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Holmes MR, Giddings MC. Prediction of posttranslational modifications using intact-protein mass spectrometric data. Analytical Chemistry 76;2004:276–282. [PubMed]
An application is described that assists in predicting the proteolytic cleavages and post-translational modifications that may account for differences between the mass of a protein measured by mass spectrometry and the calculated mass based on the known amino acid sequence. The application, called PROCLAME (Protein Cleavage and Modification Engine), is available as a web-based application at http://proclame.unc.edu. It uses a depth-first tree search, bounded by a set of rules controlled by a custom-built fuzzy logic engine, to explore a large number of combinations of possible mass-altering events.
Bader JS, Chaudhuri A, Rothberg JM, Chant J. Gaining confidence in high-throughput protein interaction networks. Nature Biotechnology 22;2004:78–85.
This paper addresses the problem of extracting biologically meaningful information from protein interaction data derived by high-throughput methods such as yeast two-hybrid screening and co-immunoprecipitation. Such data are understood to contain a high proportion of spurious interactions. The approach here is to define a quantitative confidence measure for the biological relevance of interactions based entirely on screening statistics and network topology. Additional information such as expression correlations, genetic interactions, and database annotations could provide an improved level of interaction confidence, but it is reserved here for subsequent independent validation of the purely topological confidence measures so as to avoid bias and cross-pollution. The topological properties of the network show a hierarchical organization with a mean complex size of 15 proteins and a correlation distance (the maximum number of links that indicates a significant correlation between protein pairs) of one to two links. This agrees with the correlation distance identified by database annotations and co-expression. The methods presented are expected to play an important role in analyzing the biological content of the huge genomic and proteomic data sets now emerging from model organisms and humans.
Zhang Z, Gildersleeve J, Yang Y-Y, Xu R, Loo JA, Uryu S, Wong C-H, Schultz PG. A new strategy for the synthesis of glycoproteins. Science 303;2004:371–373. [PubMed]
This paper describes the co-translational incorporation of glycosylated serine at defined positions into proteins expressed in E. coli. To achieve this, an amino acyl tRNA synthase is selected that charges its cognate tRNA with GlcNAc-modified serine and incorporates the modified residue in response to the amber codon, TAG. Because carbohydrates with free hydroxyl groups are transported poorly across cell membranes, the acetylated derivative, tri-acetyl-β-GlcNAc-serine is used instead. Incorporation of this residue into myoglobin at a defined position is demonstrated to occur with high yield and fidelity. The β-GlcNAc moiety can then be modified by galactosyltransferase to build a more complex carbohydrate. The system affords the possibility of making homogeneously glycosylated proteins in large amounts for characterization of the functional effects of glycosylation.
Clayton D, Shapovalov G, Maurer JA, Dougherty DA, Lester HA, Kochendoerfer GG. Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences, USA 101;2004:4764–4769.
The mechanosensitive integral membrane ion channel proteins, Ec-MscL from E. coli and Tb-MscL from Mycobacterium tuberculosis, are subjected to chemical synthesis by sectional synthesis methods optimized for hydrophobic peptides. The resulting products are transferred to 2,2,2-trifluoroethanol and reconstituted into lipid membranes. Fluorescence imaging shows that the proteins are localized to the membrane. Single-channel recording techniques are used to demonstrate that they exhibit conductance and pressure-dependent functions. This methodology provides an opportunity to incorporate chemically modified amino acids into the protein for use in functional studies.
Wuhrer M, Koeleman CAM, Deelder AM, Hokke CH. Normal-phase nanoscale liquid chromatography-mass spectrometry of underivatized oligosaccharides at low-femtomole sensitivity. Analytical Chemistry 76;2004:833–838. [PubMed]
A normal-phase method for the chromatographic separation of underivatized glycans is described. The method employs 75-μm ID columns of Amide-80 (Tosohaas, Montgomeryville, PA) with a flow rate of 300 nL/min. The solvent system is compatible with on-line electrospray mass spectrometry. Separation of isobaric structures is achieved, and detection sensitivities in the low fmol range attained.
Bunkenborg J, Pilch BJ, Podtelejnikov AV, Wisniewski JR. Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics 4;2004:454–465. [PubMed]
Glycoproteins are first selected by lectin affinity chromatography using concanavalin A or wheat germ agglutinin. Retained glycoproteins are then digested by endoproteinase Lys-C and deglycosylated by N-glycanase F. They are digested with trypsin prior to on-line liquid chromatography/mass spectrometry for peptide identification. The method can be used to identify sites of N-glycosylation among proteins in complex mixtures, although information about carbohydrate structures is restricted to that which can be deduced from their lectin-binding characteristics.
Matsuzaki H, Loi H, Dong S, Tsai Y-Y, Fang J, Law J, Di X, Liu W-M, Yang G, Liu G, Huang J, Kennedy GC, Ryder TB, Marcus GA, Walsh PS, Shriver MD, Puck JM, Jones KW, Mei R. Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Research 14;2004:414–425. [PubMed]
Genotyping of 11,555 SNPs is performed by allele-specific hybridization to an oligonucleotide array now commercially available from Affymetrix. Genomic DNA is subjected to restriction enzyme digestion, ligation with an adaptor, and amplification by PCR using a single primer specific for the adaptor sequence. This PCR step preferentially amplifies restriction fragments of 250–10,000 base pairs, providing a reproducible 50-fold reduction in genome complexity that is responsible for allele-specific hybridization to the array. The genotyped SNPs are spaced by an average of 210 kb (0.31 cM). The average heterozygosity for this SNP locus set is 0.38, providing a viable genotyping alternative to panels of microsatellites (STRs). Commercial arrays with much higher numbers of SNPs affording increased resolution are anticipated for release soon. The methodology provides high-throughput capability and will be useful for detecting loss of heterozygosity, for population genetics and for whole-genome association studies.
Smylie KJ, Cantor CR, Denissenko MF. Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF. Genome Research 14;2004:134–141. [PubMed]
This method for SNP analysis involves base-specific cleavage of primer extension products in which acid-labile phosphoramidite bonds replace the 5′ phosphodiester bonds where phosphoramidite-modified dTTP or dCTP is newly incorporated into the chain. After cleavage, the fragments are analyzed by MALDI-TOF mass spectrometry.
Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M, Aruleba S, Ballja V, O’Shaughnessy A, Gnoj L, Scoble K, Chang K, Westbrook T, Cleary M, Sachidanandam R, McCombie WR, Elledge SJ, Hannon GJ. A resource for large-scale RNA-interference-based screens in mammals. Nature 428;2004:427–431. [PubMed]
An extensive short hairpin RNA (shRNA) expression library has been constructed for use in gene silencing by RNA interference in mammalian cells; 9,610 human genes are targeted. Sequences have been chosen where possible to be identical to the mouse ortholog and they target 5,563 mouse genes. Each gene is targeted by between three and nine constructs in the library. Only coding regions are targeted, and each shRNA is chosen to give at least three mismatches to any other mammalian gene to minimize cross-hybridization. The abundance of any shRNA can be followed by means of a unique “bar code,” a 60-residue sequence that will hybridize to a complementary probe on a specially designed oligonucleotide microarray. The bar codes allow individual mutants to be followed in complex cell populations, for example in the context of experiments in which a growth advantage is sought. This library is expected to become a most valuable resource for functional genomics in mammalian cells.
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nature Biotechnology 22;2004:326–330.
From an analysis of the potency of 180 siRNAs targeting the products of two genes, structural correlations are made to assist in the selection of siRNA sequences. These include low G/C content, low internal structural stability at the sense strand 3′-terminus, lack of inverted repeats, and preferences for particular bases at four positions in the sense strand. By applying the complete set of criteria in the selection of siRNA sequences for silencing a panel of six human test genes, 29 of 30 selected sequences induced more than 50% silencing of their targets.
Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proceedings of the National Academy of Sciences, U.S.A. 101;2004:1892–1897.
This and the following paper are among several recent reports indicating that caution should be exercised in the interpretation of RNA interference data. In a test of the specificity of siRNA-mediated gene silencing, 10 different siRNAs targeting a single gene, multiple endocrine neoplasia, type 1 (MEN1), are tested for their effect on products of the intended gene, and on two ostensibly irrelevant genes, TP53 (p53) and CDKN1A (p21), in four different cell lines. Significant changes in the levels of p53 and p21 unrelated to the silencing of the target gene are observed, although no such changes are found in another protein, actin. Such off-target effects are speculated to be due to partial complementary sequence matches causing micro-RNA-like inhibition of translation.
Kim D-H, Longo M, Han Y, Lundberg P, Cantin E, Rossi JJ. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nature Biotechnology 22;2004:321–325.
siRNAs can be chemically synthesized, but can be produced more cheaply by the action of bacteriophage T7 DNA-dependent RNA polymerase. This paper shows that siRNAs synthesized by T7 polymerase can trigger induction of interferon α and β in a variety of cell lines. This response is mediated by the T7 initiating sequence, pppGGG, where p is phosphate and G is guanine. Treatment of T7-synthesized siRNAs with RNase T1 and then calf intestinal alkaline phosphatase to remove the 5′-triphosphate largely abolishes the interferon response, but maintains full efficacy of the siRNAs in gene silencing.
Graber A, Juhasz PS, Khainovski N, Parker KC, Patterson DH, Martin SA. Result-driven strategies for protein identification and quantitation—A way to optimize experimental design and derive reliable results. Proteomics 4;2004:474–489. [PubMed]
The combination of liquid chromatography and tandem mass spectrometry in proteomic studies has spawned useful data-dependent (result-driven) strategies in which the acquisition of MS/MS information from the LC eluate is triggered when the quality of MS data being acquired exceeds some threshold. This paper illustrates the possibilities for increasing the sophistication of such strategies when mass spectrometry is performed off-line, as when column fractions are collected for subsequent study using MALDI. For example, MS/MS spectra may be acquired only for pairs of ICAT-peptides with signal intensities significantly different from the overall mean, indicating that the expression level of the corresponding protein has changed. Minor components of a peptide mixture may be subjected to MS/MS analysis before major ones, since the latter are less likely to be lost through the cumulative effects of laser pulses. MS/MS scans may be triggered in a search-dependent manner only when MS data fail to yield unambiguous protein identifications.
Gustafsson M, Hirschberg D, Palmberg C, Jörnvall H, Bergman T. Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk. Analytical Chemistry 76;2004:345–350. [PubMed]
This paper documents an integrated microfluidic system for the concentration, desalting, and deposition of protein digests on a target for MALDI mass spectrometry. The processing is all done on a compact disk, and uses centrifugal force to move liquid through microchannels on the disk surface, finally depositing each sample onto a target area of 200 × 400 μm on the disk. Ninety-six samples can be processed in parallel. The disk is then inserted into the mass spectrometer for analysis. Routine sensitivities for protein identification down to 200 amol are demonstrated, and peptides are detected down to 50 amol. Disks and associated robotic devices are supplied commercially by Gyros AB, www.gyros.com.
Yang X, Wu H, Kobayashi T, Solaro RJ, van Beemen RB. Enhancing ionization of phosphorylated peptides during MALDI TOF mass spectrometry. Analytical Chemistry 76;2004:1532–1536. [PubMed]
Enhancement of signal strength for tryptic phosphopeptides in MALDI-TOF mass spectrometry is demonstrated with the use of 2′,4′,6′-trihydroxyacetophenone (THAP) plus diammonium citrate as the matrix instead of α-cyano-4-hydroxycinnamic acid plus diammonium citrate. Tenfold increases in signal strength are demonstrated, and benefits are observed in both positive and negative ion modes.
Wu Z, Gao W, Phelps MA, Miller DD, Dalton JT. Favorable effects of weak acids on negative-ion electrospray ionization mass spectrometry. Analytical Chemistry 76;2004:839–847. [PubMed]
Acids are commonly added to solvents to promote positive ion electrospray ionization. However, addition of base doesn’t generally facilitate deprotonation in the negative ion mode. In the context of a study of four small molecules, selective androgen receptor modulators, this paper examines the effects of various solvent additives in negative ion ESI. Low concentrations of weak acids are found to increase negative ion responses, probably because of the high gas-phase proton affinity of their anions. High concentrations of acids suppress the ESI− response owing to the propensity of the acid to accumulate at the droplet surface and displace analyte. This effect is strongest for the most hydrophilic analytes, which are most easily displaced. Acetic acid provides the optimal properties for an ESI− modifier.
Pan P, Gunawardena HP, Xia Y, McLuckey SA. Nanoelectrospray ionization of protein mixtures: solution pH and protein pI. Analytical Chemistry 76;2004:1165–1174. [PubMed]
This paper explores the possibility that selective ionization of different proteins in mixtures may occur during the electrospray process, depending on protein pI and solution pH. Although it is possible to form gas-phase protein ions of one polarity from a solution in which the protein has a net charge of the opposite polarity, the total electrospray response under such conditions is at least 10-fold lower than when the gas phase polarity is the same as the sign of the solution-phase charge. Maximal signals in the positive ion mode are observed when the solution pH is 4–5 units lower than the protein pI. In the negative ion mode, maximal signals are seen when the solution pH is 5 units higher than the protein pI. The extent of sodium ion adduction also depends strongly on the relationship between pI and pH, being greatest when the protein ion polarities are of opposite sign in solution and in the gas phase.
Kenzelmann M, Klären R, Hergenhahn M, Bonrouhi M, Gröne H-J, Schmid W, Schütz G. High-accuracy amplification of nanogram total RNA amounts for gene profiling. Genomics 83;2004:550–558. [PubMed]
In the amplification of mRNA required for gene expression profiling from small numbers of cells, the faithfulness of amplification is sensitive to the error-prone reverse transcription (RT) reaction and to the number of cycles of amplification. Here, reproducibility is improved by addition of the bacteriophage T4 DNA-binding protein T4gp32 to the RT reaction, and the use of an increased reaction temperature (50°C instead of 42°C). These changes provide better accessibility of the template to the reverse transcriptase and help reduce RNA secondary structure formation, thus diminishing the loss of 5′ end information. The reproducibility and accuracy of the improved RT method is demonstrated in an analysis of human prostate tissue cells obtained by laser-assisted microdissection.
Liu RH, Yang J, Lenigk R, Bonanno J, Grodzinski P. Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Analytical Chemistry 76;2004:1824–1831. [PubMed]
This paper describes a fully integrated, self-contained biochip device manufactured by Motorola Labs for “all-in-one” DNA analysis of complex biological samples. The device contains chambers for sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), followed by PCR amplification and electrochemical DNA microarray-based detection. Crude biological samples and reagent solutions are loaded into the device, and genetic information is the output. Use of the device for detection of putatively pathogenic bacteria from milliliters of whole blood, and SNP analysis directly from whole blood samples is demonstrated. Uses in point-of-care genetic analysis, environmental testing, and detection of biological warfare agents are anticipated.
Ishkanian AS, Malloff CA, Watson SK, deLeeuw RJ, Chi B, Coe BP, Snijders A, Albertson DG, Pinkell D, Marra MA, Ling V, MacAulay C, Lam WL. A tiling resolution DNA microarray with complete coverage of the human genome. Nature Genetics 36;2004:299–303. [PubMed]
Array-format comparative genomic hybridization (array CGH) has proven to be a valuable method for assessment of DNA copy-number variation within the genome. The technique is usually performed with BAC DNA markers positioned at intervals of 1–2 Mb. In the present paper, the power of the methodology is extended by the production of a tiling array consisting of 32,433 overlapping BAC clones covering the entire human genome. Use of this array permits comprehensive screening for very small genomic changes, and definition of their boundaries, at sub-megabase resolution.
Bignell GR, Huang J, Greshock J, Watt S, Butler A, West S, Grigorova M, Jones KW, Wei W, Stratton MR, Futreal PA, Weber B, Shapero MH, Wooster R. High-resolution analysis of DNA copy number using oligonucleotide microarrays. Genome Research 14;2004:287–295. [PubMed]
The effectiveness of an oligonucleotide array originally designed for human SNP analysis is tested for its effectiveness in detecting DNA copy number alterations. Twenty cancer cell lines are studied. Ten homozygous deletions and 12 amplifications with copy number > 4 are detected using the SNP array and all are confirmed by PCR. Lower level copy number changes are seen in 2 cell lines. Study of these by array CGH show concordant results in 44 instances. Of the 10 discordant comparisons, 8 are in regions of low SNP density. Among the cell lines tested, 94 regions of 10 Mb or greater show loss of heterozygosity but no decrease in copy number, suggesting that either mitotic recombination or loss of one parental region and compensatory duplication of the other parental copy may have occurred. Such events would not be detected by array CGH. This illustrates an advantage of acquiring genotyping data in conjunction with copy number analysis.
Canziani GA, Klakamp S, Myszka DG. Kinetic screening of antibodies from crude hybridoma samples using Biacore. Analytical Biochemistry 325;2004:301–307. [PubMed]
Surface plasmon resonance spectroscopy is used to determine kinetic and affinity constants for the interaction of antibodies and antigen in a Biacore system. Antibodies in crude samples are immobilized on the Biacore chip using an anti-Fc capture antibody, and antigen binding is then measured using a single concentration of antigen. Immobilizing the antibody minimizes complications due to avidity effects. Although antibody concentrations are not known beforehand, the data for different antibodies can be normalized based on the amount of antibody captured in each case, and a 50-fold range of antibody concentration can be accommodated. The four separate binding surfaces in a Biacore 2000 or 3000 system permit 3 antibodies to be analyzed simultaneously, keeping one surface as a control, and the capture reagent is sufficiently robust for at least 100 capture cycles with different antibodies. In this way, 100–200 antibody samples can be tested per day.
Drake AW, Myszka DG, Klakamp SL. Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods. Analytical Biochemistry 328;2004:35–43. [PubMed]
SPR spectroscopy is used as described above to measure rate constants and KD for antigen/antibody interactions with KD in the low pmol to low nmol range. For the antibodies with the slowest dissociation rates, it is necessary to monitor dissociation over a 4-h period to obtain sufficient data, but dissociation rate constants as low as 1 × 10−5 s−1 are measured. Measured values are confirmed by the independent technique of kinetic exclusion assay, indicating that mass transport and steric hindrance effects in the Biacore experiments do not significantly affect these measurements.
Shumaker-Parry JS, Campbell CT. Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy. Analytical Chemistry 76;2004:907–917. [PubMed]
Shumaker-Parry JS, Zareie MH, Aebersold R, Campbell CT. Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein– DNA interactions by surface plasmon resonance microscopy. Analytical Chemistry 76;2004:918–929. [PubMed]
SPR microscopy, or imaging SPR, is a method for measuring spatial variations in molecular interactions with resolution down to ~4 μm on an SPR-active surface of relatively large area. If the absorbate has a refractive index different from that of the solvent, its binding to the surface can be detected by changes in reflected light intensity. The first paper presents an analysis of the relationship between reflected light intensity at high contrast angles and the absolute coverage by adsorbate. The methodology is applied to the adsorption of protein onto DNA arrays on gold. The detection limit for protein adsorption is 1.2 ng/cm2 (~0.5 pg in a 200-μm spot), and the linear dynamic range is ~720 ng/cm2 of adsorbed protein. Instrument drift and changes in refractive index of the solution in the flow cell can be corrected using a nearby spot as a reference channel. The second paper presents methods for spotting 10 × 12 arrays of double-stranded DNAs onto a gold-coated glass slide for high-throughput studies of protein DNA interactions. Simultaneous monitoring of protein binding to many 200-μm features in the array is demonstrated with 1-sec time resolution and a sensitivity of < 1 pg of protein.
Cargile BJ, Bundy JL, Grunden AM, Stephenson JL Jr. Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover. Analytical Chemistry 76;2004:86–97. [PubMed]
Relative rates of turnover of proteins identified in shotgun proteomic experiments are assessed by measuring incorporation of 13C into these proteins while growing E. coli in [13C6]-glucose for a given period. Proteins are subjected to SDS-PAGE, the gel is sectioned, and bands are digested with trypsin. Peptides are subjected to LC-MS/MS for identification and determination of the extent of 13C incorporation. The proportion of newly synthesized protein is calculated from the distribution of signals within the extended isotopic envelope of each peptide, representing the incorporation of varying numbers of 13C atoms. Data are collected from as many peptides as are possible to assign to each protein. Turnover kinetic rate constants are not provided by this method.
Blonder J, Conrads TP, Yu L-R, Terunuma A, Janini GM, Issaq HJ, Vogel JC, Veenstra TD. A detergent- and cyanogen bromide-free method for integral membrane proteomics: Application to Halobacterium purple membranes and the human epidermal membrane proteome. Proteomics 4;2004:31–45. [PubMed]
A buffered solution of 60% methanol is used to extract and solubilize proteins from a preparation of Halobacterium purple membranes. Trypsin is shown to be capable of digesting proteins in this chemical environment without solvent exchange. Subsequent analysis of the peptides by LC-MS/MS results in the identification of all the predicted tryptic peptides of bacteriorhodopsin, and sufficient peptides to identify 30 other known membrane-associated proteins. This provides a simple and efficient method for the extraction, solubilization, and direct digestion of integral membrane proteins without the use of detergents, strong organic acids, or chemical cleavage reagents such as cyanogen bromide.
Zhao Y, Zhang W, Kho Y, Zhao Y. Proteomic analysis of integral plasma membrane proteins. Analytical Chemistry 76;2004:1817–1823. [PubMed]
A general method for proteomic analysis of plasma membrane proteins is presented. Cell surface proteins in viable cells are labeled with biotin and then purified by streptavidin affinity chromatography, using stringent high salt and high pH washes to remove non-specifically bound proteins from other subcellular sources. Bound proteins are eluted by SDS treatment and fractionated by SDS-PAGE prior to identification by LC-MS/MS. A preparation of this kind from a human lung cancer cell line is almost free of the cytosolic marker, actin. Of the 898 proteins identified, 781 are known plasma membrane proteins. Of these, 526 are integral membrane proteins.
Williams TL, Callahan JH, Monday SR, Feng PCH, Musser SM. Relative quantitation of intact proteins of bacterial cell extracts using coextracted proteins as internal standards. Analytical Chemistry 76;2004:1002–1007. [PubMed]
The use of signal strengths of endogenous, invariant, marker proteins for the normalization of signal strengths in LC/MS profiles of intact proteins is described. The procedure is analogous to standardization methods used to quantitate proteins during image analysis of 2D gels.
Villanueva J, Philip J, Entenberg D, Chaparro CA, Tanwar MK, Holland EC, Tempst P. Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry. Analytical Chemistry 76;2004:1560–1570. [PubMed]
Methodology for profiling peptides from human serum by MALDI mass spectrometry is described. Peptides are captured from 50 mL aliquots of serum using C8 reverse phase magnetic beads, a format that avoids problems due to sample viscosity. The peptides are then subjected to MALDI-TOF mass spectrometry. Prefractionation to remove high abundance proteins by Cibachrom blue affinity chromatography, ethanol precipitatation, or ultrafiltration through 30-kDa cut-off membranes are all found to decrease the numbers of peptides detected in the size range of interest (800–15,000 Da), and are therefore not employed routinely. Data are analyzed using standard microarray analysis programs.