Biomedical subjects

Dominic M Desiderio

Publications and source records attributed to Dominic M Desiderio.

32 records · Page 2Linked to original sources

PubMed2003

Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry.

Over the past several years, a large effort has been focused on improvements of two-dimensional (2-D) gel electrophoresis-based proteomics technology, and on development of novel approaches for proteome analysis. Here, we describe the application of an alternative strategy for the analysis of complex proteomes. The strategy combines isoelectric focusing in immobilized pH gradient strips (in-gel IEF), mass spectrometry (MS), and bioinformatics. A protein mixture is separated by in-gel IEF, and the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the tryptic peptides are subjected to liquid chromatography-nanoelectrospray-quadrupole ion-trap tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS data are used to identify the proteins through searches of a protein sequence database. Using this in-gel IEF-LC-MS/MS strategy, we have identified 127 proteins from a human pituitary. This study demonstrates the potential of the in-gel IEF-LC-MS/MS approach for analyses of complex mammalian proteomes.

Computational Biology↗

PubMed2003

A reference map of a human pituitary adenoma proteome.

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.

Adenoma↗

PubMed2003

Between-gel reproducibility of the human cerebrospinal fluid proteome.

This manuscript describes the between-gel reproducibility of the two-dimensional gel electrophoresis analysis of the human lumbar cerebrospinal fluid (CSF) proteome. This reproducibility study is a necessary component for our long-term research program that uses comparative proteomics to analyze lumbar CSF samples in a study of human idiopathic low back pain. A Protein-Plus Dodeca Cell electrophoresis apparatus and PDQuest software were used to measure the level of between-gel reproducibility of the CSF proteome. One pooled CSF sample was used to evaluate the level of within-sample, between-gel reproducibility, and a set of seven different CSF samples (CSF-1 to 7) was used to test the level of within-group and between-group variability. Differentially expressed proteins (six CSF samples versus the designated control, CSF-3) were characterized with mass spectrometry. The number of spots found in the pooled CSF sample was 490 +/- 30 (n = 10 gels); the percentage of protein spots found in those 10 gels was 92 +/- 6%, with a coefficient of variation of 6%; and a positive coefficient of correlation (r = 0.82) was found. In order to test the proof-of-principle, that set of seven CSF samples served as a test of our ability to perform reproducibility comparative proteomics, and to detect differentially expressed proteins within that set of test samples. One sample (CSF-3) served as the control for the other six to locate the differentially expressed proteins. A comparison of fifteen differentially expressed proteins found in that set of test CSF samples correlated with pathology. Matrix-assisted laser desorption/ionization-time-of-flight and electrospray ionization quadrupole ion trap mass spectrometry were used to characterize thirteen of those fifteen differentially expressed proteins. These results (reproducibility, protein characterization, set of test samples, and proof-of-principle) suggest that the analysis of human CSF two-dimensional gels can achieve a high level of within-sample and between-sample reproducibility, and that PDQuest software can measure the relative protein abundance in the human CSF proteome.

Adult↗

PubMed2003

Proteome of monocytes primed with lipopolysaccharide: analysis of the abundant proteins.

We performed a proteomic analysis of monocytes primed by lipopolysaccharide (LPS) in vitro, using two-dimensional gels stained with Coomassie blue. We found 16 proteins of approximately 500 detected that either increased or decreased in abundance as a result of priming by LPS (14 with P </= 0.05). The proteins were identified by comparing the masses of their tryptic peptides with those of all known proteins, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the SWISS-PROT database. Identities were confirmed by matching the sequence of several tryptic peptides, using liquid chromatography electrospray-ionization quadrupole ion trap mass spectrometry. There were increases in the protective enzymes superoxide dismutase and catalase, in four calgranulins, in the cytokine pre-B cell enhancing factor, and in annexin 2, macrophage capping protein, transketolase, pyruvate kinase, and serine/threonine protein kinase 10. Proteins that decreased in abundance were integrin alpha-IIB, protein disulfide isomerase, and platelet-activating factor acetylhydrolase. Many of these altered proteins have interesting functions in inflammation.

Amino Acid Sequence↗

PubMed2003

Quantification of [Dmt1]DALDA in ovine plasma by on-line liquid chromatography/quadrupole time-of-flight mass spectrometry.

The synthetic peptide [Dmt(1)]DALDA (Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine; 'super-DALDA') is a mu opioid-receptor agonist. On-line liquid chromatography/quadrupole time-of-flight mass spectrometry and the corresponding stable isotope-incorporated synthetic peptide internal standard were used to quantify [Dmt(1)]DALDA that had been extracted from ovine plasma samples. The [M+2H](2+) ion was used to construct the calibration curve, and the product ion was used for verification of the peptide. The detection sensitivity for the [Dmt(1)]DALDA [M+2H](2+) ion was 12.5 fmol and 50 fmol for the m/z 432.3 product ion. The concentration profile of [Dmt(1)]DALDA was determined from a set of ovine plasma samples. The molecular specificity of the peptide quantification was confirmed by tandem mass spectrometry (MS/MS).

Amino Acid Sequence↗

PubMed2003

Proteomics and transcriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.

In order to explore the presence of, and the potential role of, secretagogin in human pituitary adenomas, an analytical strategy that integrated comparative proteomics and comparative transcriptomics was used to detect the protein and the mRNA expression, respectively, of secretagogin in human non-functional pituitary adenomas compared to controls. Proteomics methods included two-dimensional gel electrophoresis, 2D gel image analysis, mass spectrometry [matrix-assisted laser desorption/ionization-time of flight-peptide mass fingerprinting (MALDI-TOF PMF) and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry (LC-ESI-Q-IT MS/MS)], and database analysis. Transcriptomics methods included the GeneChip microarray, image processing, and data analysis. The proteomics and transcriptomics data demonstrated that secretagogin was significantly down-regulated at the protein and mRNA levels, respectively, in the human non-functional (NF) pituitary adenomas (NF-, LH+, FSH+, and FSH+ + LH+). For the secretagogin protein, the expression level was NF- < FSH+ + LH+ < FSH+ < LH+ < Control, with a range of down-regulation of 2.2-6.9 fold in non-functional pituitary adenomas compared to controls, with a significant difference (p < 0.001). For secretagogin mRNA, the expression level was NF- < LH+ < FSH+ + LH+ < FSH+ < Control, with a range of down-regulation of 1.8-18.6 fold in non-functional pituitary adenomas compared to controls that was significant (p < 0.05). The secretagogin protein expression correlated significantly with its mRNA expression. Those results suggest that secretagogin might play a role in human non-functional pituitary adenomas. This novel finding may provide clues to clarify the basic molecular mechanisms of pituitary adenoma formation, and to identify new tumor-related markers.

Adenoma↗

PubMed2003

Heterogeneity analysis of the human pituitary proteome.

BACKGROUND: A human proteome is relatively dynamic compared with its corresponding genome. Our aim was to study the heterogeneity of a human pituitary proteome as a function of gender, age, and race. METHODS: Pituitary control tissues (n = 8) were used to extract proteins; each control tissue was analyzed (n = 3-5) with two-dimensional gel electrophoresis (2DGE) and PDQuest software. We obtained 30 high-resolution 2DGE gels and conducted a comparative analysis as a function of gender, age, and race. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry were used to characterize the protein in each differential spot. RESULTS: We detected approximately 1000 protein spots in each 2DGE map, and 51 differential spots (7 differing with gender, 17 with age, 15 with race, and 12 with the coeffect of age and race). Among those 51, we characterized 28 proteins [5 differing with gender, 8 with age, 6 with race, 8 with the coeffect of age and race, and 1 (somatotropin chain 1) with all of these]. Somatotropin was related to gender, age, and race, and prolactin was higher in females than males. The differentially expressed proteins that were related to age were mainly those proteins associated with cell growth, proliferation, differentiation, apoptosis, and death; those proteins showed no difference with gender and race. Age and race affected some proteins associated with hormone regulation (e.g., follistatin, thyroid hormone receptor beta-2, adenylate cyclase-inhibiting Galpha protein). CONCLUSIONS: A heterogeneity exists in the human pituitary proteome as a function of gender, age, and race. These findings will serve as a basis for our comparative proteomics studies of human pituitary adenomas.

Adult↗

PubMed2002

Downregulation of a unique photoreceptor protein correlates with improper outer segment assembly.

A unique photoreceptor protein has been characterized. This protein, termed XAP-1 antigen, is expressed by photoreceptors exclusively under conditions in which the outer segment membranes are properly assembled. When the retinal pigment epithelium is adherent to the underlying neural retina, the XAP-1 antigen is localized to the plasma membrane that surrounds the inner and outer segments in the areas juxtaposed to the subretinal space. A similar labeling pattern is detected in retinal pigment epithelium-deprived retinas in which assembly of nascent outer segments is supported by lactose. In retinas that undergo degeneration subsequent to the removal of the retinal pigment epithelium, the expression of this protein is completely downregulated. Immunohistochemical analyses and subcellular fractionation along with Western blot analysis, indicate that the XAP-1 antigen is a membrane-associated soluble protein. Mass spectrometric analysis indicates that the XAP-1 antigen shares homology via 12 tryptic peptide masses with the gamma-crystallin (lens structural protein) subclasses, although it does not immunolocalize to the same ocular structures as reported for the gamma-crystallins. We propose that XAP-1 antigen is a unique protein that is expressed extensively by healthy photoreceptor cells; the expression of the XAP-1 antigen exclusively by photoreceptors with organized outer segments suggests that this protein may play a critical role in outer segment assembly.

Animals↗

PubMed2002

Preliminary analysis of the mouse cerebellum proteome.

This paper reports on the initial analysis of protein expression in the mouse cerebellum with the proteomics approach. Proteins from cerebellar tissue homogenates were separated by two-dimensional gel electrophoresis, and the proteins were stained with colloidal Coomassie Blue to produce a high-resolution map of the cerebellum proteome. Selected proteins from this map were digested with trypsin, and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray quadrupole ion trap mass spectrometry. The mass spectrometric data were used to identify the proteins through searches of the SWISSPROT protein sequence database. To date, 30 prominent proteins with various functional characteristics were identified. These data will be used for future studies of differential protein expression in mouse models of neurological disorders.

Animals↗

PubMed2002

Analysis of the human lumbar cerebrospinal fluid proteome.

Idiopathic low back pain has no known cause, and the molecular basis is unknown. Neuropeptidergic systems have been previously studied, and proteomics methods have been applied in this present study. Proteomics combines high-resolution two-dimensional (2-D) gel electrophoresis, high-sensitivity mass spectrometry, and continuously expanding protein databases. Proteomics offers a comprehensive, bird's-eye view to analyze, at a systems level, all of the proteins in cerebrospinal fluid (CSF) that might contribute to idiopathic low back pain. CSF contains a high salt concentration and low protein concentration. In order to obtain a high-quality 2-D pattern, several sample preparation methods were tested to remove salts - protein precipitation with either acetone or trichloroacetic acid/acetone, or sample treatment with a Bio-Spin column. More spots were visualized on the 2-D gel of human CSF, and a relatively high protein recovery was obtained when a Bio-Spin column was used to process a human CSF sample. Sixty-one protein spots, obtained from 2-D gels with a pH range of either 3-10 or 4-7, were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-post-source decay (PSD)-MS. These 61 protein spots represent 22 proteins; six of those proteins were not annotated in any previously published 2-D maps. Those six proteins are PRO2619, pigment epithelium-derived factor, albumin homolog, kallikrein-6 precursor, DJ717I23.1, and AMBP protein precursor. These protein-mapping data will contribute to the database that will be used in the future to compare the proteomes obtained from the CSF of controls and low back pain patients, to characterize differentially expressed proteins, and to elucidate the biological markers for idiopathic low back pain.

Amino Acid Sequence↗

PubMed2002

Analysis of the proteome in the human pituitary.

The pituitary is the master endocrine gland responsible for the regulation of various physiologic and metabolic processes. Proteomics offers an efficient means for a comprehensive analysis of pituitary protein expression. This paper reports on the application of proteomics for the mapping of major proteins in a normal (control) pituitary. Pituitary proteins were separated by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient strips. Major protein spots that were visualized in the two-dimensional gel by silver staining were excised, and the proteins in these spots were digested with trypsin. The tryptic digests were analyzed by mass spectrometry, and the mass spectrometric data were used to identify the proteins through searches of the SWISS-PROT or NCBInr protein sequence databases. The majority of the proteins were identified on the basis of peptide mass fingerprinting data obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Several proteins were also characterized based on product-ion spectra measured by post-source decay analysis and/or liquid chromatography-electrospray-quadrupole ion trap mass spectrometry. To date, 62 prominent protein spots, corresponding to 38 different proteins, were identified. The identified proteins include important pituitary hormones, structural proteins, enzymes, and other proteins. The protein identification data were used to establish a two-dimensional reference database of the human pituitary, which can be accessed over the Internet (http://www.utmem.edu/proteomics). This database will serve as a tool for further proteomics studies of pituitary protein expression in health and disease.

Computational Biology↗

PubMed2002

Protein identification with Teflon as matrix-assisted laser desorption/ionization sample support.

Protein identification is a critical step in proteomics, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) plays an important role in that identification. Polytetrafluoroethylene (Teflon) was tested as a new MALDI sample support to improve protein identification. The tryptic peptides obtained from a model protein were bound to the surface of a modified MALDI sample holder via the hydrophobic interactions that occur between the Teflon surface and the peptide ion-pairs, and the affinity of alpha-cyano-4-hydroxycinnamic acid for the peptides. During that surface-binding step, the peptide mixture was also desalted and concentrated. A greater number of matched peptides and a larger sequence coverage were obtained for the proteins when Teflon was used as the sample support compared with conventional sample preparation methods and a stainless-steel surface. In addition, the characterization of a small amount of protein was improved with Teflon. Nine silver-stained protein spots obtained from 2-D gel of a human cerebrospinal fluid (CSF) proteome were identified by this method. Among the nine protein spots, peptide 6:c3c fragment and procollagen c-proteinase enhancer were not annotated in any published 2-D map of human CSF. A Teflon MALDI sample support is a low-cost, simple, and effective method that can be used to improve the quality of the MALDI mass spectrum of a complex tryptic peptide mixture, and to achieve a higher level of reliability and success in protein identification.

Animals↗

PubMedDate not supplied

Comparative proteomics analysis of human pituitary adenomas: current status and future perspectives.

This article will review the published research on the elucidation of the mechanisms of pituitary adenoma formation. Mass spectrometry (MS) plays a key role in those studies. Comparative proteomics has been used with the long-term goal to locate, detect, and characterize the differentially expressed proteins (DEPs) in human pituitary adenomas; to identify tumor-related and -specific biomarkers; and to clarify the basic molecular mechanisms of pituitary adenoma formation. The methodology used for comparative proteomics, the current status of human pituitary proteomics studies, and future perspectives are reviewed. The methodologies that are used in comparative proteomics studies of human pituitary adenomas are readily exportable to other different areas of cancer research.

Adenoma↗

PubMedDate not supplied

Proteomics analysis of phosphotyrosyl-proteins in human lumbar cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a secretion product of several different central nervous system (CNS) structures, including the choroid plexus in the ventricles. Pathological CNS processes are reflected in the protein composition of CSF. To elucidate the molecular events that occur in the homeostatic and pathological processes of the CNS, the high-throughput characterization of differentially expressed proteins, and of post-translationally modified proteins, is needed for proteomics studies of CSF. Among the post-translational modifications of proteins, phosphorylation is the most common and important mechanism for the reversible regulation of protein function. In this study, CSF phosphotyrosyl (p-Tyr)-proteins were detected with antibodies and were analyzed with proteomics methods. Three different combination methods--1D gel electrophoresis and Western blotting, immunoprecipitation and 2D gel electrophoresis, and 2D gel electrophoresis and Western blotting--were used to detect p-Tyr-proteins in human lumbar CSF samples. Six protein spots, representing four proteins on a 2D Western blot, were identified as p-Tyr-proteins with the 2D gel electrophoresis and Western blotting method. Those four p-Tyr-proteins are kallikrein-6 precursor, complement C4 gamma-chain, gelsolin, and ceruloplasmin precursor. Additionally, four other nonphosphorylated CSF proteins--beta-2-glycoprotein I precursor, fibulin-1 precursor, EGF-containing fibulin-like extracellur matrix protein 1 precursor, and angiotensinogen precursor--were characterized for the first time.

Blotting, Western↗