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This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 279 William St., Athens, GA 30607-1777. Tel: (706) 369-5945; Fax: (706) 369-5936: E-mail: ude.gcm.liam@rethgualsc; or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.
Hiatt J B, Patwardhan R P, Turner E H, Lee C, Shendure J. Parallel, tag-directed assembly of locally derived short sequence reads. Nature Methods 2010;7:119–122.
Young A L, Abaan H O, Zerbino D, Mullikin J C, Birney E, Margulies E H. A new strategy for genome assembly using short sequence reads and reduced representation libraries. Genome Research 2010;20:249–256.
These papers propose a solution to the problems created by limited read-lengths available with most next-generation sequencing methods. Although reference sequences allow short reads to be assembled for resequencing and digital profiling, long, accurate reads are required for applications such as metagenomics, de novo assembly of large genomes, haplotyping, and study of immunoglobulin diversity. Both groups propose partitioning the genome into reduced representation libraries within which assembly of short reads is relatively easy. They achieve this in different ways. Young et al. use two restriction enzymes independently to create a series of overlapping fragment libraries derived from size-selected fragments (4 size ranges, creating 8 libraries in total). Each library is sequenced independently on an Illumina Genome Analyzer and short reads are assembled to create contigs from each library for subsequent merging. Genomic paired-end reads are incorporated into the assembly process to orient the contigs. Hyatt et al. begin with long fragments (~500 bp), ligate them to tag-adjacent adaptors, and then dilute them for PCR amplification to impose a complexity bottleneck on the PCR product population. They concatemerize the products, then shear them by sonication and ligate to a breakpoint-adjacent adaptor. A second PCR amplification prepares these amplicons for sequencing. One end of each amplicon corresponds to the end of a long fragment and the other end corresponds to an internal shearing breakpoint. Breakpoint-reads may then be grouped in silico on the basis of tag-reads so that reads from the same parent long fragment may be assembled separately. These strategies are anticipated to be helpful for accurate assembly of large genomes and for metagenomic sequencing.
Rohmer M, Meyer B, Mank M, Stahl B, Bahr U, Karas M. 3-Aminoquinoline acting as matrix and derivatizing agent for MALDI MS analysis of oligosaccharides. Analytical Chemistry 2010;82:3719–3726.
The matrix still most commonly used for MALDI analysis of oligosaccharides is 2,5-dihydroxybenzoic acid (DHB), despite its restriction to positive ion mode and its inhomogeneous crystallization. An alternative matrix, 3-aminoquinoline (3-AQ), although supporting high sensitivity and displaying compatibility with both positive and negative ion modes, hasn't found favor because it forms Schiff bases (imines) with the reducing end of oligosaccharides, thereby complicating the mass spectra. The present article, however, takes advantage of this reaction to exploit benefits of the derivatization. Conditions that lead to rapid, quantitative, and reproducible formation of Schiff bases are described for use directly on the MALDI target. Precursor and product ion spectra can be acquired in both positive and negative ion modes. The matrix works well with both N2 and Nd:YAG lasers, and detection limits as low as 1 fmol are demonstrated. Post-source decay of both protonated and chlorinated ions provides sequence, linkage, and branching information for oligosaccharides. Non-reducing sugars can also be analyzed because the matrix supports high sensitivity even without derivatization. The method has advantages over on-target formation of Schiff bases with DHB/aniline, where spectral acquisition is restricted to positive ion mode, and fragmentation of [M+Na]+ ions of aniline derivatives show no advantage over underivatized oligosaccharides. Behavior with acidic sugars (sialic acids and sulfated sugars) is presently under investigation.
Berven F S, Ahmad R, Clauser K R, Carr S A. Optimizing performance of glycopeptide capture for plasma proteomics. Journal of Proteome Research 2010;9:1706–1715.
This article provides details of the optimization and performance of the solid-phase hydrazide method for capture of glycopeptides and glycoproteins from plasma. The work shows that commercially available magnetic beads perform as well as, or better than, the commonly used macroporous beads and offer advantages for automation. The performance of the technique is found to be influenced by the choice of coupling solution, the amounts of magnetic beads, and the washing procedure. Depleting the 14 most abundant proteins in plasma improves detection sensitivity for the remaining glycoproteins by about 10×, and increases the number of glycopeptides identified by 24%. Digestion of glycoproteins prior to capture gives better reproducibility than capture of intact glycoproteins but the number of glycopeptides/glycoproteins identified is less. Interestingly, even under the best conditions, spike-in experiments indicate that detection sensitivity for the method is limited to 10–100 pmol/mL (100s of nanograms/mL to 10s of micrograms/mL).
Illig T, Gieger C, Zhai G, Romisch-Margl W, Wang-Sattler R, Prehn C, Altmaier E, Kastenmuller G, Kato B S, Mewes H-W, Meitinger T, De Angelis M H, Kronenberg F, Soranzo N, Wichmann H E, Spector T D, Adamski J, Suhre K. A genome-wide perspective of genetic variation in human metabolism. Nature Genetics 2010;42:137–141.
This large-scale study addresses the question of how much of the measured variation in blood metabolite levels between individuals in a human population can be attributed to genetic variation detectable by SNP genotyping. The authors use LC-MS/MS to measure fasting levels of 163 metabolites (including amino acids, sugars, acylcarnitines, and phospholipids) in 1,809 individuals in Germany and 422 in England. They perform genotyping using the Affymetrix 6.0 GeneChip Array, from which a total of 517,480 SNPs are selected for consideration. Genotype is correlated with all measured metabolite concentrations and all possible pairwise ratios of metabolite concentrations (163×162=26,406 traits). The loci from an initial population giving the best P-values for association with any of these traits are selected for a second, independent study with different individuals from the same population, and a list of those loci giving an increase in P-value with the addition of the second data set is compiled. This list, which contains 15 loci, is finally subject to replication by study of a separate population of individuals, and associations with 9 of the 15 loci survive Bonferroni correction. When the functional role of these loci is considered, 8 of the 9 fully confirmed associations (as well as 4 of the remaining ones in the final 15) are with polymorphisms found to be located in or near genes encoding enzymes with a function known to match the metabolites (e.g., the rate-limiting step in the relevant synthesis pathway). The loci explained as much as 5.6–36.3% of the observed variance in metabolite concentrations. These results suggest that metabolomics has the capability of contributing to the understanding genetic variation in human metabolism.
Guan S, Burlingame A L. High mass selectivity for top-down proteomics by application of SWIFT technology. Journal of the American Society for Mass Spectrometry 2010;21:455–459.
The selectivity with which an ion can be isolated for gas-phase dissociation and structure analysis has an obvious effect on the integrity of the structural data when the ion of interest requires efficient separation from other ions of closely similar m/z. In many tandem mass spectrometer systems, ions are isolated by a quadrupole mass filter, an ion-trap, or by time-of-flight effects. The selectivity of these methods, however, is limited. This problem is especially significant in cases where the species to be discriminated are multiply charged, thereby decreasing the m/z interval between them. The present paper describes the deployment of a device providing enhanced isolation selectivity on a commercial Fourier transform mass spectrometer, the LTQ-FT from Thermo-Fisher Scientific. The device delivers complex ejection/isolation waveforms by means of an arbitrary waveform generator and a high speed analog switch. It uses a previously described method known as the stored waveform inverse Fourier transform (SWIFT) method. The technology is demonstrated to be capable of 0.1 m/z discrimination and succeeds in isolating the 10+ charge state of acetylated histone H4 from the unacetylated form of the protein for electron capture dissociation.
Sakakura M, Takayama M. In-source decay and fragmentation characteristics of peptides using 5-aminosalicylic acid as a matrix in matrix-assisted laser desorption/ionization mass spectrometry. Journal of the American Society for Mass Spectrometry 2010;21:979–988.
In-source decay (ISD) in MALDI-MS is a most useful method for identification of peptides and proteins. It is characterized by cleavage at backbone N-Cα bonds without loss of side-chains. This cleavage is enabled by hydrogen atoms that form peptide/protein radicals. MALDI matrices that have high hydrogen-donating ability are therefore favorable for in-source decay. The present article evaluates 5-aminosalicylic acid (5-ASA) for this purpose. This matrix is shown to have hydrogen donating ability superior to that of 2,5-dihydroxybenzoic acid, the matrix usually employed for ISD. It produces fewer interfering peaks such as multiply protonated molecules and metastable peaks and yields sharper peaks for molecular ions and ISD fragments. For mono- and di-phosphorylated peptides it produces c-ions without loss of phosphate groups or peak broadening. Its hydrogen donating ability does, however, result in reduction of disulfide bridges. It has eminently suitable characteristics as a matrix for ISD.
Wenger C D, McAlister G C, Xia Q, Coon J J. Sub-part-per-million precursor and product mass accuracy for high-throughput proteomics on an electron transfer dissociation-enabled Orbitrap mass spectrometer. Molecular and Cellular Proteomics 2010;9:754–763.
The authors present a facile method for post-acquisition mass calibration to improve mass accuracy in a linear ion-trap-Orbitrap hybrid mass spectrometer being used for electron transfer dissociation (ETD). The method makes use of the fluoranthene cations employed for ETD as the mass calibrant. With only external mass calibration, a typical LC-MS/MS run yields a mass accuracy of –3.31 ± 0.93 ppm for precursor ions and –2.32 ± 0.89 ppm for product ions. After applying the internal calibration with fluoranthene ions, the accuracy improves to –0.77 ± 0.71 ppm for precursor ions and +0.17 ± 0.67 ppm for product ions. Little impact on scan time is experienced in acquiring the data necessary for this calibration.
Smith D P, Radford S E, Ashcroft A E. Elongated oligomers in β2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proceedings of the National Academies of Sciences U.S.A. 2010;107:6794–6798.
Understanding the pathophysiological processes at work in diseases of protein misfolding and amyloid formation is likely to depend critically upon knowledge of the structure and dynamics of the oligomeric species that form prior to fibril assembly. This paper demonstrates the utility of electrospray ionization-ion mobility mass spectrometry for characterizing the complicated ensemble of structures that arise during the lag time for in vitro amyloid fibril assembly of one protein, β-2 microglobulin. Measurements of mass allow the population of monomeric, dimeric, trimeric, and larger species to be ascertained, while measurement of collisional cross-section provides information about the shape of these assemblies and the conformation of the monomers that comprise them. The dynamic properties of oligomeric species are determined by observing subunit exchange between 14N- and 15N-labeled oligomers. A picture emerges of a lag phase populated by small, elongated aggregates without larger oligomeric species, and of decreasing oligomer dynamics with increasing oligomer size within the ensemble. This study highlights the capabilities of the technology to characterize a process of substantial complexity.
Theberge A B, Whyte G, Huck W T S. Generation of Picoliter Droplets with Defined Contents and Concentration Gradients from the Separation of Chemical Mixtures. Analytical Chemistry 2010;82:3449–3453.
Droplet-based microfluidics is an emerging field that uses aqueous or organic droplets as discrete nano- or femtoliter reaction vessels separated from one another by an immiscible carrier phase. The use of droplets avoids dispersion of fluids in microfluidic channels and supports rapid mixing with reagents for enhancement of reaction rates. Droplets can be fused with other droplets, incubated, analyzed, and sorted according to contents. Microdroplets have potential for high throughput screening, and have been applied to problems in protein crystallization, organic synthesis and bioassay. The present paper describes the splitting of individual compounds eluted from UPLC into some 105 droplets. The time-varying concentration of the eluting compound, measured, for example, by fluorescence, produces droplets that continuously vary in the concentration of compound they contain, over a range spanning 2–3 orders of magnitude. The method thus compartmentalizes substances from an initial mixture according to both identity and concentration. The “product libraries” produced in this way are envisioned to find application in the analysis of mixtures from synthesis reactions, natural product extracts, peptide mixtures, and biological samples, and the concentration spread to be useful in determining dose-response curves and performing titrations.
Zhang M M, Tsou L K, Charron G, Raghavan A S, Hang H C. Tandem fluorescence imaging of dynamic S-acylation and protein turnover. Proceedings of the National Academies of Sciences U.S.A. 2010;107:8627–8632.
Like protein phosphorylation, protein S-palmitoylation is a post-translational modification that is metabolically reversible: the addition and removal of palmitoyl groups exercises spatial and temporal control over the function of the modified protein. This paper describes a method for simultaneously quantifying turnover of the palmitoyl groups and turnover of the protein. Dual pulse-chase experiments with radioactive fatty acid and methionine or cysteine analogs are conventionally used for such applications, but require lengthy detection periods of weeks or months. Here, sensitive and efficient fluorescence reporters are employed for orthogonal monitoring of protein and palmitoyl turnover. Cells are supplied with alkyne-functionalized palmitic acid that will react with an azide-functionalized fluorophore, and are supplied with azidohomoalanine, an azide-bearing methionine surrogate that will react with a different, alkyne-functionalized fluorophore. The methodology is used to characterize palmitate turnover of the Lck protein, revealing that the fatty acid cycling is accelerated upon T-cell activation and that specific inhibitors affect palmitate removal. This general approach is amenable to adaptation for study of the turnover of other dynamic protein modifications.
Onono F O, Morgan M A, Spielmann H P, Andres D A, Subramanian T, Ganser A, Reuter C W M. A Tagging-via-substrate Approach to Detect the Farnesylated Proteome Using Two-dimensional Electrophoresis Coupled with Western Blotting. Molecular and Cellular Proteomics 2010;9:742–751.
Protein prenylation (addition of farnesyl or geranylgeranyl moieties) is critical to the function of a variety of proteins, including ones of medical importance. This paper presents a convenient method for global proteomic study of farnesylated proteins. Detection is accomplished by incorporating a farnesyl analog for which an antibody suitable for use in Western blotting is available. Farnesyl groups are attached by a farnesyl transferase that uses farnesyl pyrophosphate as substrate. Here, anilinogeraniol, the alcohol precursor of the unnatural farnesyl pyrophosphate substrate analog 8-anilinogeranyl diphophate is employed to tag target proteins by the enzyme. Tagged proteins are then visualized by Western blotting of 2-D gels. The method is shown to be suitable for dynamic analysis of the farnesyl proteome.
Berger M F, Levin J Z, Vijayendran K, Sivachenko A, Adiconis X, Maguire J, Johnson L A, Robinson J, Verhaak R G, Sougnez C, Onofrio R C, Ziaugra L, Cibulskis K, Laine E, Barretina J, Winckler W, Fisher D E, Getz G, Meyerson M, Jaffe D B, Gabriel S B, Lander E S, Dummer R, Gnirke A, Nusbaum C, Garraway L A. Integrative analysis of the melanoma transcriptome. Genome Research 2010;20:413–427.
This study provides a methodological framework for systematic discovery of aberrations that contribute to carcinogenesis, including gene fusions, alternative splice isoforms, and somatic mutations. The framework integrates transcriptomic and structural genomic data by combining massively parallel sequencing of cDNA (RNA-seq) with high-resolution chromosomal copy number data. The approach is applied to melanoma and results in the identification of 11 novel melanoma gene fusions, 12 novel read-through transcripts, and a remarkably high rate of somatic mutation. Of particular methodological interest is the initial validation of the approach by paired-end RNA-seq using cDNA from a well-characterized chronic myelogenous leukemia (CML) cell line that bears the BCR-ABL1 gene fusion. Using cDNA sheared to 400–600 bp, sequencing on an Illumina Genome Analyzer II, yielded 15.5 million pairs of 51-mer reads (8.8 million purity filtered pairs) providing 1.6 Gb of total sequence. These reads are aligned to both a transcriptome and, independently, to a genome reference to allow splice junctions to be split and mapped to their genomic coordinates. Mindful of the possibility of false discovery of splice junctions due to artifacts arising during PCR in library construction, only read pairs that give unique genomic placement for each read are considered and duplicate pairs likely to have arisen during PCR are eliminated. Through this stringent filter, 4.0 million read pairs are considered for further analysis, achieving a mean sequence coverage of 4.4 X for the annotated transcriptome. Because gene fusions are recognized by the presence of read pairs mapping to different chromosomal loci, the danger of results contaminated by artifactual misalignment is a concern. Accordingly, only read pairs mapping uniquely to opposite strands of separate structural genes and providing at least one 51-bp read unambiguously spanning the junction between two exons of the genes are considered. In the case of the BCR-ABL1 gene fusion in the CML cell line, the data provided 37 distinct read pairs with ends mapping to the two genes and 23 reads spanning the effusion point. The methodology established in this study is applicable to the transcriptomic analysis of many tumor types.
Vierbuchen T, Ostermeier A, Pang Z P, Kokubu Y, Südhof T C, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 2010;463:1035–1041.
This study achieves a milestone: the direct conversion of one differentiated cell type to another without an intermediary, pluripotent cell type. The conversion here is from fibroblast (a cell type that, during embryologic development, belongs to the mesodermal lineage) to induced neuronal (iN) cell (neurons are of embryonal ectoderm lineage). The present investigators induce the conversion in fibroblasts from mouse embryos or adult tail tips. The conversion is initially performed by forcing expression of 19 transcription factor genes chosen for their specific expression in neural cell types or their roles in reprogramming to pluripotency. The conversion is monitored by detecting expression of a Tau-EGFP fusion protein, which is expressed in a neuron-specific manner in knock-in mice. The 19 transcription factors are eventually paired down to just 3: Ascl1, Brn2, and either Zic1 or Myt1l. Ascl1 alone produces cells with immature neuronal features. The conversion to iN cells occurs with the high efficiency of 20%. This effectively eliminates the possibility that differentiation of rare stem cells or precursor cells with neurogenic potential is being observed. The resulting iN cells are functional neurons that receive both excitatory and inhibitory synaptic connections from mouse neurons and form functional synapses with each other. The majority of iN cells produced are excitatory and express markers of cortical identity, although interestingly, other neuronal phenotypes are also detected. For the purposes of putative regenerative medicine, this study is important for the absence of involvement of potentially tumorigenic pluripotent stem cells.
Goodwin R J A, Scullion P, Macintyre L, Watson D G, Pitt A R. Use of a Solvent-Free Dry Matrix Coating for Quantitative Matrix-Assisted Laser Desorption Ionization Imaging of 4-Bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in Rat Brain and Quantitative Analysis of the Drug from Laser Microdissected Tissue Regions. Analytical Chemistry 2010;82:3868–3873.
For tissue-imaging studies, application of MALDI matrix in solution is potentially problematic because it risks delocalization of analytes by diffusion in the applied matrix solution prior to drying. This problem is likely to be especially acute for analytes that are small molecules. In such cases, application of dry matrix is an attractive option. The present study demonstrates that dry matrix application supports detection of one small molecule, the drug 4-bromophenyl-1,4-diazabicyclo (3.2.2)nonane-4-carboxylate. This molecule was undetectable with conventional application of matrix in solution. Dry application of α-cyano-4-hydroxycinnamic acid as matrix was accomplished by covering tissue sections on glass slides with a large excess of finely-ground matrix, then tipping off and tapping to remove loosely adhering matrix particles. This procedure was repeated 10 times, and non-adhering matrix was finally removed under a fast stream of nitrogen. The localization of the drug observed during MALDI imaging was confirmed by inspection of the product ions derived from the observed MS signal for the drug and by LC-MS analysis of samples collected from tissue sections by laser-capture microdissection. These results indicate that dry matrix application provides a useful, complementary method for MALDI imaging.
Maurer-Stroh S, Debulpaep M, Kuemmerer N, De La Paz M L, Martins I C, Reumers J, Morris K L, Copland A, Serpell L, Serrano L, Schymkowitz J W H, Rousseau F. Exploring the sequence determinants of amyloid structure using position-specific scoring matrices. Nature Methods 2010;7:237–242.
Proteins aggregate most commonly by forming assemblies of β-sheet-like structures. Despite this enrichment in β-sheet structure, most protein aggregates are amorphous and lacking overall in ordered 3-D structure. Some, however, are highly ordered and share a common diffractional signature characteristic of the so-called cross-β-sheet structure. These latter are the hallmarks of amyloid fibers. Amyloids also show biological behavior distinct from amorphous aggregates, including the subcellular locations in which they accumulate, and the mechanisms by which they are degraded. The present paper contributes to our ability to predict which sequences are likely to be strongly amyloidogenic. Because the shortest peptides that can form an amyloid nucleation core when introduced into a previously non-amyloidogenic protein are 6-residues long, a series of peptides are studied by a variety of biophysical techniques to identify those that are strongly amyloidogenic and those that are non-amyloidogenic. Using the resulting expanded list of amyloidogenic sequences, a web-based tool called Waltz is presented that uses a position-specific scoring matrix to distinguish amyloid-forming sequences from sequences forming amorphous aggregates entered by users. The tool is freely available at http://waltz.switchlab.org/. It is validated by identifying amyoidogenic sequences in proteins that use the amyloid structural motif to perform their normal functions.