensitive and high speed microfabricated devices has been reinforced by a recent initiative to construct a mammalian gene collection Strausberg et al. In one strategy Tang et al. Each silicon chip contained 36 wells with a surface area of 6. DNA fragments containing the polymorphic sites i.

One of the emerging disciplines of human genetics is the detection of DNA polymorphisms to identify the components involved in complex genetic diseases. Clearly, this seemingly small variation has led to profound differences between the two species. This corresponds to approximately 1 in every nt. The significant implications of SNP include the presence of a particular SNP allele that could be the underlying reason for a genetic disorder along with variations in protein coding sequences.

Furthermore, SNPs could be useful as genetic markers for mapping purposes and localization of important functional genes. In this approach a primer oligonucleotide was annealed to a target DNA upstream of the polymorphic location and was extended by a single base in the presence of a thermostable DNA polymerase ThermoSequenase and all four dideoxynucleotide triphosphates.

The base at the polymorphic site was easily detected by the mass added onto the primer oligonucleotide. Although DNA immobilization through biotin—streptavidin i.

Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175
Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175 Maldi Mass Spectrometry for Synthetic Polymer Analysis, Volume 175

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