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Randy J Arnold

Publications and source records attributed to Randy J Arnold.

10 recordsLinked to original sources
PubMed2006

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker.

Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase delta. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer.

Antibodies↗
PubMed2006

A computational approach toward label-free protein quantification using predicted peptide detectability.

We propose here a new concept of peptide detectability which could be an important factor in explaining the relationship between a protein's quantity and the peptides identified from it in a high-throughput proteomics experiment. We define peptide detectability as the probability of observing a peptide in a standard sample analyzed by a standard proteomics routine and argue that it is an intrinsic property of the peptide sequence and neighboring regions in the parent protein. To test this hypothesis we first used publicly available data and data from our own synthetic samples in which quantities of model proteins were controlled. We then applied machine learning approaches to demonstrate that peptide detectability can be predicted from its sequence and the neighboring regions in the parent protein with satisfactory accuracy. The utility of this approach for protein quantification is demonstrated by peptides with higher detectability generally being identified at lower concentrations over those with lower detectability in the synthetic protein mixtures. These results establish a direct link between protein concentration and peptide detectability. We show that for each protein there exists a level of peptide detectability above which peptides are detected and below which peptides are not detected in an experiment. We call this level the minimum acceptable detectability for identified peptides (MDIP) which can be calibrated to predict protein concentration. Triplicate analysis of a biological sample showed that these MDIP values are consistent among the three data sets.

Algorithms↗
PubMed2006

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach.

This study compares the total liver proteome of inbred alcohol-preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three-step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2-DE. Scanned gels of two sample groups, alcohol-exposed iP and alcohol-naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC-MS/MS. Twenty-four individual rats, 12 alcohol-naïve, and 12 alcohol-exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid beta-oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.

Alcoholism↗
PubMed2006

The discovery of labile methyl esters on proliferating cell nuclear antigen by MS/MS.

The post-translational modification of proliferating cell nuclear antigen (PCNA) has been implicated in modulating its function for over 20 years. With multiple interacting partners, PCNA is involved in processes ranging from DNA replication and repair to cell cycle control and apoptosis. The ability of PCNA to distinguish between specific binding partners in different tasks is currently of intense interest, and several post-translational modifications have been reported to modulate its function. Unfortunately, these reports have produced contradictory information on the type(s) of modification present on the molecule. Here we report a detailed structural analysis of a single acidic PCNA isoform, cancer-specific polyferating nuclear anitgen (csPCNA), isolated from breast cancer cells by 2D-PAGE and LC-MS/MS. With this approach we fully characterized the csPCNA isoform and confidently identified a single post-translational modification, methyl esterification. Interestingly, the methyl esters consistently localized to 15 specific glutamic and aspartic acid residues of csPCNA. The methyl esterification of csPCNA represents a novel type of post-translational modification in mammalian cells that could ultimately hold the key towards unlocking its diverse functions.

Amino Acid Sequence↗
PubMed2006

A machine learning approach to predicting peptide fragmentation spectra.

Accurate peptide identification from tandem mass spectrometry experiments is the cornerstone of proteomics. Although various approaches for matching database sequences with experimental spectra have been developed to date (e.g. Sequest, Mascot) the sensitivity and specificity of peptide identification have not yet reached their full potential. This is in part due to the tradeoffs between robustness and accuracy of the existing methods with respect to the non-uniform nature of peptide fragmentation and bond cleavages induced by different mass spectrometers. Accordingly, it is expected that new approaches to de novo predicting peptide fragmentation spectra will enable more accurate peptide identification. To address this problem, here we used a data-driven approach to learn peptide fragmentation rules in mass spectrometry, in the form of posterior probabilities, for various fragment-ion types of doubly and triply charged precursor ions. We show that the accuracy of our neural-network based methodology is useful for subsequent peptide database searches and that the most useful rules of fragmentation significantly differ across ion and precursor types.

Amino Acids↗
PubMed2005

Modulation of differentiation-related gene 1 expression by cell cycle blocker mimosine, revealed by proteomic analysis.

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Amino Acid Sequence↗
PubMed2005

A proteomic survey of rat cerebral cortical synaptosomes.

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

Animals↗
PubMedDate not supplied

Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver.

The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches.

Animals↗
PubMedDate not supplied

Mapping the proteome of Drosophila melanogaster: analysis of embryos and adult heads by LC-IMS-MS methods.

Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins. In total, 780 are identified in the head, and 660 are identified in the embryo. Only 307 proteins are in common to both developmental stages, indicating that there are significant differences in these proteomes. A comparison of the proteome to a database of mRNAs that are found from analysis by cDNA approaches (i.e., transcriptome) also shows little overlap. All of this information is discussed in terms of the relationship between the predicted genome, and measured transcriptomes and proteomes. Additionally, the merits and weaknesses of current technologies are assessed in some detail.

Animals↗