An FD-LC-MS/MS Proteomic Strategy for Revealing Cellular Protein Networks: A Conditional Superoxide Dismutase 1 Knockout Cells

<div><p>Systems biology aims to understand biological phenomena in terms of complex biological and molecular interactions, and thus proteomics plays an important role in elucidating protein networks. However, many proteomic methods have suffered from their high variability, resulting in only showing altered protein names. Here, we propose a strategy for elucidating cellular protein networks based on an FD-LC-MS/MS proteomic method. The strategy permits reproducible relative quantitation of differences in protein levels between different cell populations and allows for integration of the data with those obtained through other methods. We demonstrate the validity of the approach through a comparison of differential protein expression in normal and conditional superoxide dismutase 1 gene knockout cells and believe that beginning with an FD-LC-MS/MS proteomic approach will enable researchers to elucidate protein networks more easily and comprehensively.</p> </div

Simplified scheme for the proposed strategy.

<p>Using the method, protein expression changes caused by a particular stimulus are quantified. The resulting data are integrated with other data and a diagram of predicted protein networks is constructed.</p

Predicted protein networks in SOD1(−) cells.

<p>Protein networks for denaturation, refolding, decomposition, ATP-consumption and -production. Arrows indicate increased (up) and decreased (down) expression. The (+) and (−) signs indicate enhancement or suppression, respectively, of cellular processes in response to changes in protein expression. The abbreviated protein names are in Table S1. GR: glucocorticoid receptor.</p

Classification of identified proteins.

<p>Functional classification of differentially expressed proteins identified in this study. The most significant changes were observed with proteins involved in mediating protein folding. ND: No Data.</p

Schematic illustration of the FD-LC-MS/MS proteomic method.

<p>After fluorogenic derivatization, the protein mixtures are separated by HPLC, and proteins exhibiting significant differential expression are isolated and identified using nano-HPLC-MS/MS and database searching.</p

Arp4p and Arp4 containing complexes associate with the centromere and telomere

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> Cells with 13Myc-tagged Arp4 (YHO312) were grown in YPAD at 20°C for 3 h with 100 ng/ml α-factor. Cells were released by washing in YPAD and incubated in fresh YPAD medium at 20°C. Samples were taken at the time points indicated and analyzed by flow cytometry () and ChIP (). () Flow cytometry analysis of cell cycle profiles. () Input DNA and DNA coimmunoprecipitated with the anti-Myc antibody (IP) were amplified with primer sets corresponding to sequences around centromeres ( and ), the inner region of a large ORF (), a telomere (), and a sub-telomeric region (). To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. ChIP PCR products were separated by agarose gel electrophoresis. Representative data are shown. () Arp4p, Ino80p, Esa1p and Swr1p interact with and localize to , , , but not to and a subtelomeric region. Flag-tagged Arp4 (YHO311), Ino80 (YHO313), Esa1 (YHO314), Swr1 (YHO315) or untagged (YK402) cells were arrested in G2/M by treatment with nocodazole at 30°C. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-FLAG antibody (IP) were amplified with primer sets corresponding to sequences around , , , , and a subtelomeric region. The templates used were total chromatin (Input) or immunoprecipitate (IP)

The centromeric binding of kinetochore components is partially impaired in mutants

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> 3HA-tagged or untagged wild-type and cells were grown in YPAD at 23°C for 3 h with 15 μg/ml nocodazole to ensure that both populations had an equivalent cell cycle distribution since a higher proportion of cells are in G2/M phase. The culture was shifted to 37°C and incubated in the presence of nocodazole for 1 h. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-HA antibody (IP) were amplified with primer sets corresponding to sequences around centromeres (). Quantitative data were obtained by real-time PCR. To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. In each case, ChIP enrichment is expressed relative to that for a subtelomeric region of chromosome V (9716–9823). Results are expressed as the mean and SD of two independent ChIP experiments. Dashed lines indicate the background level of ChIP signal intensity in an untagged strain. () Schematic of kinetochore components. () The centromere-specific histone H3 variant Cse4p (wild-type cells: YHO805; cells: YHO825), a representative protein of the inner kinetochore Mif2p (wild-type cells: YHO806; cells: YHO826) and Ndc10p (wild-type cells: YHO807; cells: YHO827) were analyzed by ChIP at . () A representative protein of the outer kinetochore Mtw1p (wild-type cells: YHO808; cells: YHO828), Nuf2p (wild-type cells: YHO809; cells: YHO829) and Ctf3p (wild-type cells: YHO810; cells: YHO830) were analysed by ChIP at . () The cohesin component Scc1p (wild-type cells: YHO811; cells: YHO831) was analysed by ChIP at