Although some of the underlying technology for quantifying protein abundance was introduced almost thirty years ago [1,2], there has recently been a significant increase in the development of new tools. Concurrently, tools for analyzing mRNA expression are becoming more mainstream. The quantification of both of these molecular populations is not an exercise in redundancy; measurements taken from mRNA and protein levels are complementary and both are necessary for a complete understanding of how the cell works [3]. Additionally, as mRNA is eventually translated into protein, one might assume that there should be some sort of correlation between the level of mRNA and that of protein. Alternatively, there may not be any significant correlation, which, in itself, is an informative conclusion.The two commonly used high-throughput methods for measuring mRNA expression, microarrays and Affymetrix chips, have both been extensively reviewed elsewhere [4][5][6]. There are also two basic methods for determining protein abundance; either based on two-dimensional electrophoresis or on mass-spectrometric methods (Table 1). We provide a brief review of these technologies and recent efforts to determine correlations between quantified protein abundances and mRNA expression. Methods for determining protein levels Two-dimensional electrophoresisDetermining relative protein expression levels by conventional two-dimensional electrophoresis requires isoelectric focusing, SDS-polyacrylamide gel electrophoresis, staining, fixing, densitometry, and careful matching of the same spots on two or more gels. Differentially expressed spots are then excised and enzymatically digested, and the resulting peptides are identified using mass spectrometry. An attractive aspect of this approach is the low capital equipment cost, but a high level of expertise is needed to obtain reproducible gels, and two-dimensional electrophoresis is generally limited to proteins that are neither too acidic, too basic, nor too hydrophobic, and that are between 10 and 200 kDa in size, so that they are reliably separated on gels. Additionally, this approach detects only those proteins that are expressed at relatively high levels and that have long half-lives [7,8]. In one study using 40 µg yeast lysate, the average protein AbstractAttempts to correlate protein abundance with mRNA expression levels have had variable success. We review the results of these comparisons, focusing on yeast. In the process, we survey experimental techniques for determining protein abundance, principally two-dimensional gel electrophoresis and mass-spectrometry. We also merge many of the available yeast protein-abundance datasets, using the resulting larger 'meta-dataset' to find correlations between protein and mRNA expression, both globally and within smaller categories.

Type I protein arginine methyltransferases catalyze the formation of asymmetric -N G ,N G -dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300 -400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein.However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(؉/؉), FDH(؉/؊), and FDH(؊/؊) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.

We compare the performance of several classes of statistical methods for the classification of cancer based on MS spectra. These methods include: linear discriminant analysis, quadratic discriminant analysis, k-nearest neighbor classifier, bagging and boosting classification trees, support vector machine, and random forest (RF). The methods are applied to ovarian cancer and control serum samples from the National Ovarian Cancer Early Detection Program clinic at Northwestern University Hospital. We found that RF outperforms other methods in the analysis of MS data.

ABSTRACT

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant eukaryotic nuclear RNA binding protein. A1 is involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm and may modulate splice site selection. The crystal structure of A1(RBD1,2) reveals two independently-folded RNA binding domains (RBDs) connected by a flexible linker. Both RBDs are structurally homologous to the U1A(RBD1), and have their RNA binding platforms oriented in an anti-parallel fashion. The anti-parallel arrangement of the A1 RNA binding platforms suggests mechanisms for RNA condensation and ways of bringing together distant RNA sequences for RNA metabolism such as splicing or transport.

Three sites of N(G),N(G)-arginine methylation have been located at residues 205, 217, and 224 in the glycine-rich, COOH-terminal one-third of the HeLa A1 heterogeneous ribonucleoprotein. Together with the previously determined dimethylated arginine at position 193 [Williams et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670], it is evident that all four sites fall within a span of sequence between residues 190 and 233 that contains multiple Arg-Gly-(Gly) sequences interspersed with phenylalanine residues. These RGG boxes have been postulated to represent an RNA binding motif [Kiledjian and Dreyfuss (1992) EMBO J. 11, 2655-2664]. Dimethylation of HeLa A1 appears to be quantitative at each of the four positions. Arginines 205 and 224 have been methylated in vitro by a nuclear protein arginine methyltransferase using recombinant (unmethylated) A1 as substrate. This suggests A1 may be an in vivo substrate for this enzyme. Examination of sequences surrounding the sites of methylation in A1 along with a compilation from the literature of sites that have been identified in other nuclear RNA binding proteins suggests a methylase-preferred recognition sequence of Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe, with the COOH-terminal flanking glycine being obligatory. Taken together with data in the literature, identification of the sites of A1 arginine methylation strongly suggests a role for this modification in modulating the interaction of A1 with nucleic acids.

Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and from an overproduction vector derived from the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per mol of protein. A linear incorporation of three molar equivalents of p-hydroxymercuriphenylsulfonate (PMPS) results in a linear release of 1.1 mol of Zn(II) from the protein. Reversal of formation of the g32P-PMPS complex with thiol in the presence of EDTA results in a zinc-free apo-g32P. Cd(ll) and Co(ll) can be exchanged with the intrinsic Zn(ll) ion. The Cd(II) protein shows a charge-transfer band at m250 nm. The Co(II) protein shows a set of absorption bands typical of a tetrahedral Co(ll) complex (e. = 660 M-1 cm-1 at 645 nm), and two intense chargetransfer bands are present at 355 nm (e = 2250 M-1 cm-1) and 320 nm (e = 3175 M-1 cm-1). These observations are consistent with three cysteines as ligands to the Zn(ll) ion in g32P. Zn(ll) g32P undergoes precise limited proteolysis by trypsin to produce the small fragments A and B and the core, g32P-(A+B). Under identical conditions, apo-g32P is hydrolyzed rapidly beyond the g32P-(A+B) stage to produce many proteolyzed fragments. Fluorescence quenching experiments show that at low protein concentration apo-g32P has markedly altered binding affinity for poly(dT) relative to native g32P. Three of the four cysteines of g32P are found in a tyrosine-rich sequence corresponding to residues 72-116 and implicated in DNA binding by 1H NMR investigations. Zn(ll) appears to provide a conformational element contributing to DNA binding by coordinating the cysteine and possibly histidine side chains in the sequence -Cys-X3-His-X5-Cys-X2-Cys-, residues 77-90, located in the DNA binding domain of g32P.The product of gene 32 of bacteriophage T4 (g32P) is one of a class of proteins that bind to single-stranded (ss) DNA (or ss RNA) in a stoichiometric fashion and are referred to as helix-destablizing proteins or ss DNA binding proteins (SSBs) (1-4). Other widely studied members of this class include gene S protein from bacteriophage fd and SSB from Escherichia coli (5, 6). g32P is known to play key roles in DNA replication, recombination, and repair (2). g32P is thought to be able to quickly cover those transiently singlestranded regions that arise near the advancing T4 DNA replication forks and in so doing stabilize a particular ss DNA conformation that is most appropriate to serve as a substrate for other catalytic proteins (2). E. coli SSB probably functions in much the same way (6). Gene 5 protein, in contrast, acts to prevent replicative DNA synthesis by covering newly synthesized fd ss DNA until packaging into the phage coat can occur (3, 5).The molecular details of how each of these SSBs interacts with ss DNA has been probed by one-and two-dimensional 1H NMR techniques applied to the oligonucleotide complexes of these proteins (7-11). For both gene 5 protein and the g32P tryptic core, the results support a model involving intercalation of tyrosine and phenylalanine side cha...

The widespread use of mass spectrometry for protein identification has created a demand for computationally efficient methods of matching mass spectrometry data to protein databases. A search using X!Tandem, a popular and representative program, can require hours or days to complete, particularly when missed cleavages and post-translational modifications are considered. Existing techniques for accelerating X!Tandem by employing parallelism are unsatisfactory for a variety of reasons. The paper describes a parallelization of X!Tandem, called X!!Tandem, that shows excellent speedups on commodity hardware and produces the same results as the original program. Furthermore, the parallelization technique used is unusual and potentially useful for parallelizing other complex programs.