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J Biomol Tech. 2007 February; 18(1): 94–98.
PMCID: PMC2291924

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AMINO ACID ANALYSIS AND SEQUENCING

Song Y, Feng Y, LeBlanc MH, Zhao S, Liu Y-M. Assay of trace D-amino acids in neural tissue samples by capillary liquid chromatography/tandem mass spectrometry. Analytical Chemistry 78;2006:8121–8128. [PubMed]

Derivatization and separation conditions suitable for quantitating low levels of D-amino acids in tissues are developed. The methodology involves derivatizing amino acids with 7-fluoro-4-nitrobenzoxadiazole, concentrating them on a reverse-phase microcolumn, and then resolving them on a teicoplanin aglycon chiral stationary phase from Astec (Whippany, NJ). The procedure is shown to support quantitation of trace levels of D-Asp and D-Ser in tissue samples.

DNA CHARACTERIZATION AND GENOTYPING

Fiegler H, Redon R, Andrews D, Scott C, Andrews R, Carder C, Clark R, Dovey O, Ellis P, Feuk L, French L, Hunt P, Kalaitzopoulos D, Larkin J, Montgomery L, Perry GH, Plumb BW, Porter K, Rigby RE, Rigler D, Valsesia A, Langford C, Humphray SJ, Scherer SW, Lee C, Hurles ME, Carter NP. Accurate and reliable high-throughput detection of copy number variation in the human genome. Genome Research 16;2006:1566–1574. [PubMed]

Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, Fiegler H, Shapero MH, Carson AR, Chen W, Cho EK, Dallaire S, Freeman JL, Gonzalez JR, Gratacos M, Huang J, Kalaitzopoulos D, Komura D, MacDonald JR, Marshall CR, Mei R, Montgomery L, Nishimura K, Okamura K, Shen F, Somerville MJ, Tchinda J, Valsesia A, Woodwark C, Yang F, Zhang J, Zerjal T, Zhang J, Armengol L, Conrad DF, Estivill X, Tyler-Smith C, Carter NP, Aburatani H, Lee C, Jones KW, Scherer SW, Hurles ME. Global variation in copy number in the human genome. Nature 444;2006:444–454. [PubMed]

These papers document the methodology (Fiegler et al.) and the results (Redon et al.) of a project to investigate the genome-wide characteristics of copy number variation in the humans. For comparative genomic hybridization (array-CGH), a whole-genome tiling path resolution array is constructed. The array consists of 26,574 clones selected from the published “Golden Path” that was used to generate the reference human sequence. It covers 93.7% of euchromatic regions. The clones are extensively validated by fingerprinting and end sequencing, and by the use of control “add-in” hybridizations that allow direct estimation of the hybridization characteristics of each individual clone. To detect significant copy number changes, estimates of data reproducibility and false-positive rates are made by self-self hybridizations, replicate experiments, and by using independently validated copy number variants to maximize the number of variants called while keeping false positives to less than 5%. Two hundred seventy individuals from Europe, Africa, and Asia (the HapMap collection) are screened, and a total of 1,447 copy number variable regions covering 360 megabases (12% of the genome) are identified. Linkage disequilibrium patterns are discerned for many of the variants, and differences in copy number between the populations are documented. These results represent a resource for genetic study of disease.

Komura D, Shen F, Shikawa S, Fitch KR, Chen W, Zhang J, Liu G, Ihara S, Nakamura H, Hurles ME, Lee C, Scherer SW, Jones KW, Shapero MH, Huang J, Aburatani H. Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays. Genome Research 16;2006:1575–1584. [PubMed]

An algorithm for identifying copy number variants using Affymetrix Human Mapping 500K SNP genotyping arrays is described. The methodology utilizes improved intensity normalization methods, an adapted SW-ARRAY algorithm for automatic detection of candidate copy number variant regions, and a routine to identify and quantitate gains and losses relative to a confirmed diploid group. Some 1203 copy number variations are identified among the 270 HapMap samples.

Zhang Z, Zhou L, Zhao S, Deng H, Deng Q. 3-Hydroxycoumarin as a new matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of DNA. Journal of the American Society for Mass Spectrometry 17;2006:1665–1668. [PubMed]

A new matrix, 3-hydroxycoumarin (3-HC), is described for analysis of oligonucleotides by MALDI-TOF mass spectrometry. It is dissolved in a mixture of acetone and diammonium hydrogen citrate. Using a Bruker REFLEX III mass spectrometer with a nitrogen laser, the matrix supports ionization of oligonucleotides from 3 to 70 bases in length. Oligonucleotides with masses 800–6900 Da are isotopically resolved, and less than 250 amol of a 10-mer is detected on target. The matrix can be used for both positive and negative ion analyses. The matrix provides better resolution than conventional matrices such as 3-hydroxypicolinic acid.

CARBOHYDRATE AND GLYCOPROTEIN ANALYSIS

Snovida SI, Chen VC, Perreault H. Use of a 2,5-dihydroxybenzoic acid/aniline MALDI matrix for improved detection and on-target derivatization of glycans: A preliminary report. Analytical Chemistry 78;2006:8561–8568. [PubMed]

To facilitate the detection of glycans by mass spectrometry in the positive ion mode, glycans are commonly subjected to chemical modification, often by reductive or non-reductive amidation. In this paper, aniline is used to form a stable Schiff base with the non-reducing end GlcNAc residues. Upon mixing the glycan on-target with 2,5-dihydroxybenzoic acid (DHB) matrix containing aniline, the reaction takes place concurrently with drying of the spot within minutes and without application of heat. The mixed matrix improves MALDI signal strength even for detection of the underivatized glycan, and produces a finer, more homogeneous crystal layer than DHB alone.

MASS SPECTROMETRY

Frank AM, Savitski MM, Nielsen ML, Zubarev RA, Pevzner PA. De novo peptide sequencing and identification with precision mass spectrometry. Journal of Proteome Research 6;2007:114–123. [PubMed]

A new de novo sequencing algorithm is presented for the interpretation of MS/MS spectra from instruments that provide high mass accuracy, such as QTOF, OrbiTrap, and FT-ICR mass spectrometers. By making use of high product ion mass accuracy, the multiple sequence solutions that can often be obtained from single mass spectra acquired at lower levels of mass accuracy are rarely encountered, and unique solutions are normal. The availability of such unique solutions provides the capability of assigning peptide identities based on sequence search rather than pattern matching of uninterpreted spectra. The benefits from this are considerable. Because de novo sequencing becomes the heaviest computational task, search times become essentially independent of the size of the sequence database. Furthermore, sequences not in the database, such as alternatively spliced variants, fusion proteins, and frame shifts become amenable to successful identification. The de novo sequencing software is available at http://peptide.ucsd.edu/.

Na S, Paek E. Quality assessment of tandem mass spectra based on cumulative intensity normalization. Journal of Proteome Research 5;2006:3241–3248. [PubMed]

A new method for assessing the quality of an MS/MS spectrum is presented. The method is based on the use of “cumulative intensity normalization” for measuring the distribution of peak intensities in a spectrum. A quality score is calculated that permits potentially interpretable spectra to be distinguished from spectra of lesser quality. The latter can then be excluded from database searches to reduce false-positive assignment rates.

Horneffer V, Strupat K, Hillenkamp F. Localization of non-covalent complexes in MALDI preparations by CLSM. Journal of the American Society for Mass Spectrometry 17;2006:1599–1604. [PubMed]

The hexameric complex of the protein allophycocyanine is studied to elucidate conditions under which non-covalently associated protein complexes may be studied by MALDI mass spectrometry. 6-Aza-2-thiothymidine is used as MALDI matrix. The integrity of complexes is assessed by confocal fluorescence microscopy (the protein looses its fluorescence when it dissociates into subunts) as well as by mass spectrometry. Protein precipitated at the matrix crystal surface is observed by both techniques to retain its multimeric structure, but protein incorporated into the crystal lattice dissociates. These observations provide an explanation for the “first shot phenomenon,” in which only the first (or the first few) laser exposures of a given sample reveal quaternary structure.

PROTEINS—PURIFICATION AND CHARACTERIZATION

McKay AR, Ruotolo BT, Ilag LL, Robinson CV. Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. Journal of the American Chemical Society 128;2006:11,433–11,442.

The analysis of very large complexes by electrospray mass spectrometry is complicated by broadening of spectral peaks and increase in m/z values due to the formation of adducts with salts. This paper shows a correlation between peak broadening and percent increase in m/z. This correlation provides a means of estimating from experimental data the m/z values that would be obtained in the absence of salt adduction. Moreover, because methods for charge state deconvolution of electrospray spectra ordinarily used for smaller proteins don’t work well for large complexes, a method is presented for estimating charge states by fitting values to the data and determining which provides the greatest consistency of masses calculated from all the peaks in the spectrum. Using these methods, accurate mass values are shown to be obtainable for complexes as large as the 30 S ribosomal subunit and the intact ribosome of T. thermophilus (approximately 2.3 MDa).

Soderblom EJ, Goshe MB. Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis. Analytical Chemistry 78;2006:8059–8068. [PubMed]

Chemical cross-linkers designed to facilitate the analysis of cross-linked peptides by mass spectrometry are described. The cross-linkers contain an aspartyl-proline bond, which is cleaved more readily by collision-induced dissociation than most other peptide bonds. Using an LCQ Deca ion-trap mass spectrometer, the mass of cross-linked pairs of peptides is first measured under conditions that do not cleave the cross-linker. Then, by applying a potential offset between the skimmer lens and the multipole region of the mass spectrometer, fragmentation of the cross-linker is induced. The separated peptides may then be analyzed individually to establish their identity and hence to determine the position of the cross-link.

Stead JA, Keen JN, McDowall KJ. The identification of nucleic acid–interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay. Molecular and Cellular Proteomics 5;2006:1697–1702. [PubMed]

The electrophoretic mobility shift assay method is incorporated into a 2D electrophoresis protocol to assist in the identification of proteins that specifically bind to DNA sequences of interest. Upon detecting the presence of a protein that binds a labeled oligonucleotide in a standard electrophoretic mobility shift assay, native gel electrophoresis of the protein mixture is performed in tube gels in the presence and absence of the oligonucleotide. The tube gels are then fused to the top of the stacking portion of SDS-polyacrylamide gels for the second dimension of a two-dimensional separation. Proteins migrating differently in the presence and absence of the oligonucleotide are then subjected to protein identification as putatively specific oligonucleotide-binding species.

Benita Y, Wise MJ, Lok MC, Humphrey-Smith I, Oosting RS. Analysis of high-throughput protein expression in Escherichia coli. Molecular and Cellular Proteomics 5;2006:1567–1580.

For a set of 547 proteins (average length, 76 amino acids), correlations are sought between features of amino acid sequence and the degree of success achieved in high-throughput expression in E. coli. Seventy-seven percent of the proteins in the study were expressed at levels detectable by general protein staining of bacterial lysates after SDS-PAGE, and 59% were of the expected molecular weight. Although no algorithm predicting success with good sensitivity and specificity emerges from the results, indicators of unsuccessful expression are high hydrophobicity, high pI, low sequence complexity, low disorder, and high β-sheet propensity.

PROTEOMICS

Valentine SJ, Plasencia MD, Liu X, Krishnan M, Naylor S, Udseth HR, Smith RD, Clemmer DE. Toward plasma proteome profiling with ion mobility–mass spectrometry. Journal of Proteome Research 5;2006:2977–2984. [PubMed]

Without any attempt to remove abundant proteins beforehand, plasma is digested with trypsin and the peptides are fractionated by strong cation exchange and reverse-phase chromatography. Ion mobility separation is then used during mass spectrometric analysis to further disperse peptides in the gas phase. This procedure reduces spectral congestion and thus increases dynamic range without any penalty in throughput. Seven hundred thirty-one high-confidence peptide assignments are recorded from a single 3-h experiment, allowing 438 proteins to be identified. The technique is of interest to investigators seeking to profile large numbers of individuals in bio-marker discovery studies.

Nielsen ML, Savitski MM, Zubarev RA. Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics. Molecular and Cellular Proteomics 5;2006:2384–2391. [PubMed]

Using a program called ModifiComb, which clusters together MS/MS spectra derived from a single peptide sequence but displaying various modifications (including unexpected and novel ones), the incidence of modifications in tryptic peptides encountered during proteomic analyses is estimated. The average number of modified peptides per unmodified peptide is 2.4. The number for a single abundant protein, actin, is 6.5. Eleven of the peptides from actin have 10 or more modified forms each. This very high incidence of modified peptides results from the combinatorial association of numerous types of modification, even though each modification may be present at sub-stoichiometric levels. As dynamic range is expanded, the incidence of modified peptides rises to levels that cannot be ignored, not least because modified peptides generally show a high rate of spurious matching with unmodified sequence libraries, including reversed-sequence libraries. Among the strategies for dealing with this problem, that of including numerous possible modifications in database searches causes an explosion of false-positive assignments unless threshold acceptance scores are elevated to a point at which false negatives in turn become problematic. A variety of alternative stratagems is evaluated.

Meiring HD, Soethout EC, Poelen MCM, Mooibroek D, Hoogerbrugge R, Timmermans H, Boog CJ, Heck AJR, de Jong APJM, van Els CACM. stable isotope tagging of epitopes. Molecular and Cellular Proteomics 5;2006:902–913. [PubMed]

Methodology for distinguishing peptides induced by viral infection from endogenous peptides is described for use in the study of antigen presentation on MHC Class I molecules. Proteins synthesized during viral infection are metabolically labeled by incorporation of isotope-tagged amino acids from the culture fluid. Labeled cells are mixed with an equal number of cells cultured in normal medium; peptides are eluted from immunoprecipitated MHC molecules, and then analyzed by two-dimensional LC-MS. The data allow both identification of virally induced peptides and absolute quantification of epitope expression.

Dowell JA, Heyden WV, Li L. Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: Enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC. Journal of Proteome Research 5;2006:3368–3375. [PubMed]

The study of neuropeptides is complicated by rapid proteolytic degradation of neural proteins post mortem and associated contamination of neuropeptide fractions by protein fragments. As an alternative to the use of focused microwave irradiation to inactivate proteases, brains are here rapidly removed and snap frozen for cryostatic dissection. Tissue samples are removed from the freezer and boiling water is added immediately. The temperature is maintained at 100°C for 10 min. The water is decanted and saved, and the tissue is re-extracted with 0.25% acetic acid. The resulting peptide mixtures show little contamination from protein degradation products. Fifty-six peptides from known neuropeptide precursors are identified after two-dimensional liquid chromatography and mass spectrometry.

Donoghue PM, McManus CA, O’Donoghue NM, Pennington SR, Dunn MJ. CyDye immunoblotting for proteomics: Co-detection of specific immunoreactive and total protein profiles. Proteomics 6;2006:6400–6404. [PubMed]

In 2D electrophoresis, the difficulty of aligning proteins specifically detected by immunoblotting and the complex spot patterns revealed by total protein staining is addressed in this paper by the use of the newly developed ECL Plex-CyDye immunoblot detection system from GE Healthcare. Protein samples are labeled with charge-matched cyanine CyDye reagents Cy3 and Cy5, then separated by 2D electrophoresis, and transferred to a membrane by electroblotting. The membrane is probed with an antibody of choice, which is then localized with wavelength-specific ECL Plex fluor-labeled secondary antibodies. This allows both the immunoreactive protein(s) and the total protein profile to be visualized concurrently.

Kane LA, Yung CK, Agnetti G, Neverova I, Van Eyk JE. Optimization of paper bridge loading for 2-DE analysis in the basic PH region: Application to the mitochondrial subproteome. Proteomics 6;2006:5683–5687. [PubMed]

Modifications are described to a new method for loading samples onto IPG strips that helps minimize protein streaking. The method is similar to cup loading, but the sample is transferred to the strip not from a cup but from a small piece of absorbant paper applied to the strip surface. In the present variant of the method, filter paper is used and applied to the anodal end of the strip. Sample loading occurs overnight, with the electrodes placed at the far end of the filter paper bridge and at the cathodal end of the IPG strip. The bridge is then removed and the electrodes are repositioned to the ends of the IPG strip for continued sample resolution. Better resolution on 2D gels than that achieved by cup loading is observed over a wide range of protein loads. Proteins retained by the paper bridge while loading onto basic pH range IPG strips can be reloaded onto acidic range strips to cover a broader pH range.

MICROARRAYS

Glazer M, Fidanza JA, McGall GH, Trulson MO, Forman JE, Suseno A, Frank CW. Kinetics of oligonucleotide hybridization to photolithographically patterned DNA arrays. Analytical Biochemistry 358;2006:225–238. [PubMed]

The binding behavior of DNA targets to surface-bound probes remains incompletely understood, yet is important for designing and optimizing array applications. In this paper, adsorption and desorption rates, as well as equilibrium binding constants, are measured for arrays manufactured by the photolithographic process used at Affymetrix, in which the density of initiation sites for probe synthesis is high but the stepwise yield is poor, producing a low density of full-length probes. Hybridization is investigated both at 22°C and at the commonly used 45°C. Kinetic constants are found to agree with those measured on arrays made by other methods. The adsorbed target density is much lower than expected for ideal Langmuir-type adsorption, but can be fitted by the Sips model, which allows for a distribution of binding energies. Probe-probe interactions, steric effects, and electrostatic effects may contribute to this behavior. Binding kinetics display further non-ideal behavior in showing an overshoot at 22°C and a secondary rise at 45°C. The overshoot behavior is attributed to adsorption of multiple targets to approximately 30% of the probes, followed by displacement by the one target that binds to the maximum number of bases on the probe. These results are of interest with respect to improvements in the design of arrays concerning probe density, length distribution, and surface properties, and with respect to the desire to shorten hybridization assay times.

Angenendt P, Kreutzberger J, Glökler J, Hoheisel JD. Generation of high-density protein microarrrays by cell-free in situ expression of unpurified PCR products. Molecular and Cellular Proteomics 5;2006:1658–1666. [PubMed]

High-density protein microarrays are produced from DNA template molecules or indeed from unpurified PCR products by in situ transcription and translation. The DNA template is first spotted onto the array surface, and then a cell-free transcription and translation mixture is transferred to the same spot. Yields of protein comparable to 300 μg/mL of spotted protein are demonstrated. The methodology is utilized for in situ expression of 384 randomly chosen clones.

FUNCTIONAL GENOMICS AND PROTEOMICS

Berger MF, Philippakis AA, Qureshi AM, He FS, Estep PW III, Bulyk ML. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nature Biotechnology 24;2006:1429–1435.

An experimental platform for determining the DNA binding specificity of transcription factors is described. A protein-binding microarray presenting all possible DNA sequence variants of 10-base-pair binding sites is constructed. For this purpose, a single-stranded oligo-nucleotide array is converted to a double-stranded array by primer extension from a primer sequence common to all probes. The binding specificities of five transcription factors are determined with the system.

Hartmann CH, Klein CA. Gene expression profiling of single cells on large-scale oligonucleotide arrays. Nucleic Acids Research 34;2006:e143. [PubMed]

A PCR-based amplification method that avoids distortion of transcript abundance is presented, and is tested on single cells rather than diluted total RNA. A major increase in correlation between the results from single cells and standard total RNA samples is realized by isolating mRNA with biotinylated poly-T peptide nucleic acids bound to streptavidin beads. This results in a four- to fivefold increase in signal strength compared to oligo-dT beads, and a 10-fold improvement in dynamic range. Use of a cDNA synthesis primer containing oligo-dT(24) instead of oligo-dT(15) also improves the results. The method is tested on single cells with low mRNA content, including epithelial cells, dendritic cells, and hematopoietic stem cells. Quantitative PCR is employed to confirm that relative transcript abundance is preserved. The method requires no special equipment, is easy to perform, and is robust.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities