Attempts 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.

Affymetrix and spotted oligonucleotide microarrays were used to assess global differential gene expression comparing normal human melanocytes with six independent melanoma cell strains from advanced lesions. The data, validated at the protein level for selected genes, confirmed the overexpression in melanoma cells relative to normal melanocytes of several genes in the growth factor/receptor family that confer growth advantage and metastasis. In addition, novel pathways and patterns of associated expression in melanoma cells not reported before emerged, including the following: (a) activation of the NOTCH pathway; (b) increased Twist expression and altered expression of additional transcriptional regulators implicated in embryonic development and epidermal/mesenchymal transition; (c) coordinated activation of cancer/testis antigens; (d) coordinated down-regulation of several immune modulation genes, in particular in the IFN pathways; (e) down-regulation of several genes implicated in membrane trafficking events; and (f) down-regulation of growth suppressors, such as the Prader-Willi gene NECDIN, whose function was confirmed by overexpression of ectopic Flag-necdin. Validation of differential expression using melanoma tissue microarrays showed that reduced ubiquitin COOH-terminal esterase L1 in primary melanoma is associated with worse outcome and that increased expression of the basic helix-loop-helix protein Twist is associated with worse outcome. Some differentially expressed genes reside on chromosomal regions displaying common loss or gain in melanomas or are known to be regulated by CpG promoter methylation. These results provide a comprehensive view of changes in advanced melanoma relative to normal melanocytes and reveal new targets that can be used in assessing prognosis, staging, and therapy of melanoma patients.

Type I protein arginine methyltransferases catalyze the formation of asymmetric ω-N ^G,N ^G-dimethylarginine residues by transferring methyl groups fromS-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 glutathioneS-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrateS-adenosyl-l-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.

Motivation: Novel methods, both molecular and statistical, are urgently needed to take advantage of recent advances in biotechnology and the human genome project for disease diagnosis and prognosis. Mass spectrometry (MS) holds great promise for biomarker identification and genome-wide protein profiling. It has been demonstrated in the literature that biomarkers can be identified to distinguish normal individuals from cancer patients using MS data. Such progress is especially exciting for the detection of early-stage ovarian cancer patients. Although various statistical methods have been utilized to identify biomarkers from MS data, there has been no systematic comparison among these approaches in their relative ability to analyze MS data. Results: 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. Supplementary information:http://bioinformatics.med.yale.edu/proteomics/BioSupp1.html

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

CDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplified DNA fragment encoding an internal peptide sequence of glycoprotein D (gpD) protein. The open reading frame of the 1267-bp cDNA clone indicated that gpD protein was composed of 338 amino acids, predicting a M(r) of 35,733, which was the same as a deglycosylated gpD protein. Portions of the predicted amino acid sequence, matched with six CNBr/pepsin peptides obtained from affinity-purified gpD protein. In ELISA analysis, an anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of 9 membrane-spanning alpha-helices. In bone marrow RNA blot analysis, the gpD cDNA detected a 1.27-kb mRNA in Duffy-positive but not in Duffy-negative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffy-positive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.