Created: 1st December 2000, last updated: 30th December 2000, © 2000 ABRF
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 A. Slaughter, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794; Tel: (901) 495-4844; Fax: (901) 495-2945; email: clive.slaughter@stjude.org; or to any member of the editorial board. Article summaries reflect the reviewers' opinions and not necessarily those of the Association.
Sechi S, Chait BT. A method to define the carboxy terminal of proteins. Anal Chem 2000;72:3374-3378.
Proteins are first digested with lysylendopeptidease. Anydrotrypsin, a catalytically inert derivative of trypsin in which serine-195 is converted to dehydroalanine, is used to bind peptides containing a lysine (or arginine) residue at their C-termini. The peptide derived from the C-terminus of the intact protein, lacking a C-terminal lysine or arginine, is not retained by anhydrotrypsin and is collected for subsequent analysis. Matrix-assisted laser desorption/ionization (MALDI) is used to identify the peptide, and tandem mass spectrometry is used to sequence it.
Charlwood J, Skehel JM, Camilleri P. Analysis of N-linked oligosaccharides released from glycoproteins separated by two-dimensional gel electrophoresis. Anal Biochem 2000;284:49-59.
Protocols are described for analysis of N-linked carbohydrates on glycoproteins that have been subjected to two-dimensional gel electrophoresis. Glycans are released from the proteins by digestion with protein N-deglycosidase F (PNGase F), derivatized with 3-acetamido-6-aminoacridine, and analyzed by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight mass spectrometry. Enzymic release of the glycan is demonstrated using in-gel digestion of the excised gel spot, with or without prior tryptic digestion, and also using glycoproteins electroblotted to polyvinylidene difluoride membranes.
Taguchi R, Hayakawa J, Takeguchi Y, Ishida M. Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry. J Mass Spectrom 2000;35:953-966.
A strategy for analysis of phospholipids extracted from cultured cells is described. The strategy uses capillary liquid chromatography combined with tandem mass spectrometry in a quadrupole mass analyzer. More than 500 species of phospholipids are included by the method. Positive molecular ions, negative molecular ions, positive fragment ions, and negative fragment ions are monitored, consecutively, in 11-second cycles. Positive molecular ions indicate choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, and phosphatidylethanolamine. Negative molecular ions indicate acidic phospholipids such as phosphatidylinositol. Positive fragment ions indicate diradylglycerol and derivatives of 1-alkyl and 2-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine and phosphorylcholine from choline-containing phospholipids. Negative fragment ions indicate ions from the acyl fatty acid and phosphorylcholine, phosphorylethanolamine, and cyclic inositol phosphate.
Premstaller A, Oberacher H, Huber CG. High-performance liquid chromatography-electrospray ionization mass spectrometry of single- and double-stranded nucleic acids using monolithic capillary columns. Anal Chem 2000;72:4386-4393.
High-performance liquid chromatography (HPLC) columns are most commonly filled with microparticulate stationary phases, but these suffer from the relatively large void volumes between the packed particles and the slow diffusion of solutes into and out of the stagnant mobile phase present in the pores of the separation medium. These problems are alleviated by using stationary phases consisting of a continuous rod of rigid, porous polymer with no interstitial volume and only internal porosity. This report uses such "monolithic" stationary phases, packed into capillary columns, to improve the separation of oligonucleotides. A mobile phase using 100-mM triethylammonium acetate as an ion pairing reagent is employed, and elution is performed with a gradient of acetonitrile. The method is adapted for online electrospray mass spectrometry by using 25-mM triethylammonium bicarbonate as the ion pairing reagent, with only a small reduction in chromatographic performance. Acetonitrile is supplied postcolumn to enhance the elecrospray process. Good-quality mass spectra of femtomole amounts or 3-mers to 80-mers are presented showing little cation adduction. Double-stranded DNA fragments from 51 to 587 base pairs are separated and detected.
Xu G, Enderle H, Liebich H, Lu P. Study of normal and modified nucleosides in serum by RP-HPLC. Chromatographia 2000;52:152-158.
A high-performance liquid chromatography (HPLC) method is employed for separating nucleoside mixtures containing both modified and unmodified components that are present in serum and urine of normal individuals and cancer patients. The method uses a KH2PO4 buffer (pH 4.65), and elution is performed with a 45-minute period of a gradient of methanol. The nucleosides are detected by their absorption at 260 and 280 nm.
Puapaiboon U, Jai-nhuknan J, Cowan JA. Rapid and direct sequencing of double-stranded DNA using exonuclease III and MALDI-TOF MS. Anal Chem 2000;72:3338-3341.
A strategy for sequencing double-stranded DNA by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is reported. The strategy uses exonuclease III to degrade phosphodiester bonds from the 3'-end of the double-stranded DNA. The enzyme requires the addition of appropriate divalent cations. A cation exchange column is then used in conjunction with an ammonium citrate additive to reduce formation of alkali salt adducts.
Timperman AT, Aebersold R. Peptide electroextraction for direct coupling of n-gel digests with capillary LC-MS/MS for protein identification and sequencing. Anal Chem 2000;72:4115-4121.
An electrophoretic method for extracting proteins from in-gel digests is described for use in mass spectrometric protein identification. A small-scale cell is fabricated for electroextraction, and peptides are trapped on a strong cation-exchange resin housed in a fused silica capillary tube. Immobilized in this way, they can be stored for several days without loss. The design of the system is suitable for direct interfacing with capillary liquid chromatography in tandem liquid chromatography/mass spectrometry analyses. Significant enhancements in sensitivity are realized for digests of proteins at the low nanogram level (ie, the limits of sensitivity of silver staining).
Doucette A, Craft D, Li L. Protein concentration and enzyme digestion on microbeads for MALDI-TOF peptide mass mapping of proteins from dilute solutions. Anal Chem 2000;72:3355-3362.
A method for digesting proteins available in solutions of low concentration is presented for the acquisition of peptide mass fingerprint data. The protein is first concentrated by adsorption to POROS R2 reverse-phase polymeric beads (Applied Biosystems, Foster City, CA). The beads may then be washed to remove contaminants. The bound proteins are subsequently digested with trypsin. The beads retain peptides during digestion and may be deposited directly on a target for matrix assisted laser desorption/ionization (MALDI). On-target washing may also be performed to remove substances inhibiting ionization. The method provides high-quality peptide mass fingerprint data for protein samples of only 100 nM.
Zuo X, Speicher DW. Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;21:3035-3047.
Protein recoveries are systematically evaluated at the various steps of separation by two-dimensional gel electrophoresis using immobilized pH gradients (IPGs) for the isoelectric focusing stage. The Multiphor and IPGphor systems from Amersham Pharmacia Biotech (Uppsala, Sweden) are used for the separation of metabolically radiolabeled proteins from Escherichia coli. With the Multiphor system, in which sample application to the IPG strips and subsequent electrofocusing are performed in separate vessels, sample application from cups gives better protein recoveries than sample loading by IPG rehydration. In the IPGphor system, in which sample loading and electrofocusing are performed in the same vessel, the rehydration method gives better recoveries. In all cases, carrier ampholytes in the sample buffer increase protein recoveries. Thiourea does not affect protein recoveries, but does improve protein resolution during electrofocusing.
Zuo X, Speicher DW. A method for global analysis of complex proteomes using sample prefractionation by solution isoelectofocusing prior to two-dimensional electrophoresis. Anal Biochem 2000;284:266-278.
Increasing the protein load on immobilized pH gradient (IPG) strips in an attempt to improve the number of visible spots resolved by two-dimensional electrophoresis is limited by the tendency of proteins to aggregate or precipitate when high protein loads are used, causing smearing in the isoelectric focusing dimension. The use of solution-phase isoelectric focusing to minimize such smearing is discussed, and an isoelectric focusing apparatus for prefractionating proteins for this purpose is described.
Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci USA 2000;97:9390-9395
It is widely assumed that two-dimensional gel electrophoresis, coupled with mass spectrometric identification of spots, enables a broad coverage of the proteome. Consistent with this view is the observation that the number of spots observed by two-dimensional electrophoresis approaches the number of proteins predicted for a given organism or tissue. This study examines an arbitrarily selected section from a narrow pH range (4.9-5.7) two-dimensional display of yeast proteins in which more than 1500 spots are visualized by silver staining. Fifty spots were digested with trypsin and analyzed by liquid chromatography combined with tandem mass spectrometry, and a total of 39 different proteins were identified from a region where the yeast genome is predicted to encode 57 gene products. However, these represent only the proteins predicted on the basis of codon usage to be the most abundant. Furthermore, only 14 of the proteins identified were predicted to appear in the analyzed section. Additionally, single genes produced multiple spots, and the products of up to six genes were found to make up a single spot, presenting problems for both quantitative protein expression comparisons and database-matching studies. It is concluded that the method is unsuited for the analysis of low-abundance proteins and that statements about the feasibility and straightforwardness of proteome analysis based on the two-dimensional electrophoresis mass spectrometry strategy should be rethought. [This article has also received comment by Smith RD. Probing proteomics--seeing the whole picture. Nat Biotechnol 2000;18:1041-1042.]
Ghaemmaghami S, Fitzgerald MC, Oas TG. A quantitative, high-throughput screen for protein stability. Proc Natl Acad Sci USA 2000;97:8296-8301
The use of mass spectrometric measurements of hydrogen/deuterium exchange to compare the rates of denaturation of different but related proteins is described. Matrix-assisted laser desorption/ionization (MALDI) is employed to measure the rates of exchange of amide hydrogen atoms with deuterium in solution as a function of denaturant concentration to provide a measure of protein stability. The method permits large numbers of protein samples to be screened in a short time, does not demand that the proteins of interest are pure or available in large quantities, and is forgiving of the presence of denaturants. Mutants of the lambda repressor are studied to illustrate the utility of the method, and the stabilization of maltose-binding protein on binding to maltose is also demonstrated.
Lee MT, Kuo FC, Whitmore GA, Sklar J. Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations. Proc Natl Acad Sci USA 2000;97:9834-9839
This study investigates the inherent variability in gene expression data and assess the extent to which replication in an experiment produces more consistent and reliable findings. The results show that any single microarray output is subject to substantial variability, even though, by design, variability as a result of multiple preparations of probe, arrays on different slides, or arrays generated at different times is not admitted. A single output yields numerous misclassifications. Replications are not consistent and therefore produce different lists of expressed genes. Modeling the random variation in gene expression indicates that the probability that mRNA in the sample tissue either fails to be represented as probe or fails to hybridize to the cDNAs on the slide may be as large as 5% (false-negatives). The probability that ghost genes are expressed may be as large as 10% (false-positives). When microarray data from several replicates are combined, however, a more accurate picture of gene expression is produced. It is recommended that at least three replicates be included in the design of experiments using cDNA microarrays.
Fenyö D. Identifying the proteome: software tools. Curr Opin Biotechnol 2000;11:391-395.
This article reviews the various software tools that are available on the internet for searching protein sequence databases using mass spectral information. Both peptide mapping data listing the mass values of proteolytic peptides and fragment ion data derived by collisional dissociation of peptides to yield sequence-informative ions are included. Attention is paid to the methods used by the various tools to rank protein candidates and the methods by which the quality of identifications are assessed.