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J Biomol Tech. 2006 April; 17(2): 187–192.
PMCID: PMC2291773

ARTICLE WATCH

AMINO ACID ANALYSIS

Miao H, Rubakhin SS, Sweedler JV.Subcellular analysis of d-aspartate. Analytical Chemistry 77;2005:7190–7194. [PubMed]

The neurotransmitter d-aspartate is assayed in subregions of individual neurons of aplesia using a capillary electrophoretic method. Special conditions are required to achieve the required dissection and analytical sensitivity. Cells are incubated briefly in 30% glycerol prior to microdissection to preserve cell morphology with little spatial spreading of analytes. Amino acid derivatives are made by reaction with naphthalene-2,3-dicarboxaldehyde for detection by laser-induced fluorescence. Chiral separation is achieved by a micellar electrophoresis procedure. The presence of d-Asp is demonstrated in the neuronal processes of individual cells. The methods are applicable to the analysis of spatial and temporal changes in a variety of small molecules on a similarly small scale.

Sandlin ZD, Shou M, Shackman JG, Kennedy RT. Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters. Analytical Chemistry 77;2005:7702–7707. [PubMed]

A microfluidic device is used for electrophoretic analysis of glutamate sampled continuously from rat brain with a coupled microdialysis probe. Amino acids are derivatized with o-phthaldialdehyde in the device for detection by laser-induced fluorescence. Separations are achieved with up to 156,000 theoretical plates within 20 sec. By serial injection from the probe into the resolving channel, the dialysate is analyzed at 130-sec intervals, enabling monitoring of rapid changes. The method can be extended to the analysis of other small molecules and to the monitoring of other organs, bioreactors, and environmental sampling.

DNA CHARACTERIZATION AND GENOTYPING

Neely LA, Patel S, Garver J, Gallo M, Hackett M, Mclaughlin S, Nadel M, Harris J, Gullans S, Rooke J. A single-molecule method for the quantitation of microRNA gene expression. Nature Methods 3;2006:41–46. [PubMed]

The short nature of microRNAs (miRNAs) makes them difficult to detect by conventional techniques. A new method is presented that extends the sensitivity with which miRNAs are detected and quantitated. The method makes use of locked nucleic acids (LNAs)—nucleic acid analogs that are conformationally locked in a C3′-endo/N-type sugar conformation by an O2′ to C4′ methylene linkage that confers increased stability of duplexes between LNA-containing oligonucleotides and standard nucleic acids. Two spectrally distinguishable, fluorescent LNA-DNA oligonucleotide probes are hybridized to different regions of the miRNA of interest. The two-fluorophore-labeled miRNAs are then directly counted in a single-molecule detection instrument that counts coincident bursts of photon emission from the doubly tagged molecules as they flow through a detection capillary. The method detects miRNAs at a concentration of 500 fM, and has a linear dynamic range of 103. This methodology is used to quantitate the expression of 45 human miRNAs in 16 different tissues. The results confirm and extend knowledge of the distribution of these miRNAs.

Raymond CK, Subramanian S, Paddock M, Qiu R, Deodato C, Palmieri A, Chang J, Radke T, Haugeen E, Kas A, Waring D, Bovee D, Stacy R, Kaul R, Olsen MV. Targeted, haplotype-resolved resequencing of long segments of the human genome. Genomics 86;2005:759–766. [PubMed]

A production-scale method is presented for re-sequencing targeted segments of DNA for which a reference sequence is available, in a haplotype-resolved manner. A fosmid cloning system is used, which combines the high stability of BACs with the ease of library construction of cosmids. To distinguish between parental haplotypes, the method uses single-nucleotide polymorphisms, microsatellite markers, and insertion-deletion polymorphisms to differentiate the target region into haplotype-specific tiling paths. The method is used here to re-sequence 400 kbp from the human CFTR region from 15 individuals. It is anticipated to be useful in searching for causal mutations in regions of genomes identified by linkage or association studies, for analyzing polymorphisms in highly variable regions that are difficult to amplify consistently by PCR methods, and for studies of haplotype structure.

CARBOHYDRATES, GLYCOLIPIDS AND GLYCOPROTEINS

Qiu R, Regnier FE. Comparative glycomics of N-linked complex-type glycoforms containing sialic acid in human serum. Analytical Chemistry 77;2005:7225–7231. [PubMed]

Sialylated glycopeptides in tryptic digests of serum proteins are selected by lectin affinity chromatography using Sambucus nigra agglutinin. They are then further fractionated into bi-antennary and tri/tetra-antennary pools based on differential affinity to a second lectin, concanavalin A. Peptides in these pools are then isotopically labeled by acetylation, remixed, and deglycosylated with PNGase F. Identification and relative quantitation is performed by mass spectrometry. Sialylated glycopeptides are shown to contain more bi-antennary than tri/tetra-antennary glycans, but the relative amounts vary among the separate glycosylation sites on the same glycoprotein.

Naka R, Kamoda s, Ishizuka A, Kinoshita M, Kakehi K. Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chromatography. Journal of Proteome Research 5;2006:88–97. [PubMed]

Methodology for comprehensive characterization of the ensemble of cell-surface N-glycan structures is presented. N-glycans are released from the cell surface with N-glycoamidase F and labeled with 2-aminobenzoic acid. They are then fractionated into pools containing different numbers of sialic acid residues by affinity chromatography on a serotonin column. Desialidation is then performed with neuraminidase, and carbohydrates are further fractionated by chromatography on an Amide-80 column (Tosoh) for structural analysis by MALDI-TOF mass spectrometry. The results demonstrate differences in glycoform structure between different tumor cell lines, presumably reflecting differences in expression of the corresponding glycotransferase enzymes.

METABONOMICS

Jackson SN, Wang HJ, Woods AS. In situ structural characterization of phosphatidylcholines in brain tissue using MALDI-MS/MS. Journal of the American Society of Mass Spectrometry 16;2005:2052–2056.

Phosphorylcholine (PC) species are detected and structurally characterized by MALDI mass spectrometry in cryostatically sectioned tissue. The matrix 2,6-dihydroxyacetophenone (DHA) is applied by spotting a solution in 50% ethanol. Identification and positional assignment of acyl chains are confirmed by MS/MS analysis of lithium adducts formed by incorporating 100 mM lithium chloride in the matrix solution. The methodology can be extended to imaging of the distribution of other classes of phospholipids and gangliosides in tissues.

Want EJ, O’Maillie G, Smith CA, Brandon TR, Uritboonthai W, Qin C, Trauger SA, Siuzdak G. solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry. Analytical Chemistry 78;2006:743–752. [PubMed]

In the study of serum metabolites, proteins tend to dominate chromatographic analyses and to suppress signals from compounds of lower abundance. Deproteination can be performed using organic solvents (e.g., methanol, acetonitrile), lowering the pH, or by heat denaturation. In this paper, 14 protein precipitation methods are systematically evaluated on the basis of the number of metabolite mass spectral features detected, the type of metabolites extracted based on hydrophobicity, and the amount of protein remaining. The methods varied widely in efficacy. However, one of the simplest procedures, methanol precipitation followed by centrifugation, gave the lowest protein interference, the most comprehensive metabolite profile, and the most reproducible results.

Smith CA, Want EJ, O’Maillie G, Abagyan R, Siuzdak G. XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Analytical Chemistry 78;2006:779–787. [PubMed]

An essential step in processing data for comparative analysis is matching peaks representing the same analyte from different samples. A method called XCMS (an acronym for various forms, X, of chromatography mass spectrometry) is described that incorporates nonlinear retention time alignment, matched filtration, peak detection, and peak matching without using added internal standards. The software can handle large data sets and permits discovery of novel differences between two sample groups. XCMS is freely available under open-source license at http://metlin.scripps.edu/download/.

MASS SPECTROMETRY

Yates JR, Cociorva D, Liao L, Zabrouskov V. performance of a linear ion trap–Orbitrap hybrid for peptide analysis. Analytical Chemistry 78;2006:493–500. [PubMed]

Use of a new hybrid mass spectrometer combining a linear ion trap with an Orbitrap analyzer is evaluated for study of the complex peptide mixtures encountered in shotgun proteomics. Mass resolution in excess of 40,000 and mass accuracies better than 2 ppm are demonstrated. This level of performance facilitates peptide assignment and elimination of false positives, and is expected to help de novo sequence analysis.

Tuytten R, Lemière F, Witters E, Van Dongen W, Slegers H, Newton RP, Van Onckelen H, Esmans EL. Stainless steel electrospray probe: A dead end for phosphorylated organic compounds? Journal of Chromatography A 1104;2006:209–221. [PubMed]

This study documents the propensity of stainless steel components of liquid chromatography systems to interact with phosphorylated compounds, including nucleotides, oligonucleotides, phosphopeptides, phospholipids, and phosphorylated sugars. Stainless steel can retain and trap such molecules via an IMAC-type mechanism. This may be an important source of analyte loss in studies of such compounds.

Hofstadler SA, Drader JJ, Schink A. Selective ion filtering by digital thresholding: A method to unwind complex ESI-mass spectra and eliminate signals from low molecular weight chemical noise. Analytical Chemistry 78;2006:372–378. [PubMed]

Larger, more highly charged ions produce a greater detector response than low-molecular-weight, singly charged ions. Use is made of this observation in a digital thresholding scheme applied to individual scans prior to their co-addition, with the effect that signals due to low-molecular-weight ions are filtered from those of high-molecular-weight ions. With this approach, detection of 33-kDa PCR amplicons in the negative ion mode is demonstrated in the presence of very high levels of low-molecular-weight contaminants. Multiply charged ions of carbonic anhydrase are also detected in positive ion mode in the presence of an interfering detergent. Conversely, multiple spectra acquired at different thresholds are subtracted from one another to yield spectra of low-molecular-weight species with improved peak shape and mass accuracy

Schlosser G, Pocsfalvi G, Huszár E, Malorni A, Hudecz F. MALDI-TOF mass spectrometry of a combinatorial peptide library: Effect of matrix composition on signal suppression. Journal of Mass Spectrometry 40;2005:1590–1592. [PubMed]

This study illustrates the ability of changes in MALDI matrix composition to relieve suppression effects in analyte mixtures. As a model test mixture, a combinatorial peptide library of pentapeptides with sequence TQTXT is tested, where X represents 19 different amino acids. In α-cyano-4-hydroxycinnamic acid (CCA), only the arginine peptide, TQTRT, is detected with good intensity. When ammonium citrate is added, the situation is even worse, with the arginine peptide the only component detected. However, much improved ion yields are observed with a mixture of CCA and 2,5-dihydroxybenzoic acid (DHB). Addition of K+ as a cationizing agent to CCA produces optimal spectra with relative ion intensities similar to those observed by electrospray ionization.

Wa C, Cerny R, Hage DS. Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: Analysis of human serum albumin as a model. Analytical Biochemistry 349;2006:229–241. [PubMed]

Using human serum albumin as a model protein, methods for maximizing sequence coverage by MALDI mass spectrometry are explored. A matrix mixture of CCA and DHB gives higher sequence coverage than CCA alone. When used with a tryptic digest, this mixed matrix yields a coverage of 52%. However, when the results of digestion with endopeptidase Lys-C and endopeptidase Glu-C are added to those with trypsin, the coverage increases to 73%. Finally, when the peptides are fractionated on C-18 ZipTips, the coverage reaches 97%.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Hanna C, Gjerde D, Nguyen L, Dickman M, Brown P, Hornby D. Micro-scale open-tube capillary separations of functional proteins. Analytical Biochemistry 350;2006:128–137. [PubMed]

A conceptually simple technique is described for purification and concentration of native proteins and protein complexes in open fused-silica capillary tubes. The walls of the tubing are chemically derivatized for binding to the protein of interest, and sample is pumped through with a syringe. After washing protein bound to the tube walls, elution is performed by introducing eluting solvent in a volume that is small compared to the internal volume of the tube, moving the liquid plug back and forth within the tube, and finally pumping it out for subsequent analysis. Hundred-fold concentration is achieved with samples of less than 1 mL initial volume. The derivatized tubes are used disposably to prevent carry-over.

Kameyama K, Minton AP. Rapid quantitative characterization of protein interactions by composition gradient static light scattering. Biophysical Journal 90;2006:2164–2169. [PubMed]

Methodology is described for quantitative analysis of self- and hetero-association equilibria in a solution containing two interacting proteins. A programmable dual-syringe pump delivers the interacting species to a tee junction for mixing to produce a solution of time-varying composition. A peristaltic pump delivers buffer into the flow stream to modify conditions under which association takes place, again on a programmable basis. The flow of interacting species is then spit between an absorbance detector and a multiangle light-scattering detector. The method is shown to resolve both self- and hetero-association equilibria occurring in the same solution, and to characterize the self-association of a protein that forms chains of indefinite length. Data acquisition is rapid. In one case, an experiment lasting less than 15 min produced information equivalent to that obtained via sedimentation equilibrium that would have required several days to record in an analytical ultracentrifuge.

Talkington MWT, Siuzdak G, Williamson JR. An assembly landscape for the 30s ribosomal subunit. Nature 438;2005:628–632. [PubMed]

Methodology is presented for characterizing the pathways by which complex macromolecular machines are assembled, and is applied to the self-assembly of the 30S ribosomal subunit. 16S ribosomal RNA from E. coli is incubated with a mixture of the 21 30S ribosomal proteins uniformly labeled with 15N. At various time points, binding of the 15N proteins is chased with an excess of unlabeled (14N) proteins. Completely formed 30S subunits are allowed to form, and are purified. Mass spectral analysis then yields 15N/14N ratios that provide information about the stage in the assembly pathway at which each of the subunits is recruited into the complex. By purifying mature particles, complexity due to the presence of mis-assembled complexes is avoided. The method is suitable in principle for any self-assembling system.

Noda T, Sagara H, Yen A, Takada A, Kida H, Cheng RH, Kawaoka Y. Architecture of ribonucleoprotein complexes in infuenza A virus particles. Nature 439;2006:490–492. [PubMed]

This paper uses transmission electron microscopy of serial sections through cells to characterize the assembly of a particle that forms in a membrane-dependent manner—the infuenza A virus. The virion contains eight single-stranded RNA segments associated with nucleoproteins (RNPs) and three polymerase subunits. The RNPs are shown to be organized in a distinct pattern in which the individual RNPs are suspended from the interior of the viral membrane envelope at the cell surface, oriented perpendicular to the budding tip. The findings suggest an ordered assembly process rather than random incorporation of RNPs into virions.

PROTEOMICS

Heller M, Ye M, Michel PE, Morier P, Stalder D, Jünger MA, Aebersold R, Reymond F, Rossier JS. Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides. Journal of Proteome Research 4;2005:2273–2282. [PubMed]

Peptide assignments returned by the PHENYX or SEQUEST search engines in a shotgun proteomics study are subjected to validation by comparing the predicted isoelectric point and hydrophobicity of the assigned peptide sequence with the experimentally observed behavior of the peptide during the separations that precede mass spectrometry. Peptides are subjected first to fractionation by isoelectic focusing, and then by reverse-phase chromatography, providing analytical information about isoelectric point and hydrophobicity, respectively. A Visual-Basic macro is used to calculate pI values and predict reverse-phase retention times of assigned peptides. The method is designed to substantially reduce false-positive identification rates in shotgun proteomics. The predictive macro is available from DiagnoSwiss SA at moc.ssiwsongaid@ofni.

Steen H, Jebanathirajah JA, Rush J, Morrice N, Kirschner MW. Phosphorylation analysis by mass spectrometry: Myths, facts, and the consequences for qualitative and quantitative measurements. Molecular and Cellular Proteomics 5;2006:172–180. [PubMed]

Among the reasons commonly cited for the problematic nature of mass spectrometric analysis of phosphopeptides are: (1) increased hydrophilicity of phosphopeptides with attendant loss during loading on reverse-phase columns; (2) selective suppression of phosphopeptide ionization in the presence of unphosphorylated peptides; and (3) lower ionization/detection efficiency of phosphopeptides compared with their unphosphorylated counterparts. The present paper reports the results of an experimental survey of phosphopeptides by electrospray mass spectrometry designed to test these arguments. Contrary to expectations, the results show no general increase in the risk of losing phosphopeptides during sample loading for reverse-phase separations, or suppression of ionization by non-phosphorylated peptides. Furthermore, no general evidence for lower ionization/detection efficiency was obtained. These results suggest that the main difficulty in phosphopeptide analysis is the frequently substoichiometric nature of the modification.

Ptacek J, Devgan G, Michaud G, Zhu H, Zhu X, Fasolo J, Guo H, Jona G, Breitkreutz A, Sopko R, Mccartney RR, Schmidt MC, Rachidi N, Lee S-J, Mah AS, Meng L, Stark MJR, Stern DF, De Virgilio C, Tyers M, Andrews B, Gerstein M, Schweitzer B, Predki PF, Snyder M. Global analysis of protein phosphorylation in yeast. Nature 438;2005:679–684. [PubMed]

The specificities of 82 of the 122 yeast protein kinases are documented. The kinases are purified and each is incubated in the presence of [γ-33P]ATP with an array containing 4400 different yeast proteins. The arrays are subjected to autoradiography to determine kinase specificity. Four thousand phosphorylation events involving 1325 proteins are documented. The results are assembled into a phosphorylation map for yeast.

Csizmók V, Szöllösi E, Friedrich P, Tompa P. A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins. Molecular and Cellular Proteomics 5;2006:265–273. [PubMed]

Intrinsically unstructured proteins (IUPs) have highly flexible conformations in the unbound state, but acquire definable structures when bound to other proteins. They are believed to be numerous and their unstructured nature is thought to be of regulatory importance in allowing them to bind to and activate/de-activate various target proteins differentially according to physiological requirements. This paper describes a technique based on 2D electrophoresis for recognizing and characterizing IUPs. Cell lysates are enriched in IUPs by making use of their general resistance to precipitation when heated. The heat-treated proteins are then subjected to native gel electrophoresis in the first dimension and to electrophoresis in 8 M urea in the second. IUPs migrate on the diagonal in this system, whereas globular proteins that unfold in urea migrate off the diagonal. Several new IUPs are identified by mass spectrometry.

MICROARRAYS

Elo LL, Lahti L, Skottman H, Kyläniemi M, Lahesmaa R, Aittokallio T. Integrating probe-level expression changes across generations of Affymetrix arrays. Nucleic Acids Research 33;2005:e193. [PubMed]

Alternative methods for combining data on gene expression changes across successive generations of Affymetrix GeneChips with different probe collections are evaluated in terms of the comparability of gene expression changes and the consistency of the lists of differentially expressed genes they yield, specifically when sample size is reduced. The superiority of combination based on probe-level information to that based on manufacturer-defined probe sets is illustrated, and a new combination method based on probe-level sequence matching is described. Data from mis-targeted, nonspecific, and conflicting probes are disregarded, and alternative probe sets based on the remaining verified probes are defined. Expression statistics are first calculated separately for each probe using perfect-match intensities that are quantile normalized and log transformed, and then the expression statistics are averaged over probes in the alternative probe sets.

Little SE, Vuononvirta R, Reis-Filho JS, Natrajan R, Iravani M, Fenwick K, Macay A, Ashworth A, Pritchard-Jones K, Jones C. Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA. Genomics 87;2006:298–306. [PubMed]

Studies of alterations in copy number of genes in tumor DNA and the utility of these studies in diagnosis, prognosis, and treatment response have hitherto focused on samples flash-frozen at the time of surgical tissue removal. This paper explores the use of tumor DNA from formalin-fixed, paraffin-embedded (FFPE) archival specimens for use in array comparative genome hybridization (CGH) studies. The GenomePlex whole genome amplification (WGA) kit from Rubicon Genomics/Sigma-Aldrich is shown to provide reproducible and accurate array CGH profiles for FFPE tumor samples using as little as 5 ng of starting DNA. Copy number changes based on hybridization to BAC clones with 0.9-Mb resolution are measured throughout the genome with no chromosomal bias.

FUNCTIONAL GENOMICS AND PROTEOMICS

Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M. Silencing of microRNAs in vivo with “antagomirs.” Nature 438;2005:685–689. [PubMed]

To better understand the function of endogenous miRNAs in vivo, a class of chemically modified, single-stranded RNA analogs complementary to miRNAs are developed. Upon intravenous injection into mice, these molecules, called antagomirs, produce marked reductions in the corresponding miRNA levels.

Gillette WK, Esposito D, Frank PH, Zhou M, Yu L-R, Jozwik C, Zhang X, McGowan B, Jacobowitz DM, Pollard HB, Hao T, Hill DE, Vidal M, Conrads TP, Veenstra TD, Hartley JL. Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression. Molecular and Cellular Proteomics 4;2005:1647–1652. [PubMed]

Not all genes are amenable to high-throughput protein expression and purification. Testing each gene individually for its ability to produce high levels of soluble, purified protein is labor intensive and costly. This paper describes an alternative, simpler methodology that combines recombinational cloning of pools of sequenced ORFs to produce pools of the cognate proteins, and two-dimensional gel electrophoresis to identify the genes that express well.

Siddiqui AS, Khattra J, Delaney AD, Zhao Y, Astell C, Asano J, Babakaiff R, Barber S, Beland J, Bohacec S, Brown-John M, Chand S, Charest D, Charters AM, Cullum R, Dhalla N, Featherstone R, Gerhard DS, Hoffman B, Holt RA, Hou J, Kuo BY, Lee LL, Lee S, Leung D, Ma K, Matsuo C, Mayo M, McDonald H, Prabhu AL, Pandoh P, Riggins GJ, de Algara TR, Rupert JL, Smailus D, Stott J, Tsai M, Varhol R, Vrljicak P, Wong D, Wu MK, Xie YY, Yang G, Zhang I, Hirst M, Jones SJ, Helgason CD, Simpson EM, Hoodless PA, Marra MA. A mouse atlas of gene expression: large-scale digital gene-expression profiles from precisely defined developing c57Bl/6J mouse tissues and cells. Proceedings of the National Academy of Sciences, U.S.A. 102;2005:18,485–18,490.

To systematically identify the genes expressed in precisely defined cell and tissue types, serial analysis of gene expression (LongSAGE) is used to develop gene expression profiles throughout development in a total of 200 mouse cells and tissues. Some 8.55 million LongSAGE 21-bp tags are generated from 72 libraries, each library from a different mouse tissue. The data provide a detailed description of changes in gene expression levels during development. The majority of previously identified transcripts are covered, plus transcripts from some 24,000 previously undescribed loci. The data and software tools are available at http://cgap.nci.nih.gov/SAGE.

Uhlén M, Bjorling E, Agaton C, Szigyarto CA, Amini B, Andersen E, Andersson AC, Angelidou P, Asplund A, Asplund C, Berglund L, Bergstrom K, Brumer H, Cerjan D, Ekstrom M, Elobeid A, Eriksson C, Fagerberg L, Falk R, Fall J, Forsberg M, Bjorklund MG, Gumbel K, Halimi A, Hallin I, Hamsten C, Hansson M, Hedhammar M, Hercules G, Kampf C, Larsson K, Lindskog M, Lodewyckx W, Lund J, Lundeberg J, Magnusson K, Malm E, Nilsson P, Odling J, Oksvold P, Olsson I, Oster E, Ottosson J, Paavilainen L, Persson A, Rimini R, Rockberg J, Runeson M, Sivertsson A, Skollermo A, Steen J, Stenvall M, Sterky F, Stromberg S, Sundberg M, Tegel H, Tourle s, Wahlund E, Walden A, Wan J, Wernerus H, Westberg J, Wester K, Wrethagen U, Xu LL, Hober S, Ponten F. A human protein atlas for normal and cancer tissues based on antibody proteomics. Molecular and Cellular Proteomics 4;2005:1920–1932. [PubMed]

The Human Proteome Resource program announces a new immunohistological atlas of the distribution of some 700 proteins in 48 normal human tissues and 20 different cancers. The image data have been acquired using a panel of mono-specific, polyclonal antisera, each elicited against a selected fragment of protein sequence expressed with an epitope tag for purification. Polyclonal antisera from animals immunized with these protein fragments are immunoaffinity purified, and then used in high-throughput protein expression and localization studies on tissue microarrays from which the images in the atlas are generated. The database can be accessed at http://www.proteinatlas.org/, and consists of approximately 400,000 high-resolution images. Interestingly, very few proteins are expressed in just a single tissue or organ, even though many of the proteins analyzed have previously been described as tissue specific. See also a paper covering related ground: Nilsson P et al., Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling. Proteomics 5;2005:4327–4337 [PubMed] . Work in which the antibody library is used for serum and plasma biomarker discovery appears in Janzi M et al., Serum microarrays for large scale screening of protein levels. Molecular and Cellular Proteomics 4;2005:1942–1946. [PubMed]

BIOINFORMATICS

Allison DB, Cui X, Page GP, Sabripour M. Microarray data analysis: From disarray to consolidation and consensus. Nature Reviews Genetics 7;2006:55–65.

This article represents a distillation of the statistical literature on methodology for microarray analysis. A guide is provided for the alternative methods applicable to each stage in the microarray analysis procedure: experimental design, data processing (including normalization, transformation, data filtering, background subtraction, etc.), statistical inference (involving testing statistical hypotheses) and/or classification (supervised or unsupervised), and validation (confirming the validity of conclusions). Investigators are encouraged to pick a method from those available for each stage, but there is often insufficient information on which to base dogmatic choices between the alternatives. Among the important needs highlighted are for the development of methods for microarray quality control, standardized testing platforms, and data sets for which the true structure is known, which can be used for testing proposed analytical methods. Also recommended are the collection of empirical evidence about the performance of sample pooling, and consideration of how best to use resampling-based inference.


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