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J Biomol Tech. 2003 September; 14(3): 238.
PMCID: PMC2279947

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AMINO ACID SEQUENCING AND COMPOSITION ANALYSIS

Higbee A, Wong S, Henzel WJ. Automated sample preparation using vapor-phase hydrolysis for amino acid analysis.Analytical Biochemistry 2003;318:155–158. [PubMed]

The design for an automated workstation that prepares vials for vapor phase hydrolysis with hydrochloric acid is described. The workstation alternately evacuates and purges with nitrogen gas using conditions that avoid bumping of the HCl. The device represents an improvement for the preparation of samples for amino acid composition analysis.

BIOINFORMATICS

Lai EC, Tomancak P, Williams RW, Rubin GM. Computational identification of Drosophila microRNA genes. Genome Biology 2003;4:R42. [PubMed]

Considerable interest is presently being focused on microRNAs (miRNAs), 21–22 nucleotide, non-coding RNAs that are believed to play roles in translational regulation. This paper describes a computational method for identifying miRNA genes based on (a) the derivation of miRNAs from precursor transcripts of 70–100 nucleotides with an extended stem-loop structure, (b) the conservation of these genes between species, and (c) the characteristic pattern of their evolutionary divergence. The method is called miRseeker. It identifies 75% of previously known miRNAs in Drosophila, and indicates 48 novel miRNA candidates. Expression of many of these is demonstrated. The Drosophila genome is estimated to contain 110 miRNA genes, or 1% of the number of genes encoding proteins. MiRseeker may be accessed at http://www.fruitfly.org/seq_tools/miRseeker.html.

CARBOHYDRATES, GLYCOLIPIDS AND GLYCOPROTEINS

Zhang H, Li X-J, Martin DB, Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nature Biotechnology 2003;21:660–666.

Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M, Hirabayashi J, Kasai K-I, Takahashi N, Isobe T. Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins. Nature Biotechnology 2003;21:667–672.

These two groups adopt similar approaches to identification of proteins bearing N-linked carbohydrate on a proteomic scale, quantitative profiling of protein glycosylation and localization of glycosylation sites. Zhang et al. selectively immobilize glycoproteins by oxidation of hydroxyl groups on sugar side chains to aldehydes with periodate, then covalently bind them to hydrazide beads. Kaji et al. immobilize glycoproteins with a lectin. Both groups then digest immobilized proteins with trypsin, and wash away unbound peptides. The glycopeptides remaining immobilized are then released by digestion with peptide-N-glycosidase F (PNGase F). Zhang et al. accomplish the relative quantitation of peptides in sample pairs through labeling peptide N-terminal residues by reaction with light or heavy forms of succinic anhydride, and measuring signal strengths and fragmentation patterns by tandem mass spectrometry. Kaji et al. instead perform isotopic labeling by PNGase F digestion in the presence of H218O or water of natural isotopic abundance, then mixing these digests prior to mass spectrometry. See also the News and Views commentary: Freeze H. Mass spectrometry provides sweet inspiration. Nature Biotechnology 2003;21:627-629.

Wenk MR, Lucast L, Di Paolo G, Romanelli AJ, Suchy SF, Nussbaum RL, Cline GW, Shulman GI, McMurray W, De Camilli P. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nature Biotechnology 2003;21:813–817.

Measurement of phosphatidyl inositol phosphate (PIP) and phosphatidyl inositol bisphosphate (PIP2), low abundance lipids involved in intracellular signaling, is performed by electrospray ionization mass spectrometry. Triethyl ammonium acetate or piperidine are included in the solvent to support detection of these ions. Precursor ion scanning of head-group fragments is then used to assess pool sizes of species differing in fatty acid composition.

DNA CHARACTERIZATION AND GENOTYPING

Hardenbol P, Banér J, Jain M, Nilsson M, Namsaraev EA, Karlin-Neumann GA, Fakhrai-Rad H, Ronaghi M, Willis TD, Landegren U, Davis RW. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nature Biotechnology 2003;21:673–678.

SNP loci are recognized by “padlock probes,” linear oligonucleotides whose free ends hybridize to immediately adjacent sequences on target DNA. The polymorphic nucleotide position to be tested fills the gap between these two adjacent sequences. Unlabeled dATP, dCTP, dGTP or dTTP are added to separate aliquots of the hybridization mixture, and DNA ligase incorporates the nucleotide(s) complementary to the target, thus closing the circle in the probe molecule. Exonucleases then digest remaining linear probes. The circular probes are then linearized, released, and amplified by PCR using a common primer pair. Each probe contains a detection tag sequence for which a cognate oligonucleotide is present on an Affymetrix GenFlex Tag Array. The probe is hybridized to four identical arrays, one for each gap fill reaction, and quantitated by a fluorescent label incorporated into the probe. The padlock probes provide sufficient specificity to analyze SNPs directly, without previous amplification of the target sequences. Most importantly, the system can be multiplexed by adding more probes to the mixture, one for each SNP locus. The present study yields more than 1,000 genotypes simultaneously, and multiplexing to 10,000 is anticipated.

MASS SPECTROMETRY

Schmidt A, Karas M, Dülcks T. Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI? Journal of the American Society of Mass Spectrometry 2003;14:492–500.

Nanoelectrospray (nanoESI) displays several important advantages over conventional electrospray (ESI) besides economy of analyte consumption. These include ability to spray from solutions containing no organic solvent, detection of very hydrophilic compounds such as carbohydrates, and reduced sensitivity to signal suppression by salts and surface-active molecules. This paper systematically documents the effect of spray needle orifice diameter, and hence flow rate (in the absence of pumping), on ion signals for mixtures of compounds with different physical properties (detergent/oligosaccharide and oligosaccharide/peptide). The suppression effects in these mixtures are pronounced even at flow rates as low as 50 nL/min, but disappear at flow rates of a few nanoliters per minute. The results are of practical consequence for the design of ESI experiments in which signal suppression is an important issue.

Kast J, Gentzel M, Wilm M, Richardson K. Noise filtering techniques for electrospray quadrupole time of flight mass spectra. Journal of the American Society of Mass Spectrometry 2003;14:766–776.

Software to increase the signal-to-noise ratio of ion signals in electrospray spectra is described. Noise levels are reduced by Fourier transformation. Signal-to-noise ratio is then further enhanced by using an algorithm that distinguishes signals from random noise based on the higher than random probability that elevated ion counts will be found in adjacent detection channels. This algorithm is applied cyclically.

Breci LA, Tabb DL, Yates III JR, Wysocki VH, Cleavage N-terminal to proline: Analysis of a database of peptide tandem mass spectra. Analytical Chemistry 2003;75: 1963–1971. [PubMed]

In collision-induced dissociation, the intensity of fragment ions due to cleavage N-terminal to proline residues is relatively high. This so-called “proline effect” is examined for doubly charged tryptic peptides analyzed in a quadrupole ion-trap mass spectrometer. The magnitude of the effect is greatest for Xxx-Pro bonds where Xxx is His, Asp, Val, Ile or Leu, and least when Xxx is Gly or Pro. The results for His and Asp are understandable in terms of the strong cleavage C-terminal to these residues, which is further amplified by the Pro. The results with aliphatic amino acids are interpreted in terms of local conformational effects. Allowance for the magnitude of the proline effect in different sequences may improve the outcome of database searching.

Benesch JLP, Sobott F, Robinson CV. Thermal dissociation of multimeric protein complexes by using nanoelectrospray mass spectrometry. Analytical Chemistry 2003;75: 2008–2214.

A source that controls the temperature of the solution in a nanoESI probe is designed and constructed to study heat-induced changes in proteins and non-covalent protein complexes. The transition temperature measured for the unfolding of lysozyme is shown to be in good accord with the value measured by fluorescence spectroscopy. The system is also used to study the dissociation of a small heat shock protein, which makes the transition from a large multisubunit complex into smaller oligomers.

METABONOMICS

Allen J, Davey HM, Broadhurst D, Heald JK, Rowland JJ, Oliver SG, Kell DB. High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nature Biotechnology 2003;21:692–696.

“Metabolic footprinting” is the term here given to the mass spectrometric monitoring of extracellular metabolites in spent culture medium. This approach to assessing the metabolic status of cells is simpler than monitoring intracellular metabolites because it avoids the problems of metabolite turnover, the need to quench metabolism, and the requirement to separate intracellular material from the extracellular space. Features revealed by electrospray mass spectrometry are subjected to clustering and machine learning procedures based on genetic programming. The results permit discrimination of yeast strains differing in single gene deletions. The biochemical basis of unknown defects may be approached by identifying the metabolites involved.

Matuszewski BK, Canstanzer ML, Chavez-Eng CM. Strategies for assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry 2003;75:3019–3030. [PubMed]

“Matrix effects” in HPLC-MS/MS experiments are reductions or enhancements in analyte ion intensity occurring as a result of co-eluting, undetected components of a biological sample. This paper prescribes methods for measuring matrix effects, based on comparing MS/MS response of the pure analyte with the resonse given by analyte at the same concentration spiked into a biological sample post extraction. The need to evaluate variability of the matrix effect in multiple independent samples is emphasized, and the utility of chemically identical, isotopically labeled, internal standards in compensating for matrix effects is detailed. This article will be of interest to all involved in quantitative LC-MS/MS, including proteomics applications.

Go EP, Prenni JE, Wei J, Jones A, Hall SC, Witkowska E, Shen Z, Siuzdak G. Desorption/ionization on silicon time-of-flight/time-of-flight mass spectrometry. Analytical Chemistry 2003;75:2504–2506. [PubMed]

Desorption/ionization on silicon (DIOS), is a matrix-free technique in which analyte molecules are trapped within a porous silicon surface from which they are desorbed and ionized with a laser. Used in conjunction with time-of-flight mass analysis, the absence of interference from matrix at low mass makes DIOS particularly suitable for analysis of small molecules. It is here shown also to be suitable for protein identification by MS and MS/MS of tryptic peptides.

PROTEINS — PURIFICATION AND CHARACTERIZATION

McCarthy J, Hopwood F, Oxley D, Laver M, Castagna A, Righetti PG, Williams K, Herbert B. Carbamylation of proteins in 2-D electrophoresis—Myth or reality? Journal of Proteome Research 2003;2:239–242. [PubMed]

Carbamylation of protein amino groups in the urea/thiourea solutions used during protein solubilization and processing for 2-D gel electrophoresis has long been recognized as a potentially source of serious artefacts. Experiments described here indicate that carbamylation is unlikely to be problematic during isoelectric focusing or electrophoresis in the presence of urea, but may become serious under poorly controlled conditions of sample preparation and storage. Close attention to temperatures during these steps is recommended.

Herbert B, Hopwood F, Oxley D, McCarthy J, Laver M, Grinyer J, Goodall A, Williams K, Castagna A, Righetti PG. β-elimination: an unexpected artefact in proteome analysis. Proteomics 2003;3:826–831. [PubMed]

β-elimination (desulfuration) of cysteine residues, resulting in the loss of H2S, is observed to occur during isoelectric focusing of proteins in the alkaline region. The dehydroalanine formed in this reaction in turn promotes peptide bond cleavage at the affected position. This process can be obviated by alkylation of cysteines prior to isoelectric focusing. See also: Ma S, Caprioli RM, Hill KE, Burk RF. Loss of selenium from selenoproteins: conversion of selenocysteine to dehydroalanine in vitro. Journal of the American Society of Mass Spectrometry 2003;14:593–600. This paper documents analogous modification of proteins at selenocysteine residues.

Rogers M, Graham J, Tonge RP. Using statistical image models for objective evaluation of spot detection in two-dimensional gels. Proteomics 2003;3:879–886. [PubMed]

Synthetic data sets are created to simulate 2-D gel data, using statistical models of the shape, intensity, size, spread and location of spots encountered in real 2-D gel data sets. The synthetic data are used to evaluate various commercially available gel image analysis packages in terms of sensitivity and accuracy in the interpretation of patterns displaying variation in signal-to-noise ratio and degree of spot overlap. This evaluation strategy removes ambiguity and uncertainty in the interpretation of the images, because the manner in which the test data were generated is completely defined, enabling evaluation without subjectivity.

Werner WE. Run parameters affecting protein patterns from second dimension electrophoresis gels. Analytical Biochemistry 2003;317:280–283. [PubMed]

Vertical smearing of protein spots during the second dimension of 2-D electrophoresis is sometimes pronounced, especially for high molecular weight proteins. This paper reduces this tendency by increasing the concentration of buffer components in the cathode reservoire, and by performing electrophoresis at elevated temperatures, e.g. 30°C.

Thompson AJ, Hart SR, Franz C, Barnouin K, Ridley A, Cramer R. Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin β-elimination and Michael addition. Analytical Chemistry 2003;75:3232–3243. [PubMed]

Serine and threonine phosphorylation of proteins is studied by combining immobilized metal ion affinity chromatography (IMAC), β-elimination of phospho-serine or -threonine to yield dehydroalanine or dehydro-2-amino butyric acid, and then Michael addition with reagents such as 2-aminoethanethiol to produce residues that can readily be identified in subsequent analysis. The complementary use of these procedures minimizes their individual disadvantages, including poor recovery of multiply phosphorylated proteins from IMAC, binding of non-phosphorylated proteins to IMAC, and β-elimination of O-linked glycosylated as well as phophorylated residues.

Burke BJ, Regnier FE. Stopped-flow enzyme assays on a chip using a microfabricated mixer. Analytical Chemistry 2003;75:1786–1791. [PubMed]

A microfabricated system for rapid enzyme kinetic studies is described and tested. It is based on a micromixer of 200 μm length and 100 pL volume, through which streams of reagents can be passed by electroosmotic flow in a period of less than 1 sec with efficient mixing. The entire system has a volume of about 6 nL, 103–105 times smaller than the volume used for conventional enzyme assays.


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