Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses

PMeS: Prediction of Methylation Sites Based on Enhanced Feature Encoding Scheme

Protein methylation is predominantly found on lysine and arginine residues, and carries many important biological functions, including gene regulation and signal transduction. Given their important involvement in gene expression, protein methylation and their regulatory enzymes are implicated in a variety of human disease states such as cancer, coronary heart disease and neurodegenerative disorders. Thus, identification of methylation sites can be very helpful for the drug designs of various related diseases. In this study, we developed a method called PMeS to improve the prediction of protein methylation sites based on an enhanced feature encoding scheme and support vector machine. The enhanced feature encoding scheme was composed of the sparse property coding, normalized van der Waals volume, position weight amino acid composition and accessible surface area. The PMeS achieved a promising performance with a sensitivity of 92.45%, a specificity of 93.18%, an accuracy of 92.82% and a Matthew’s correlation coefficient of 85.69% for arginine as well as a sensitivity of 84.38%, a specificity of 93.94%, an accuracy of 89.16% and a Matthew’s correlation coefficient of 78.68% for lysine in 10-fold cross validation. Compared with other existing methods, the PMeS provides better predictive performance and greater robustness. It can be anticipated that the PMeS might be useful to guide future experiments needed to identify potential methylation sites in proteins of interest. The online service is available at http://bioinfo.ncu.edu.cn/inquiries_PMeS.aspx

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

Several amino acids were found to undergo progressive age-dependent racemisation in the lifelong proteins of normal human lenses. The two most highly racemised were Ser and Asx. By age 70, 4.5% of all Ser residues had been racemised, along with >9% of Asx residues. Such a high level of inversion, equivalent to between 2 and 3 d - amino acids per polypeptide chain, is likely to induce significant denaturation of the crystallins in aged lenses. Thr, Glx and Phe underwent age-dependent racemisation to a smaller degree. In model experiments, d - amino acid content could be increased simply by exposing intact lenses to elevated temperature. In cataract lenses, the extent of racemisation of Ser, Asx and Thr residues was significantly greater than for age-matched normal lenses. This was true, even for cataract lenses removed from patients at the earliest ages where age-related cataract is observed clinically. Racemisation of amino acids in crystallins may arise due to prolonged exposure of these proteins to ocular temperatures and increased levels of racemisation may play a significant role in the opacification of human lenses

The serine-rich N-terminal region of Arabidopsis phytochrome A is required for protein stability.

Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6-12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation

Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry

Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labelingThis work was supported by the Spanish Ministry of Science and Innovation (nos.BFU2009-06933 and BFU2012-32045), by PLAN E and Fondo Europeo de Desarrollo Regional (FEDER), by the Spanish program Consolider-Ingenio 2010 (grant no. CSD 2007-0020) and by grant no. SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. and J.A. are recipients of Institució Catalana de Recerca i Estudis Avançats (ICREA) /nAcademia Awards (Generalitat de Catalunya