Protein Spot Patterns Research Articles

Evaluation of Six Protocols for Protein Extraction from Rice Young Panicles byTwo-Dimensional Electrophoresis

ABSTRACT For most reported proteomics approaches, protein extraction and sample preparation are of crucial importance for optimal results. However, extraction of protein from crop plant tissues remains a great challenge due to low protein content and abundant secondary metabolites that prominently interfere with isoelectric focusing and subsequent proteomic analysis. Up until now, no attempts are focused on comparison of protein extraction procedures from rice young panicles. To establish a high-efficiency protein extraction protocol suitable for two-dimensional electrophoresis (2-DE) in rice young panicles, six protocols for protein preparation were evaluated: (1) Phenol extraction; (2) Mg/ Nonidet P-40 (NP-40) extraction; (3) Tris-Base/acetone precipitation; (4) SDS extraction; (5) trichloroacetic acid (TCA)/acetone/phenol extraction; (6) TCA/acetone precipitation. The study explicitly demonstrates that TCA/ acetone/phenol method provides a high-enhanced protein extraction efficacy from rice young panicles than other protocols in terms of the higher quality of 1-DE gel, the maximum number (450) of well-resolved protein spots, greater resolution and spot abundance. In addition, these methods also generated remarkably different twodimensional gel electrophoresis protein spot patterns. Twenty-nine of 30 visible differential extracted proteins were identified by MALDI-TOF-MS/MS analysis and were divided into eight categories according to molecular function. Accumulated data suggested that different extraction methods respectively necessitate certain special plant tissues due to different physicochemical properties of each protocol. Overall, this study, which is presented in this paper, will facilitate to providing a cornerstone of comparative proteomic analysis from rice young panicles, including other complicated plant tissues.

Improved Alignment and Quantification of Protein Signals in Two-Dimensional Western Blotting.

Western blotting is widely used for protein identification and quantification in research applications, but different protein species, resulting from alternative splicing and post-translational modifications, can often only be detected individually by two-dimensional gel electrophoresis and immunodetection by Western blotting (2D-WB). The additional separation by isoelectric focusing enables the detection of different protein species with the same specific antibody. Reliable assignment of signals from antibody-based detection to the total protein spot pattern of the original gel image is a challenge in 2D-WB, often resulting in ambiguous results. We therefore propose a reliable strategy for assignment of antibody signals from 2D-WB to the total protein spot pattern, using an imaging workflow in combination with a straightforward and easily reproducible image alignment strategy. The strategy employs vector-based alignment of protein spots and image contours in a stepwise manner. Our workflow is compatible with various protein visualization techniques, including prelabeling of proteins and poststaining of gels and membranes, as well as with chemiluminescent and fluorescent detection of bound antibody. Here, we provide a detailed description of potential applications and benefits of our workflow. We use experimental test settings with gold-standard stressors in combination with multiple staining and detection methods, as well as spike-in recombinant proteins. Our results demonstrate reliable attribution of signals to very similar heat shock proteins, phosphorylation patterns, and global analysis of proteins modified with O-linked N-acetylglucosamine (O-GlcNAc).

A novel method for quantifying traction forces on hexagonal micropatterned protein features on deformable poly-dimethyl siloxane sheets

Many methods exist for quantifying cellular traction forces, including traction force microscopy and microfabricated post arrays. However, these methodologies have limitations, including a requirement to remove cells to determine undeflected particle locations and the inability to quantify forces of cells with low cytoskeletal stiffness, respectively. Here we present a novel method of traction force quantification that eliminates both of these limitations. Through the use of a hexagonal pattern of microcontact-printed protein spots, a novel computational algorithm, and thin surfaces of polydimethyl siloxane (PDMS) blends, we demonstrate a system that:•quantifies cellular forces on a homogeneous surface that is stable and easily manufactured.•utilizes hexagonal patterns of protein spots and computational geometry to quantify cellular forces without need for cell removal.•quantifies cellular forces in cells with low cytoskeletal rigidity.

Effective protein extraction from mycelium and fruiting body of Auricularia auricula for proteomics studies

ABSTRACTAuricularia auricula is one of the most popular edible mushrooms in China. Limited information is available regarding genes and proteins related to A. auricula development from mycelium to mature fruiting body and response to environmental stimuli. With the development of proteomics, this technology platform for addressing this problem is available. A critical step of a good proteome is protein extraction which can gives the best quality and a wide coverage of total proteins. To establish an effective protein extraction protocol suitable for proteomics analysis in mycelium and fruiting body of A. auricula, four previously reported protein extraction protocols including direct extraction, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation and phenol-based extraction, were compared based on the protein yield and 2-DE patterns. The efficiency of the four methods was evaluated by comparing protein yield and 2-DE patterns. Of the four methods, both TCA precipitation and phenol-based extraction method for A. auricula mycelia protein extraction yielded the good protein separation pattern and higher number of protein spots in 2-DE analysis. Furthermore, phenol-based extraction is superior to the other tested methods for A. auricula fruiting body proteome analysis .

Messenger RNas : their utilization and degradation during pollen germination and tube growth

During pollen germination and tube growth at least 230 new proteins are synthesized, as determined by <sup>35</sup>S-methionime labeling and two dimensional gel electrophoretic analysis of the labeled proteins. The same number and pattern of protein spots is seen whether or not actinomycin D is included in the, medium, indicating that the mRNAs present in the unger-minated pollen grain and those newly synthesized code for the same proteins. The genetic program during at least the latter part of pollen maturation prior to anthesis and that during pollen germination and tube growth thus appears to be similar if not identical. During the first hour of pollen tube growth about 500/0 of the protein synthesis that occurs utilizes previously synthesized mRNAs. The remaining 50% occurs on newly made mRNAs. The ungerminated mature pollen grain contains 196 pg of RNA and approximately 6 X 10<sup>6</sup> molecules of poly(A)+ RNA, i.e. mRNAs. The rate of protein synthesis corrected for internal pool changes in the labeled amino acid used (<sup>3</sup>H-leucine) is highest during the first 15 min of pollen tube growth. The rate decreases rapidly thereafter for the next 45 min. Concurrent with the reduction in rate of protein synthesis there is a reduction in the poly(A) content of the pollen RNA and in the amount of poly(A) per pollen, grain. The total RNA per pollen grain, however, appears not to change during this period.

Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods-trichloroacetic acid/acetone, Mg/NP-40, and phenol/ammonium acetate-were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low-abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP-40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.

Comparative proteomic analysis of the sun- and freeze-dried earthworm Eisenia fetida with differentially thrombolytic activities

The dried earthworm is a traditional thrombolytic medicine in East Asia. Its thrombolytic mechanism has been extensively studied. However, the effects of drying process on thrombolysis were rarely investigated. Herein, we compared the thrombolytic activity of earthworm Eisenia fetida processed by sun-drying to that by freeze-drying. Fibrin plate and blood clot lysis assays showed that freeze-dried earthworms gave dramatically higher fibrinolytic and thrombolytic activities than the sun-dried earthworms. To address the thrombolytic difference, comparative proteomic analysis was carried out using fibrin zymography and two-dimensional gel electrophoresis (2-DE). The freeze- and sun-dried earthworms generated remarkably different 2-DE protein spot patterns. A total of 126 differential protein spots were detected, 83 of them were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching with 13 quantitative changes and 70 qualitative changes. Five of these differential proteins were identified as fibrinolytic proteases (lumbrokinases), responsible for dissolving fibrin, the main protein component of thrombus. The total abundance of these fibrinolytic proteases in the freeze-dried earthworms was significantly higher, consistent with the results of fibrin zymography. Therefore, the higher concentration of fibrinolytic enzymes along with their broad substrate specificity explained the stronger fibrinolytic and thrombolytic activities of the freeze-dried earthworms. This study suggests that freeze-drying represents an improved processing method for earthworm as the thrombolytic therapy in the future. Thrombosis has become one of the biggest concerns all over the world. The dried earthworms have been intensively used as thrombolytic agents. Its thrombotic mechanism has been studied by the modern pharmacological researches. However, the drying procedure of the earthworm and its effects on the thrombolysis were rarely investigated. The present study compared the thrombolytic effects of the freeze-dried and the normal dried earthworm E. fetida. To better understand the underlying mechanisms for differential thrombolytic effects, the fibrin zymography and the two-dimensional gel electrophoresis (2-DE) were employed to identify sets of differential proteins. Therefore, this study provides not only the comparative proteomic analysis but also molecular mechanism underlying the differential thrombolytic effects.

Web-Based Tools for the Interpretation of Chain-Like Protein Spot Patterns on Two-Dimensional Gels

Two-dimensional gel electrophoresis is a standard tool of proteomic analysis, consisting of first dimensional protein separation according to the respective proteins isoelectric point, and the second dimensional separation according to the proteins molecular weight. In these gels, “chain-like” protein spots of equal molecular weight but different isoelectric points are common observations. When identified by mass spectrometry, these chain-like protein spots are identified as the same protein entry in the databases, thus representing “protein species” in the proteomic literature. In this review, we will discuss the factors responsible for the occurrence of multiple protein species, which mostly depend on posttranslational modifications (PTMs) such as phosphorylation and glycosylation and others. Additionally, we will show how internet-based prediction tools can be used for comparing the theoretical models to the actual gel pattern. This may present useful tools for the validation of biomarkers in health and disease.

Proteomic Comparison of Two Invasive Polychaete Species and Their Naturally Occurring F1-hybrids

The mud worm genus Marenzelleria is highly invasive and is therefore studied intensively. In recently invaded habitats, sympatric populations of the sibling species Marenzelleria viridis and Marenzelleria neglecta are found. In these secondary contact zones, hybridization occurs frequently, revealing incomplete reproductive isolation between these recently diverged species. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometric methods were applied for a comparative analysis of these species and their F(1)-hybrids. Nineteen proteins were identified by cross-species identification strategies. A low degree of interindividual variability within either species allowed characterizing qualitative species-specific differences in 2-DE spot patterns as well as in peptide maps. Species-specific peptides were found in tryptic digests of various proteins, such as glyceraldehyde-3-phosphate dehydrogenase, troponin C, gelsolin-like protein, and peroxiredoxin-1. F(1)-hybrids of M. viridis and M. neglecta showed additivity of protein spot patterns, and the presence of both parental traits was confirmed by mass spectrometric data. This study is one of few dealing with global protein expression in polychaetes and is the first proteomic description of natural F(1)-hybrids in polychaetes. It furthermore indicates the feasibility of proteomic methods for analyses of speciation in Marenzelleria siblings as well as of hybridization events in secondary contact zones in general.

Proteomic Analysis of Four Brazilian MON810 Maize Varieties and Their Four Non-Genetically-Modified Isogenic Varieties

Profiling techniques have been suggested as a nontargeted approach to detect unintended effects in genetically modified (GM) plants. Seedlings from eight Brazilian maize varieties, four MON810 GM varieties and four non-GM isogenic varieties, were grown under controlled environmental conditions. Physiological parameters (aerial part weight, main leaf length, and chlorophyll and total protein contents) were compared, and some differences were observed. Nevertheless, these differences were not constant among all GM and non-GM counterparts. Leaf proteomic profiles were analyzed using two-dimensional gel electrophoresis (2DE) coupled to mass spectrometry, using six 2DE gels per variety. The comparison between MON810 and its counterpart was limited to qualitative differences of fully reproducible protein spot patterns. Twelve exclusive proteins were observed in two of four maize variety pairs; all of these leaf proteins were variety specific. In this study, MON810 leaf proteomes of four varieties were similar to non-GM counterpart leaf proteomes.

Proteomic analysis of endometrium in fertile women during the prereceptive and receptive phases after luteinizing hormone surge

In fertile women the patterns of proteome during the prereceptive (day 2 after LH surge) and receptive (day 7 after LH surge) phases were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. A total of 31 differentially expressed proteins (17 up-regulated and 14 down-regulated) were identified between the two phases, which is helpful in understanding the biological mechanisms underlying human endometrial receptivity.

Protein extraction from the earthworm Eisenia fetida for 2‐DE

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2-DE. Sample preparation is a critical step in a 2-DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2-DE. The methods generated remarkably different 2-DE protein spot patterns. We conclude that trichloroacetic acid (TCA)-A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA-A gives good distinction, more bands in 1-DE gels, and the most number of protein spots in 2-DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2-DE gels, analyzed them using MALDI-TOF/TOF MS, and tentatively identified them. The classic TCA-A method proved to be most useful as a standard method of extracting proteins from E. fetida.

A Large Number of Protein Expression Changes Occur Early in Life and Precede Phenotype Onset in a Mouse Model for Huntington Disease

Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

Proteomic Analysis of Human Cervical-Vaginal Fluids

The pathophysiology of vaginal conditions is still ill-defined at a molecular level. Because the proteome of the human cervical-vaginal fluid (CVF) has not been reported to date, we undertook the identification of proteins present in the cell-free fraction of these fluids. Proteins were separated bidimensionally (2-D) by isoelectrofocusing (pH 3-11) followed by SDS-polyacrylamide electrophoresis. The proteins of 147 spots were identified by matrix-assisted laser desorption/ ionization-time-of-flight-mass spectrometry (MALDI-TOF/TOF). This approach was supplemented by immunoassays for markers of neutrophils (myeloperoxidase, MPO; neutrophil gelatinase-associated lipocalin, NGAL/HNL) and eosinophils (eosinophil cationic protein: ECP) and by immunoblotting (lactoferrin, calgranulins A and B and annexins A1 and A3. Nearly half of the proteins (69/147) and protein fragments detected were found to be plasma components, on the basis of which the human CVF can be broadly considered a plasma transudate. Although the pattern of protein spots was very similar for all fluids analyzed, a relative overabundance of major plasma proteins such as albumin, transferrin, immunoglobulins, apolipoproteins, alpha-1-acid glycoprotein 1, and calgranulins was associated with the presence of a high number of polymorphonuclear leukocytes in the lavages from which those cell-free fluids had been obtained. Instead, fluids from women experiencing vulvovaginal candidiasis did not show differences in the protein maps compared with asymptomatic individuals. Neutrophil and eosinophil granule secretion proteins were also detected in variable amounts in the lavage fluids by both immunoassay and immunoblotting, indicating polymorphonuclear cell activation.

Establishing a Blueprint for CED-3-dependent Killing through Identification of Multiple Substrates for This Protease

Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that approximately 3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts.

Identification of a Spiroplasma citri hydrophilic protein associated with insect transmissibility

With the aim of identifying Spiroplasma citri proteins involved in transmission by the leafhopper Circulifer haematoceps, protein maps of four transmissible and four non-transmissible strains were compared. Total cell lysates of strains were analysed by two-dimensional gel electrophoresis using commercially available immobilized pH gradients (IPGs) covering a pH range of 4-7. Approximately 530 protein spots were visualized by silver staining and the resulting protein spot patterns for the eight strains were found to be highly similar. However, comparison using PDQuest 2-D analysis software revealed two trains of protein spots that were present only in the four transmissible strains. Using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and a nearly complete S. citri protein database, established during the still-ongoing S. citri GII-3-3X genome project, the sequences of both proteins were deduced. One of these proteins was identified in the general databases as adhesion-related protein (P89) involved in the attachment of S. citri to gut cells of the insect vector. The second protein, with an apparent molecular mass of 32 kDa deduced from the electrophoretic mobility, could not be assigned to a known protein and was named P32. The P32-encoding gene (714 bp) was carried by a large plasmid of 35.3 kbp present in transmissible strains and missing in non-transmissible strains. PCR products with primers designed from the p32 gene were obtained only with genomic DNA isolated from transmissible strains. Therefore, P32 has a putative role in the transmission process and it could be considered as a marker for S. citri leafhopper transmissibility. Functional complementation of a non-transmissible strain with the p32 gene did not restore the transmissible phenotype, despite the expression of P32 in the complemented strain. Electron microscopic observations of salivary glands of leafhoppers infected with the complemented strain revealed a close contact between spiroplasmas and the plasmalemma of the insect cells. This further suggests that P32 protein contributes to the association of S. citri with host membranes.

Proteomic Analysis of Mitochondrial Protein Turnover: Identification of Novel Substrate Proteins of the Matrix Protease Pim1

ATP-dependent oligomeric proteases are major components of cellular protein quality control systems. To investigate the role of proteolytic processes in the maintenance of mitochondrial functions, we analyzed the dynamic behavior of the mitochondrial proteome of Saccharomyces cerevisiae by two-dimensional (2D) polyacrylamide gel electrophoresis. By a characterization of the influence of temperature on protein turnover in isolated mitochondria, we were able to define four groups of proteins showing a differential susceptibility to proteolysis. The protein Pim1/LON has been shown to be the main protease in the mitochondrial matrix responsible for the removal of damaged or nonnative proteins. To assess the substrate range of Pim1 under in vivo conditions, we performed a quantitative comparison of the 2D protein spot patterns between wild-type and pim1Delta mitochondria. We were able to identify a novel subset of mitochondrial proteins that are putative endogenous substrates of Pim1. Using an in organello degradation assay, we confirmed the Pim1-specific, ATP-dependent proteolysis of the newly identified substrate proteins. We could demonstrate that the functional integrity of the Pim1 substrate proteins, in particular, the presence of intact prosthetic groups, had a major influence on the susceptibility to proteolysis.

Proteomic Profiling of Accessory Structures from the Mouse Sperm Flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.

Systematic Evaluation of Sample Preparation Methods for Gel-Based Human Urinary Proteomics: Quantity, Quality, and Variability

We performed systematic evaluation of 38 protocols to concentrate normal human urinary proteins prior to 2D-PAGE analysis. Recovery yield and pattern of resolved protein spots were compared among different methods and intra-/inter-individual variabilities were examined. Precipitation with 90% ethanol provided the greatest protein recovery yield (92.99%), whereas precipitation with 10% acetic acid had the least protein recovery (1.91%). In most of precipitation protocols, the higher percentage of applied organic compounds provided the greater recovery yield. With a fixed concentration at 75%, the urine precipitated with acetonitrile had the greatest number of protein spots visualized in 2D gel, whereas the acetic-precipitated sample had the smallest number of spots. For the intra-individual variability, the first morning urine had the greatest amount of total protein but provided the smallest number of protein spots visualized. Excessive water drinking, not caffeine ingestion, caused alterations in the urinary proteome profile with newly presenting spots and also proteins with decreased excretion levels. As expected, there was a considerable degree of inter-individual variability. Coefficients of variation for albumin and transferrin expression were greatest by inter-individual variables. Male urine had greater amount of total protein but provided smaller number of protein spots compared to female urine. These data offer a wealth of useful information for designing a high-quality, large-scale human urine proteome project.

New Data Analysis and Mining Approaches Identify Unique Proteome and Transcriptome Markers of Susceptibility to Autoimmune Diabetes

Non-obese diabetic (NOD) mice spontaneously develop autoimmunity to the insulin producing beta cells leading to insulin-dependent diabetes. In this study we developed and used new data analysis and mining approaches on combined proteome and transcriptome (molecular phenotype) data to define pathways affected by abnormalities in peripheral leukocytes of young NOD female mice. Cells were collected before mice show signs of autoimmunity (age, 2-4 weeks). We extracted both protein and RNA from NOD and C57BL/6 control mice to conduct both proteome analysis by two-dimensional gel electrophoresis and transcriptome analysis on Affymetrix expression arrays. We developed a new approach to analyze the two-dimensional gel proteome data that included two-way analysis of variance, cluster analysis, and principal component analysis. Lists of differentially expressed proteins and transcripts were subjected to pathway analysis using a commercial service. From the list of 24 proteins differentially expressed between strains we identified two highly significant and interconnected networks centered around oncogenes (Myc and Mycn) and apoptosis-related genes (Bcl2 and Casp3). The 273 genes with significant strain differences in RNA expression levels created six interconnected networks with a significant over-representation of genes related to cancer, cell cycle, and cell death. They contained many of the same genes found in the proteome networks (including Myc and Mycn). The combination of the eight, highly significant networks created one large network of 272 genes of which 82 had differential expression between strains either at the protein or the RNA level. We conclude that new proteome data analysis strategies and combined information from proteome and transcriptome can enhance the insights gained from either type of data alone. The overall systems biology of prediabetic NOD mice points toward abnormalities in regulation of the opposing processes of cell renewal and cell death even before there are any clear signatures of immune system activation.