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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 about articles they feel is important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 279 William St., Athens, GA 30607-1777, USA; Tel.: (706) 369-5945; Fax: (706) 369-5936; E-mail: email@example.com; or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.
Shimbo K, Yahashi A, Hirayama K, Nakazawa M, Miyano H. Multifunctional and highly sensitive precolumn reagents for amino acids in liquid chromatography/tandem mass spectrometry. Anal Chem 2009;81:5172–5179.
The use of mass spectrometry for quantification of substances is now so pervasive and instrumentation so readily available that even in the case of molecules, effectively quantified by traditional chemical methods, the development of alternative mass spectral methods is of importance. The high sensitivity of mass spectrometry may also afford advantages over older methods. Here, a new tertiary amine reagent for pre-column derivatization of amino acids is described. It is suitable for separation of the amino acid derivatives by reverse-phase HPLC and for on-line electrospray ionization. The reagent, p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS), forms a ureide bond with amino acids under mild conditions, and this bond can be selectively cleaved by collision-induced dissociation to yield a reporter fragment ion that is quantifiable by precursor ion-scanning. The method is used for 13C metabolic flux measurements, and with deuterium-labeled reagent, for relative quantification of amino acid concentrations in pairs of samples.
Deng J, Shoemaker R, Xie B, et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol 2009;27:353–360.
Ball MP, Li JB, Gao Y, et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat Biotechnol 2009;27:361–368.
These two groups have developed closely similar approaches to studying the relationship of cytosine methylation to gene expression based on the use of bisulfite conversion and high-throughput sequencing. In an attempt to avoid prohibitively expensive global sequencing of large mammalian genomes for this purpose, they seek to target regions that show different methylation patterns related to expression change in comparisons of fibroblasts and induced pluripotent stem cells. Both groups use “padlock probes” to sample selected regions of the genome. Ball et al. focus on selected regions without regard to their enrichment in CpG dinucleotides, and Deng et al. focus on CpG islands as regions known to have high levels of methylation. Additionally, Ball et al. use a complementary approach of counting cuts produced by the methylation-sensitive enzyme, HpaII. The results indicate a high level of commonality in methylation patterns in the two cell types. Interestingly, in addition to an association between high levels of expression and decreased promoter methylation, they both observe increased levels of methylation in the gene body in genes expressed at high levels.
Pushkarev D, Neff NF, Quake SR. Single-molecule sequencing of an individual human genome. Nat Biotecnol 2009;27:847–850.
This publication represents a new milestone in the development of sequencing technology: the use of single-molecule methods to sequence an individual human genome. Single-molecule sequencing has the advantages of not requiring amplification, ligation, or cloning in sample preparation, thus promising benefits in throughput, accuracy, and evenness of coverage. The present study uses the first commercial release of a single-molecule sequencer, the HeliScope, from Helicos BioSciences. This instrument allows one to follow the sequencing-by-synthesis of approximately 1 billion individual molecules during the course of 1 week. One operator with a single instrument acquired data for 28× average coverage of the genome in four runs (a total of approximately 4 weeks). The average read-length was 32 bp. The rate of production of mappable data was approximately 2.5 billion bases per day. Errors were dominated by deletions (2%), followed by insertions (1.2%) and substitutions (0.38%). This study presents identification of approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1%, as validated in sampling by Sanger sequencing, and 99.8% concordance with SNP genotyping arrays. It also describes 752 regions of variation in copy number detected by coverage depth alone.
Orlando R, Lim J-M, Atwood JA, et al. IDAWG: metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J Proteome Res 2009;8:3816–3823.
As a procedure analogous to the stable isotope labeling with the amino acids in cell culture (SILAC) method for relative quantification of proteins from cultured cells, a simple strategy for relative quantification of glycans from cultured cells is described here. It uses biosynthetic labeling with 15N-glutamine. The amide side-chain of glutamine is the sole metabolic source of nitrogen for the synthesis of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and sialic acid. By incorporating 15N-glutamine in culture media of mouse embryonic stem cells, nearly complete incorporation of the heavy isotope into N-linked and O-linked glycans is shown to occur, resulting in the addition of 1 Da per amino sugar. In addition to enabling relative quantification, the heavy atom label assists in the interpretation of fragment ion spectra of glycans. Its Georgia developers named the procedure Isotopic Detection of Amino Sugars with Glutamine (IDAWG). The method requires no extra cost for specially depleted media, as glutamine is generally omitted from culture media anyway, owing to its thermal instability.
Begley P, Francis-Mcintyre S, Dunn WB, et al. Development and performance of a gas chromatography—time-of-flight mass spectrometry analysis for large-scale nontargeted metabolomic studies of human serum. Anal Chem 2009;81:7038–7046.
To establish methods for long-term metabolic studies and to create a benchmark dataset against which to compare profiles from individuals in diverse states of health and disease, a project called the Human Serum Metabolome Project (http://www.husermet.org/) has been undertaken by the University of Manchester, AstraZeneca, and GlaxoSmithKline. The project is aimed at creating a benchmark dataset of comprehensive serum metabolite profiles derived from samples collected from over 5000 individuals over a 3- to 5-year period. The analytical method chosen for the work is based on gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). Standard operating procedures have been determined. Protein is removed from the serum samples by precipitation with methanol/water (3/1, v/v), and then oxime/trimethylsilylation (TMS) derivatives of the metabolites are made by optimized procedures. The GC-MS analysis time is 25 min/sample. Using this methodology, short-term and long-term (5 months) reproducibility of the analyses is reported here to be excellent. Use of these standard operating procedures, along with the use of suitable quality control standards, is recommended for long-term, untargeted metabolite studies.
Second TP, Blethrow JD, Schwartz JC, et al. Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures. Anal Chem 2009;81:7757–7765.
This paper documents improvements in the performance of the new Thermo Scientific LTQ Velos linear ion-trap mass spectrometer as compared with its forerunner, the LTQ XL. The new instrument incorporates ion funnel technology in the source design to replace the tube lens and skimmer. This improves the efficiency of ion transmission into the mass analyzer. The result is a fivefold increase in signal intensity. A new ion trap, consisting of two cells separated by a single aperture lens to allow differential pumping, is operated at higher pressure in the first chamber and lower in the second. In the first cell, the higher pressure and the application of isolation waveforms allow ion trapping to be performed with substantially improved mass discrimination, reducing background in MS/MS analysis from precursor ions of closely similar mass-to-charge ratio (m/z). The lower pressure in the second cell allows higher mass resolution for a given scan speed. Finally, a new method for regulating ion fill-time is deployed, replacing pre-scans with ion abundance measurements made in the preceding full MS scan. The cumulative effect of these changes is to decrease the duration of a typical full MS scan from 140 ms to 116 ms and decrease the time for acquisition of a typical MS/MS spectrum from 310 to 131 ms, while improving the quality of the MS/MS spectra. In analysis of a complex proteome, detection of low-abundance peptides is thereby improved, and proteome penetration is enhanced substantially.
Makarov A, Denisov E, Lange O. Performance evaluation of a high-field Orbitrap mass analyzer. J Am Soc Mass Spectrom 2009;20:1391–1396.
Just as Fourier transform ion cyclotron resonance mass spectrometers show performance improvements by applying trapping magnetic fields of increasing strength, so should the performance of an electrostatic trap such as the Orbitrap improve by applying higher trapping voltages. In this report, the performance of an Orbitrap with redesigned field structure and greater field strength is described. The maximum full-width at half-maximum (FWHM) resolving power is shown to be greater than 350,000 at mass-to-charge ratio (m/z) 524 and 600,000 at m/z 195. Of particular interest, the instrument shows very little sensitivity to space charging inside of the mass analyzer. However, the instrument is more demanding than the standard Orbitrap with regard to alignment of the ion optics, residual pressure, and variation of performance with tuning parameters. These characteristics necessitate further development of manufacturing infrastructure and automated tuning routines before the instrument can be deployed commercially.
Pei J, Li Q, Lee MS, Valaskovic GA, Kennedy RT. Analysis of samples stored as individual plugs in a capillary by electrospray ionization mass spectrometry. Anal Chem 2009;81:6558–6561.
For many years, high-throughput clinical, industrial, and environmental assays have made use of continuous/segmented flow analysis, in which successive samples are supplied for analysis through a single tube separated by plugs of air. Recently, this idea has been evolving rapidly, and it is now possible to perform sampling, splitting, reagent addition, concentration, and dilution on sample plugs separated by air or immiscible liquids in microfluidic systems. The present paper shows that electrospray ionization-mass spectrometry (ESI-MS) can readily be used to analyze such sample plugs pumped directly into a fused silica nanospray emitter. A Teflon tube of 75 or 150 μm i.d. is filled with Fluorinert FC-40 oil and dipped into sample solutions stored in a 96-well plate, withdrawing a desired volume of sample, removing the tube, introducing a desired volume of air, and then repeating the cycle until all of the samples have been loaded. A syringe pump controls fluid movements. The plugs are then pumped into an emitter tip for analysis. Samples of 13 nL are separated by 3 mm air gaps and are analyzed at a rate of 0.8/s. Carryover between samples is low, and reproducibility of signal strength is good. The limit of detection for leucine-enkephalin is 1 nM. Efficiency is maximized, as all of the sample withdrawn from the well is used in the mass spectrometer, and the duty cycle is rapid, as the time spent rinsing between samples is minimal. Application for this method is envisioned in high-throughput screening of label-free reactions and reactions performed in plugs, off-line coupling of separations to ESI-MS, and clinical diagnostics.
Mureev S, Kovtun O, Nguyen UT, Alexandrov K. Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol 2009;27:747–752.
Cell-free protein expression systems have long contributed to the study of protein function, drug screening, and diagnostics. The best-studied system is based on Escherichia coli, but systems based on rabbit reticulocyte lysate, wheat germ extract, and insect cell extract are commonly used for studies of eukaryotic proteins, despite the difficulties of scaling them up or down and of manipulating their source organisms genetically. Here, several advances are made in the methodology for cell-free synthesis of proteins from transcription templates generated by PCR. First, adding poly(A) sequences upstream of the initiator AUG codon and incorporating weakly stabilized hairpin structures downstream of the initiator codon promote engagement of the translation machinery and circumvents the need for 5′-capping of mRNA and numerous translation factors. Fortunately, the added sequences support translation initiation in all cell-free protein synthesis systems tested, so they apparently minimize species specificity. In the present study, this permitted the establishment of a new eukaryotic cell-free system based on the protozoan, Leishmania tarentolae, which is amenable to genetic manipulation and large-scale, inexpensive fermentation. Second, in this system, by using upstream interfering RNAs, the translation of endogenous mRNAs is prevented. The new system is shown to support the generation of protein libraries from unpurified PCR products and to yield up to 300 μg/mL recombinant protein in 2 h from 50 mL culture. The ability to coexpress multiple proteins facilitates the study of protein–protein interactions.
Barrera NP, Isaacson SC, Zhou M, et al. Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat Methods 2009;6:585–587.
Here, a mass spectrometric method, previously described by the same group for elucidating the subunit stoichiometry and lipid-binding interactions of an integral membrane protein, is shown to be generally applicable to diverse members of that challenging protein class. The method involves desorbing the protein from detergent solution into the gas phase protected by detergent micelles and then stripping off the detergent by collision-induced dissociation to allow study of the remaining protein with cofactors and substrates attached. In the present paper, they study two ABC transporter proteins, MacB and LmrCD, and two proton-driven antiporters, MexB and EmrE. The subunit structure of three of these is unknown, and that of the fourth, EmrE, is controversial. Through successful use of the methodology in these cases, the procedure had been developed into a robust approach. It is also relatively sensitive, typically requiring less than 10 pmol of complex.
Addona TA, Abbatiello SE, Schilling B, et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 2009;27:633–641.
Multiple reaction monitoring (MRM) with stable isotope dilution mass spectrometry has emerged as a method of choice for confirmation of candidate protein biomarkers for disease in biological fluids. The method is capable of accurately and reproducibly quantifying proteins within single laboratories and can be multiplexed to allow tens of proteins to be quantified per liquid chromatography/mass spectrometry (LC/MS) run. However, transferability among laboratories has not been investigated. Here, the Clinical Proteomic Technology Assessment of Cancer network of the National Cancer Institute, representing eight laboratories, reports a survey of inter- and intra-laboratory performance of MRM quantification for seven target proteins added to human plasma. The study was conducted in three phases of increasing dependence on local conditions for sample preparation and analysis. In the first phase, plasma was digested, and peptides were added to unfractionated serum before distribution (peptide concentration range, 1–500 fmol/μL, for comparison with isotope-labeled peptides at 50 fmol/μL). In the second phase, proteins containing the same peptides were digested and then added to the digested plasma prior to distribution. In the third phase, the intact proteins were added to undigested plasma, and participating laboratories were required to digest the samples independently. Inter-laboratory coefficients of variation (CVs) in Phase 3, although poorer than those generally required for clinical assays, were found to be consistent with the demands for biomarker verification. Sample preparation contributed more to CV than instrumental variability. Some peptides performed poorly, most often because of interference from the plasma digest matrix. This study demonstrates reproducibility for proteins down to the low μg/mL range. For proteins of yet lower abundance, prefractionation of plasma will be required, and the control of variability introduced by the extra processing steps will require confirmation in further experimental studies.
Bell AW, Deutsch EW, Au CE, et al. A HUPO test sample study reveals common problems in mass spectrometry-based proteomics. Nat Methods 2009;6:423–430.
Liquid chromatography-mass spectrometry (LC-MS)-based proteomic methods have a reputation for poor reproducibility: The proteins, which are identified during analyses in different laboratories or even within the same laboratory, often show variability. The possible causes include stochastic sampling effects, systematic bias as a result of use of different methodologies, or erroneous protein identification. To help identify the main causes of irreproducibility, the Human Proteome Organization (HUPO) has conducted a multi-site survey of 27 laboratories, to which was sent an equimolar mixture of 20 highly purified proteins. Each protein contained one or more unique tryptic peptides of 1250 ± 5 Da with sequences chosen to test the effect of crowding in precursor ion mass spectra. Each laboratory was asked to identify all of the proteins (20) and all of the unique peptides of 1250 Da (22) using a methodology of their own choice. All laboratories made their raw data available to the study designers. Initially, only seven of the 27 laboratories reported all 20 proteins correctly, and only one laboratory reported all of the tryptic peptides of 1250 Da. Interestingly, however, centralized inspection of the raw data indicated that data permitting identification of all 20 proteins and most of the 22 peptides had in fact been acquired by every lab. This suggested that problems with the database and search engines, particularly the ability to distinguish among different identifiers of the same protein, were prevalent. The organizers then communicated to the participants potential sources of misidentification, Once these problems were corrected, participants realized almost perfect identification scores. False-positive identifications, derived from environmental contamination and carryover from previous samples, were also common. The study indicates the need to make raw data from proteomic studies available for meta-analysis and for refinement of software tools. The sample in this study was, of course, much simpler than that of most proteomes, but the ability to achieve 100% success in identifying the proteins and peptides in the present sample would promote further trust in real-world data.
Gupta N, Pevzner PA. False discovery rates of protein identifications: a strike against the two-peptide rule. J Proteome Res 2009;8:4173–4181.
It has long been standard operating practice in proteomics to regard proteins identified on the basis of two or more peptide assignments of experimentally acquired product ion spectra as being more reliable than proteins identified on the basis of just one such assignment (so-called “one-hit wonders”). The present article critically examines this practice and concludes that it should be abandoned. The authors show that we are more likely to identify a protein with two mediocre hits by chance alone (as measured by hits generated in assignment to a decoy database) than with a single high-scoring peptide hit. The “two-hit” rule reduces the number of protein identifications in the target database more than in the decoy database and results in more false discovery rates than when single-hit proteins are not discarded. The authors also point out that optimizing false discovery rates for peptides does not necessarily optimize the false discovery rate for proteins. The reason is that improvements in peptide discovery often yield further peptides from proteins that have already been discovered (i.e., proteins with multiple peptide assignments), whereas the corresponding increase in assignment to decoy databases raises the false discovery rate. The authors recommend that publications report protein-level false discovery/positive rates as well as peptide-level error rates.
Chen L, Page GP, Mehta T, Feng R, Cui X. Single nucleotide polymorphisms affect both cis- and trans-eQTLs. Genomics 2009;93:501–508.
Single nucleotide polymorphisms (SNPs) that produce mismatching between gene expression microarray probes and their cognate RNA targets may weaken hybridization signals. This paper discusses the effects of SNPs on Affymetrix GeneChip probe set summaries, which are based on 11–16 probes for each gene. In gene expression quantitative trait locus (eQTL) studies, these probe set summaries are used for mapping genetic factors that control the level of gene expression, considered as quantitative traits. Such eQTL studies identify cis-QTLs (in which the QTL gene maps to the same locus as the structural gene affected) and trans-QTLs (in which the QTL gene maps to a different locus). Removing probes containing SNPs is shown not only to affect probe-level eQTL results dramatically but also significantly to change probe set summaries, especially for probe sets with two or more SNP-containing probes. It is recommended that such probes be removed from the analysis to improve the performance of eQTL mapping.
Newell EW, Klein LO, Yu W, Davis MM. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat Methods 2009;6:497–499.
Hadrup SR, Bakker AH, Shu CJ, et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods 2009;6:520–526.
Cytotoxic T lymphocytes (CTLs) function in the elimination of virally infected cells, tumor cells, and some bacteria and parasites. They do so by using T-cell receptors (TCRs) to recognize viral or other peptides bound to host Class I MHC proteins (pMHC) on the surface of infected target cells. CTLs clonally express an extensive repertoire of different TCRs with different peptide/pMHC specificity. Distinct CTL populations may be recognized and sorted by flow cytometry with the use of MHC:peptide tetramers—soluble versions of pMHC molecules consisting of four identical pMHC units each with peptide bound (to provide enhanced CTL-binding avidity) and linked to a fluorochrome for detection. This methodology has been limited in its application to clinical samples by the small number of different fluorochromes that can be used to detect different CTL specificities at one time. The present two papers extend the multiplexing potential of the technique by using a combinatorial approach. A given pMHC:peptide pair is conjugated not just to one fluorochrome but separately to two distinguishable fluorochromes so that CTLs of the cognate specificity are labeled by the unique combination of these two fluorescent markers as they bind to TCR molecules on the surface of the same cell. By using N different fluorochromes in this way, the number of different CTL specificities that can be distinguished is 2N − 1. Newell et al. use four standard organic fluorochromes to detect 15 specificities, and Hadrup et al. use eight fluorochromes (six quantum dots and two standard fluorochromes) to detect 25 specificities. The latter study uses the methodology to analyze CTL responses to known and potential melanoma-associated antigens in the peripheral blood of patients with melanoma. This approach is anticipated to find a plethora of uses, including diagnostic, multiplexed screening for a pathogens and tumors.
Mátés L, Chuah MKL, Belay E, et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 2009;41:753–761.
DNA transposons are mobile genetic elements that feature inverted terminal repeats that contain binding sites for a transposase, the transpopase being necessary for transposition. Normally encoded within the transposon, the transposase can be supplied in trans, leaving the transposon available to deliver a cargo gene for integration into a host cell genome. Sleeping Beauty (SB) is a synthetic transposable element that has been engineered from a transposon originally found in fish. It is active in a wide range of vertebrates, including humans. In the present study, a library of SB transposase mutants was generated by in vitro evolution using DNA shuffling and was screened for elevated activity. Highly active combinations of variants were assembled and yielded a hyperactive transposase (SB100X) with 100× enhancement in efficiency compared with SB. This new transposase is anticipated to be a useful reagent for transgenesis, insertional mutagenesis, and possibly gene therapy. It yields 35–50% stable transfer of a marker gene into human CD34+ cells enriched in hematopoietic stem cells, and transplantation of these cells into immunodeficient mice produces long-term, multilineage, hematopoietic reconstitution, a result not achieved previously. The enzyme also mediates long-term expression of factor IX introduced into mouse liver in vivo. These results emulate what is possible with viral transduction but may circumvent some or all of the problems with viral vectors for gene therapy applications, including immunogenicity of the vector or target cells, and technical and regulatory issues with viral vectors in clinical trials.
Malm J, Giannaras D, Riehle MO, Gadegaard N, Sjövall P. Fixation and drying protocols for the preparation of cell samples for time-of-flight secondary ion mass spectrometry analysis. Anal Chem 2009;81:7197–7205.
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an imaging technique capable of detecting and imaging inorganic and organic compounds at submicrometer resolution. Interpretation of the data from biological samples depends on the understanding and control of sample preparation. The effects of alternative fixation methods (glutaraldehyde fixation and cryofixation) and drying methods (freeze-drying and alcohol-substitution drying) on human fibroblast cells are documented here using TOF-SIMS, scanning electron microscopy, and interference reflection microscopy. Cryofixation followed by freeze-drying is found to preserve intactness of cell membranes, cell morphology, and sodium/potassium ion gradients across the plasma membrane. Washing with aqueous ammonium formate before cryofixation reduces salt accumulation during subsequent drying. Glutaraldehyde fixation produces finer structures on the cell surface than cryofixation in scanning electron microscopy and similar lipid distributions in TOF-SIMS but destroys sodium/potassium ion gradients. Alcohol drying tends to remove membrane phospholipids, but by using osmium tetroxide as a second fixative, this effect can be lessened. These results will be of interest to investigators planning to use the remarkable resolving power of TOF-SIMS imaging to solve biological problems.
Piehowski PD, Davey AM, Kurczy ME, et al. Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution. Anal Chem 2009;81:5593–5602.
This paper undertakes to provide a model for the interpretation of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data acquired in the study of features that approach the minimum pixel size imposed by the focus of the primary ion beam used for desorbing secondary ions for mass analysis. TOF-SIMS images are compiled by counting relatively rare events: the arrival of ions at a detector. As a result, a uniform surface will not appear uniform, as pixel intensities are expected to follow a binomial distribution. Pixel-to-pixel heterogeneity is shown here to be modeled accurately using the Poisson distribution. This model is used to interpret TOF-SIMS images of the distribution of lipids on the surface of RBL-2H3 mast cells, where lipid rafts of sizes smaller than a single pixel are hypothesized to occur. The data provide evidence that the distribution of lipids is indeed inhomogeneous, consistent with the existence of such lipid rafts.
Novere NL, Hucka M, Mi H, et al. The systems biology graphical notation. Nat Biotechnol 2009;27:735–741.
This Perspective piece describes the confusion and ambiguity engendered by the lack of a standard visual language for biochemical interaction networks, inter- and intracellular signaling, and gene regulation. The authors propose Systems Biology Graphical Notation, a graphical language developed jointly by biochemists, modelers, and computer scientists. It makes use of three complementary, largely nonoverlapping languages—process diagram, entity relationship, and activity flow diagram—to represent the various networks of biochemical interactions in a standard, unambiguous way.