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Frisco S, Choi S-W, Dolnikowski GG, Selhub J. A method to assess genomic DNA methylation using high-performance liquid chromatography/electrospray ionization mass spectrometry. Analytical Chemistry2002;74:4526–4531. [PubMed]
Methylation of cytosines in CpG dinucleotides is an important mechanism for transcriptional regulation in eukaryotes. This and the next paper describe new methods for measuring cytosine methylation in genomic DNA. The present work uses on-line LC/MS. DNA is subjected to quantitative hydrolysis and complete removal of residual RNA. The four major DNA bases and 5-methyl-2′-deoxycytidine are then resolved isocratically by reverse-phase chromatography. Cytosine and methyl cytosine are quantitated relative to isotopically labeled internal standards using electrospray ionization. The method is suitable for use on 1 μg of genomic DNA.
Balog RP, deSouza EP, Tang HM, DeMasellis GM, Gao B, Avila A, Gaban DJ, Mittelman D, Minna JD, Luebke KJ, Garner HR. Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays. Analytical Biochemistry2002;309:301–310. [PubMed]
This paper evaluates a microarray approach to highly parallelized assessment of DNA methylation for interrogation of large numbers of sites simultaneously. Using established procedures, DNA is treated with sodium bisulfite to convert unmethylated cytosines to deoxyuracil, which behaves like thymidine in enzymatic template-directed polymerization. Methylated cytosines are unreactive to sodium bisulfite and behave like cytosine. The strategy of detecting these sequence differences as differences in hybridization to features on custom oligonucleotide microarrays is adopted. Co-hybridization with a reference sample having a different label provides internal standards. The degree of methylation in different samples may thus be compared on a site-by-site basis. The specificity and sensitivity of the method are evaluated.
Angell YM, Alsina J, Albericio F, Barany G. Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides. Journal of Peptide Research2002; 60:292–299. [PubMed]
Many vendor-recommended activation protocols for use in Fmoc solid-phase peptide synthesis may produce substantial levels of racemization at susceptible residues such as cysteine. The present report recommends using less polar solvents during coupling, avoiding the preactivation step with in situ activation, and employing weaker or more hindered bases such as 2,4,6-trimethylpyridine (collidine). The effects of a range of base additives and coupling reagents are tested, and practical protocols yielding low levels of racemization are described.
Wilson NL, Schulz BL, Karlsson NG, Packer NH. Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins. Journal of Proteome Research2002;1:521–529. [PubMed]
Schulz BL, Packer NH, Karlsson NG. Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis. Analytical Chemistry2002;74:6088–6097. [PubMed]
These two papers from the same group outline a method for analysis of the carbohydrate content of glycoproteins and mucins in serum fractionated by two-dimensional gel electrophoresis. Serum is first depleted of fibrinogen, immunoglobulin G, and albumin to permit less abundant proteins to be analyzed electrophoretically. Following separation, the proteins are electroblotted to PVDF paper. N-linked carbohydrate is released intact from membrane-bound glycoproteins by PNGase F, and O-linked carbohydrate is released intact by reductive β-elimination. The carbohydrate moieties are resolved on columns of graphitized carbon and studied by LC/MS using electrospray ionization.
Hansen RK, Broadhurst RW, Skelton PC, Arkin IT. Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry. Journal of the American Society of Mass Spectrometry2002;13:1376–1387.
Conditions are described for studying hydrogen/ deuterium exchange of the transmembrane fragment of the M2 protein of Influenza A incorporated into lipid vesicles or detergent micelles. Small unilamellar vesicles are prepared by solubilizing dimyristoyl phosphatidylcholine and the peptide in aqueous solution with n-octyl-β-d-glycopyranoside, then removing the detergent by dialysis. Vesicles are concentrated by lyophilization, followed by thawing at a temperature above the liquid crystal transition temperature of the lipid. Standard electrospray parameters are carefully optimized to produce peptide ions. Detection of hydrophobic peptides in the presence of a large molar excess of Triton X-100 is also demonstrated. This paper will be of interest to investigators studying membrane–protein interactions and to mass spectrometrists studying proteins solubilized with detergents.
Coon JJ, Harrison WW. Laser desorption–atmospheric pressure chemical ionization mass spectrometry for the analysis of peptides from aqueous solutions. Analytical Chemistry2002;74:5600–5605. [PubMed]
Coon JJ, Steele HA, Laipis PJ, Harrison WW. Laser desorption–atmospheric pressure chemical ionization: a novel ion source for the direct coupling of polyacrylamide gel electrophoresis to mass spectrometry. Journal of Mass Spectrometry2002;37:1163–1176. [PubMed]
The first paper describes development of atmospheric pressure MALDI in which desorption and ionization are decoupled. An infrared laser is first used to desorb neutral molecules from a peptide solution in a water/glycerol mixture. Desorbed molecules are then subjected to atmospheric pressure chemical ionization with a corona discharge. This decoupling, in an atmospheric pressure source, opens the possibility of conveniently using a variety of new desorption matrices for MALDI. The second paper demonstrates the use of acrylamide as the matrix in an experiment to scan a protein gel treated with trypsin. Although sensitivities are presently modest, this work represents a proof of principle with implications for a variety of future developments.
Chrisman PA, McLuckey SA. Dissociations of disulfide-linked gaseous polypeptide/protein anions: ion chemistry with implications for protein identification and characterization. Journal of Proteome Research2002;1:549–557. [PubMed]
Collisional activation of protein ions generated by electrospray in the negative ion mode is studied in an ion-trap instrument. Cleavage of disulfide bridges is a strongly preferred process. This is followed by a facile cleavage of the peptide bond on the N-terminal side of cysteine residues at which the C–S bond has been cleaved, yielding c and z–S product ions. This reaction is not observed in beam-type instruments. The specificity of the cleavage makes it useful for protein identification by database searching and for localization of post-translational modifications.
Shan L, Anderson DJ. Gradient chromatofocusing. Versatile pH gradient separation of proteins in ion-exchange HPLC: characterization studies. Analytical Chemistry2002;74:5641–5649. [PubMed]
A new version of the chromatofocusing technique is described. The pH gradient is generated by combining solvents of different pH in an HPLC gradient mixing system. Inexpensive low-molecular-weight buffer components are used rather than the polymeric ampholyte buffers. The ion exchanger employed is Mono P. Advantages of the method compared with conventional chromatofocusing include an increased range of buffer concentrations and improved ability to optimize conditions for particular separations. Performance is better than the conventional chromatofocusing technique and better than salt gradient ion-exchange chromatography on Mono P.
Ober RJ, Caves J, Ward ES. Analysis of exponential data using a noniterative technique: application to surface plasmon experiments. Analytical Biochemistry2003;312: 57–65. [PubMed]
A new approach to the analysis of experimental data of exponential type is described and applied to surface plasmon resonance data acquired with a BIAcore instrument. The approach relies on advances that have been made in the signal processing field using “subspace” algorithms. Accurate estimation of association and dissociation rate constants is demonstrated, avoiding problems with currently used iterative algorithms and with linear regression of logarithm-transformed data. The new method is not restricted to 1:1 interactions and can deal readily with negative data points. A similar approach is possible for analysis of exponential data from other experiments, including liquid-phase NMR, fluorescence lifetime, and sedimentation equilibrium. The software is available by contacting the authors.
Adam GC, Sorensen EJ, Cravatt BF. Trifunctional chemical probes for the consolidated detection and identification of enzyme activities from complex proteomes. Molecular and Cellular Proteomics2002;1:828–835. [PubMed]
Affinity reagents that covalently modify active sites of enzymes in complex proteomes are useful reagents for monitoring changes in expression of enzymes in altered physiological and pathological states in an activity-based manner. Detection of expression changes is often conducted with fluorescent probes, whereas purification and identification of the enzymes utilize biotinylated probes. The present paper describes the synthesis and application of a trifunctional probe that incorporates both a fluorescent group for detection and a biotin group for isolation, linked to a sulfate ester reactive group for labeling a particular enzyme class. See also the review by the same authors: Chemical strategies for functional proteomics. Molecular and Cellular Proteomics 2002;1:781–790.
Nuwaysir EF, et al. Gene expression analysis using oligonucleotide arrays produced by maskless photolithography. Genome Research2002;12:1749–1755. [PubMed]
The quality of in situ chemical synthesis of oligonucleotides on glass surfaces using the Texas Instruments digital light processor for maskless fabrication is examined. The production of oligonucleotide microarrays containing 195,000 features is demonstrated. The utility of such arrays for gene expression analysis is evaluated, and the effect of oligonucleotide length is addressed. This technology is of interest to investigators wishing to perform experiments with high-density custom arrays.