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Pham V, Tropea J, Wong S, Quach J, Henzel WJ. High-throughput protein sequencing. Analytical Chemistry 2003;75:875–882. [PubMed]
The design of an autosampler for use on the Applied Biosystems Procise sequencer is described. The autosampler replaces one of the standard glass reaction vessels and holds up to six separate samples. Modifications to the sequencer’s programming are presented to increase the efficiency of the coupling reaction and reduce internal cleavages due to residual trifluoroacetic acid in the standard system.
Schultz CL, Moini M. Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization–mass spectrometry. Analytical Chemistry 2003;75:1508–1513. [PubMed]
A partial separation of underivatized protein amino acids is described for online detection by electrospray mass spectrometry. The separation is of 35- min duration, and on-column detection limits are in the low femtomol range. The separation of 11 underivatized l-amino acids from their d-enantiomers is also demonstrated by using a 30-mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the chiral selector/background electrolyte.
Rombel IT, Talaat AM, Johnston SA. Shotgun library construction in a day. BioTechniques 2003;34:244–250. [PubMed]
Procedures are described for the rapid production of random genomic libraries suitable for expression library immunization, in which DNA fragments are screened for their ability to protect animals against infection by particular pathogens. The procedures use starting amounts of genomic DNA in the nanogram range, and can be completed in a single day. The quantitative representation of the genes in the resulting libraries are assessed using spotted microarrays.
Ishikawa T, Hayashida Y, Hirayasu K, Ozawa K, Yamamoto N, Tanaka T, Matsuura S. Use of transcriptional sequencing in difficult to read areas of the genome. Analytical Biochemistry 2003;316:202–207. [PubMed]
The methodology involves the use of a DNA-directed RNA polymerase, T7 RNA polymerase incorporating the F644Ymutation, to transcribe into cRNA the DNA primer to be sequenced. This enzyme is able to utilize templates with GC-rich sequences, and requires a double-stranded template instead of the single-stranded template used by DNA polymerase. A sequencing ladder is obtained by terminating RNA synthesis with occasional incorporation of fluorescent dye-labeled 3′-deoxynuceotides. Efficient sequencing through GC-rich regions and regions with putative hairpin structures is demonstrated.
Marchand S, Hajdari P, Hackman P, Udd B, Richard I. Touch-down method for high-performance sequencing of polymerase chain reaction products. Analytical Biochemistry 2003;315:270–272. [PubMed]
This method minimizes the effort of designing optimal custom primers and identifying the best conditions of annealing temperature for each combination of primers and template DNA in a large sequencing project involving direct sequencing of PCR products. The cycling reactions for both the amplification and sequencing steps are performed under conditions of progressively decreasing annealing temperatures (the “touch down” approach). Despite minimal attention to primer design, a very high success rate is observed.
Jiang Y, Hofstadler SA. A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry. Analytical Biochemistry 2003;316:50–57. [PubMed]
The method uses Millipore AX (anion exchange) ZipTips in an automated protocol that includes tip pretreatment to wet the resin, sample loading, rinsing, and sample elution. It is suitable for manual operation with a standard p10 pipette, or in a fully automated microtiter plate format, and is accomplished in 20 min. The method removes salts, low molecular weight polymeric materials, and oligonucleotide primers that would otherwise dominate the spectra.
Kawasaki N, Itoh S, Ohta M, Hayakawa T. Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography /tandem mass spectrometry. Analytical Biochemistry 2003;316:15–22. [PubMed]
Oligosaccharides were separated for online electrospray mass spectrometry using a 0.2 × 150-mm capillary column of 5 μm graphitized carbon. The mobile phase was 5 mM ammonium acetate, pH 9.6, and elution was performed with an acetonitrile gradient. Sugar mapping with both capillary LC/mass spectrometry and capillary LC/tandem mass spectrometry could be performed to yield structural information.
Hart C, Schulenberg B, Steinberg TH, Leung W-Y, Patton WF. Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 2003;24:588–598. [PubMed]
The performance of a new fluorescent stain for glycoproteins, Pro-Q Emerald 488, is documented. It is 8–16-fold more sensitive than the colorimetric stain, acid fuchsin sulfite, and detects 5–18 ng of glycoprotein per gel band, depending on the nature and degree of glycosylation. Gels may be counter-stained with SYPRO Ruby to visualize total protein. Pro-Q Emerald 488 is commercially available from Molecular Probes, Inc.
Williams TL, Andrzejewski D, Lay JO Jr, Musser SM. Experimental factors affecting the quality and reproducibility of MALDI TOF mass spectra obtained from whole bacteria cells. Journal of the American Society for Mass Spectrometry 2003;14:342–351. [PubMed]
Conditions of sample preparation and mass analysis are systematically evaluated for their effect on the analysis of intact bacteria by MALDI-TOF mass spectrometry. In this particular study, optimal conditions included sterilization of the cells in 70% ethanol, use of α-cyano-4-hydroxycinnamic acid as MALDI matrix dissolved in 50:50 acetonitrile:water with 2.5% trifluoroacetic acid, use of a Nd:Yag laser for ionization, and analysis of serial dilutions of the bacteria to ensure that the optimal concentration is identified. Although it is expected that details of spectra may vary considerably between different laboratories for the same bacterial strain, investigators seeking an aid to rapid species identification may find this technique useful.
Ge Y, ElNaggar M, Sze SK, Oh HB, Begley TP, McLafferty FW, Boshoff H, Barry CE III. Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry. Journal of the American Society for Mass Spectrometry 2003;14:253–261. [PubMed]
Because proteins secreted by Mycobacterium tuberculosis may be involved in disease pathogenesis and present potential diagnostic and vaccine candidates, this paper explores the use of accurate measurement of protein mass by electrospray ionization / Fourier transform mass spectrometry for identification of these proteins. Identifications based on matches with mass values expected for proteins encoded by the fully sequenced mycobacterial genome are almost invariably unreliable. This is because of extensive posttranslational modification of these particular proteins, including loss of signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Nevertheless, for the purposes of identification, electron capture dissociation provides especially informative information about chemical structure.
Schröder E, Jönsson T, Poole L. Hydroxyapatite chromatography: Altering the phosphate-dependent elution profile of protein as a function of pH. Analytical Biochemistry 2003;313:176–178. [PubMed]
This paper demonstrates how the retention of proteins by the long-used chromatographic sorbent, hydroxyapatite, can be modified and controlled by altering the pH of the mobile phase. When elution is performed with gradients of phosphate concentration, the usable pH range is circumscribed by the poor buffering capacity of phosphate outside the range 6.0–7.5. To extend this range, Hepes or Mes buffers are added, and advantage taken of the delay in phosphate-dependent elution induced by decreasing the pH.
Sodhi R, Rajput S. Method for dialysis of samples in microliter volumes. Analytical Biochemistry 2003;315:141–142. [PubMed]
A simple devise for the dialysis of microliter volumes is described. It is readily made from a 1.5-mL microcentrifuge tube by piercing holes in the side walls of the tube, and placing a dialysis membrane between the tube and its lid to create a sample chamber in the recess inside the lid. The assembly is floated in dialysis solution, and bubbles may be removed by tilting the tube.
Wu X, Liu H, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP. Immunofluoresent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nature Biotechnology 2003;21:41–46.
Jaiswal JK, Mattoussi H, Mauro JM, Simon SM. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nature Biotechnology 2003;21:47–51.
These two reports are examples of work currently emerging that begins to fulfill the promise of quantum dot technology as a substitute for chemical fluorophores and visible fluorescent proteins. Quantum dots are semiconducor nanocrystals that absorb radiation over a broad spectral range but emit it in a very narrow band, at a wavelength governed precisely by the size to which the crystals are manufactured. Developments in surface coatings of these particles have helped overcome previous problems with toxicity, nonspecific binding, and quenching of emission. Wu et al. used multicolor quantum dots for simultaneous labeling of target molecules on the cell surface, cytoplasm, and nucleus of cultured cells. Jaiswal et al. labeled live cells with quantum dots of different color, enabling observation of their growth as distinct cell populations for over a week. See also the following reviews: Jovin TM. Quantum dots finally come of age.Nature Biotechnology 2003;21:32–33; Seydel C. Quantum dots get wet. Science 2003;300:80–81 [PubMed].
Adkins JN, Varnum SM, Auberry KJ, Moore RJ, Angell NH, Smith RD, Springer DL, Pounds JG. Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry. Molecular and Cellular Proteomics 2002;1:947–955. [PubMed]
Pieper R, Su Q, Gatlin CL, Huang S-T, Anderson NL, Steiner S. Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome. Proteomics 2003;3:422–432. [PubMed]
Rothemund DL, Locke VL, Liew A, Thomas TM, Wasinger V, Rylatt DB. Depletion of the highly abundant protein albumin from human plasma using the Gradiflow. Proteomics 2003;3:279–287. [PubMed]
These three papers exemplify the development of approaches to overcome the problem of sensitively detecting low abundance proteins in sources, notably plasma, that contain a few very high abundance proteins. This is important in the proteomic search for disease- or treatment-related markers. Adkins et al. remove immunoglobulins with protein A/G prior to LC/tandem mass spectrometry. Pieper et al. remove multiple proteins by immunoaffinity subtraction prior to 2D gel electrophoresis. Each group demonstrates large increases in the number of low abundance proteins subsequently detected. Rothemund et al. remove albumin by preparative electrophoresis and subsequently detect another protein, previously masked by albumin, using MALDI-TOF mass spectrometry.
Svensson M, Sköld K, Svennigsson P, Andren PE. Peptidomics-based discovery of novel neuropeptides. Journal of Proteome Research 2003;2:213–219. [PubMed]
Using brain tissue homogenized by sonication and then passed through a centrifugal filter with nominal molecular weight limit of 10,000 Da, LC/MS and LC/MS/MS are used to detect 550 endogenous neuropeptides, including both novel and previously described molecular species. The results are viewed in two- and three-dimensional image maps.
Wang J, Hu L, Hamilton SR, Coombes KR, Zhang W. RNA amplification strategies for cDNA microarray experiments. BioTechniques 2003;34:394–400. [PubMed]
The results of expression analysis using two alternative strategies for amplifying RNA by in vitro transcription are compared. The amplification methods differ in the method by which the dsDNA template for the RNA polymerase is prepared. The first is a combination of reverse transcription with conventional second-strand cDNA synthesis. The second is a combination of the switch mechanism at the 5′ end of RNA templates and reverse transcription, followed by PCR. Both amplification methods are found to give reproducible results, and perform similarly with regard to the identification of differentially expressed genes. However, more RNA is obtained from the same amount of starting material by second-strand cDNA synthesis than by the template-switching amplification protocol, an advantage when the amount of starting material is limited.
Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovski I, Liu Z, Vinsavich A, Trush V, Quackenbush J. TM4: A free, open-source system for microarray data management and analysis. BioTechniques 2003;34:374–378. [PubMed]
TM4, a software suite for managing and analyzing microarray gene expression data is described. It consists of four applications, Microarray Data Manager (MADAM), TIGR_Spotfinder, Microarray Data Analysis System (MIDAS) and Multiexperiment Viewer (MeV). MySQL, a database compliant with Minimal Information About a Microarray Experiment (MIAME) is also included. The suite is freely available at http://www.tigr.org/software.
Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Research 2003;31:e15. [PubMed]
A new method—log scale robust multi-array analysis (RMA)—is described for summarizing data (“probe level data”) from the set of probes representing each individual gene in an Affymetrix GeneChip array. The performance of the method is assessed using spike-in studies and a dilution study. RMA performs better than both the MAS 5.0 software available from Affymetrix and the dChip software from Harvard. It significantly reduces within-replicate variance, provides more consistent estimates of fold-change, and provides higher specificity and sensitivity when using fold-change analysis to detect differential expression. RMA is freely available at http://www.bioconductor.org.
Kusnezow W, Jacob A, Walijew A, Diehl F, Hoheisel JD. Antibody microarrays: An evaluation of production parameters. Proteomics 2003;3:254–264. [PubMed]
Peluso P, Wilson DS, Do D, Tran H, Venkatasubbaiah M, Quincy D, Heidecker B, Poindexter K, Tolani N, Phelan M, Witte K, Jung LS, Wagner P, Nock S. Optimizing antibody immobilization strategies for the construction of protein microarrays. Analytical Biochemistry 2003;312:113–124. [PubMed]
These papers both evaluate the effect on performance of antibody microarrays of parameters concerning array production. Kusnezow et al. study modification of the glass surface, the kind and length of cross-linkers, the composition and pH of the spotting buffer, blocking reagents, and antibody concentration and storage procedures. Peluso et al. use surface plasmon resonance to investigate the effect of orientation of the antibodies on the surface. These studies will be of interest to investigators undertaking work with protein expression using microarray technology.
Frazer KA, Elnitski L, Church DM, Dubchak I, Hardison RC. Cross-species sequence comparisons: A review of methods and available resources. Genome Research 2003;13:1–12. [PubMed]
This review summarizes the methods used and the computational resources available for determining which genes in different species bear an orthologous relationship one to another, i.e., are derived from the same gene in the last common ancestral species, and thus may be expected to have the same function. The identification of orthologs is an important strategy for genome annotation.
Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN. GoMiner: A resource for biological interpretation of genomic and proteomic data. Genome Biology 2003;4:R28. [PubMed]
Gene ontology (GO) organizes genes into hierarchical categories based on biological processes, molecular function, and subcellular organization. GoMiner is a program suite developed for the biological interpretation of microarray gene expression data. The enrichment of under- and over-expressed genes in different GO categories is calculated relative to the genes represented on the array as a whole. Results are displayed in “directed acyclic graph” or equivalent tree structure formats, and statistical calculations are performed. The software is available free of charge at http://discover.nci.nih.gov/gominer or http://www.miblab.getech.edu/gominer.