Biomedical subjects

Dominic M Desiderio

Publications and source records attributed to Dominic M Desiderio.

At least 19 recordsLinked to original sources

PubMed2005

Stabilization of ubiquitous mitochondrial creatine kinase preprotein by APP family proteins.

Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, the physiological role of APP and its family members is still unclear. To gain insights into APP function, we used a proteomic approach to identify APP interacting proteins. We report here for the first time a direct interaction between the C-terminal region of APP family proteins and ubiquitous mitochondrial creatine kinase (uMtCK). This interaction was confirmed in vitro as well as in cultured cells and in brain. Interestingly, expression of full-length and C-terminal domain of APP family proteins stabilized uMtCK preprotein in cultured cells. Our data suggest that APP may regulate cellular energy levels and mitochondrial function via a direct interaction and stabilization of uMtCK.

Amino Acid Sequence↗

PubMed2005

Toward a global analysis of the human pituitary proteome by multiple gel-based technology.

We describe a comprehensive method for the analysis of complex proteomes, multiple gel-based technology (MGT), which combines in-gel IEF-LC-MS/MS and SDS-PAGE-LC-MS/MS strategies. MGT was applied to the analysis of the proteome of human pituitary tissue. A total of 1449 proteins were uniquely identified. To our knowledge, this is the most comprehensive proteomic analysis of human pituitary tissue to date. Categorization of the identified proteins revealed that MGT provides an excellent and largely unbiased access to proteins with diverse characteristics, including low-abundance proteins, membrane proteins, and proteins with extremes in pI and MW.

Adult↗

PubMed2005

Proteomics analysis of human cerebrospinal fluid.

Cerebrospinal fluid (CSF) is secreted from several different central nervous system (CNS) structures, and any changes in the CSF composition will accurately reflect pathological processes. Proteomics offers a comprehensive bird's eye view to analyze CSF proteins at a systems level. This paper reviews the variety of analytical methods that have been used for proteomics analysis of CSF, including sample preparation, two-dimensional liquid and gel electrophoresis, mass spectrometry, bioinformatics, and non-gel methods. The differentially expressed CSF proteins that have been identified by proteomics methods are discussed.

Cerebrospinal Fluid Proteins↗

PubMed2005

Human cerebrospinal fluid peptidomics.

Cerebrospinal fluid (CSF) has frequently been extensively studied to explore several different central nervous system (CNS) disorders because it contains proteins, enzymes, hormones, neuropeptides and neurotransmitters that play critical regulatory roles in many different physiological processes. Individual neuropeptidergic systems in CSF have been studied. In theory, peptidomics offers a bird's-eye, comprehensive and systems-level approach to analyze all of the peptidergic systems that have been expressed in CSF at any given time. In this study, low molecular mass (M(r) < 5 kDa) peptides were isolated by ultrafiltration. The isolated peptides, with or without trypsin digestion, were preferentially enriched with a solid-phase extraction cartridge, and the peptides were separated with capillary liquid chromatography and analyzed with on-line quadrupole time-of-flight mass spectrometry (MS). In this proof-of-principle study, the 20 representative MS-characterized peptides were shown to be derived from 12 proteins, among which four proteins, amyloid-like protein 1, secretogranin I, granin-like neuroendocrine peptide precursor and neurosecretory protein VGF, have been shown elsewhere to be either associated with CNS disorders or to play a central role in the CNS. The long-term goals of this peptidomics study are to monitor the changes (amount; modifications) of CSF peptides, clarify the aberrant processing of large intact protein precursors, elucidate the molecular mechanisms of CNS disorders and find biomarkers. This analytical method is effective for the analysis of the human lumbar CSF peptidome.

Adult↗

PubMed2005

Proteomics analysis of prefractionated human lumbar cerebrospinal fluid.

Cerebrospinal fluid (CSF) is produced by the chorioid plexus in the ventricles. It surrounds the brain and bone marrow, and reflects several different disorders of the central nervous system (CNS). Proteomics has been used to analyze CSF in order to discover disease-associated proteins and to elucidate the basic molecular mechanisms that either cause, or result from, CNS disorders. However, some disease-associated proteins are of low-abundance and are difficult to detect. A low total-protein concentration, a high amount of albumin and immunoglobins, and a wide dynamic range (several orders of magnitude) of protein concentration cause several difficulties in the identification of low-abundance CSF proteins. In this study, advantage was taken of the range of different hydrophobic properties of CSF proteins, and a reversed-phase solid-phase extraction (SPE) cartridge was used to prefractionate human lumbar CSF proteins into three separate fractions prior to two-dimensional gel electrophoresis resolution of the proteome. A portion of the high-abundance CSF proteins were removed from two (eluted with 35% and 50% acetonitrile) of the three fractions. Some trace CSF proteins were preferentially enriched in the two fractions, and many proteins were detected in the two-dimensional (2-D) gels of the two fractions. Among the novel proteins identified, sixty-two protein spots that represent forty-two proteins were characterized. Most of the proteins have not been annotated in any previous 2-D map of human CSF, and several have been implicated in CNS diseases. The prefractionation of CSF proteins with SPE, followed by proteomics analysis, provides a new method to explore low-abundance, disease-specific CSF proteins.

Cerebrospinal Fluid Proteins↗

PubMed2005

Proteomics analysis of growth hormone isoforms in the human pituitary.

In order to elucidate the roles of human growth hormone (hGH) in the normal (control) pituitary and in adenomas, the hGH isoforms in the human pituitary were analyzed with two-dimensional gel electrophoresis, immobilized metal affinity column (Ga(+3)) chromatography, mass spectrometry (MS), and bioinformatics. Twenty-four hGH-containing proteins, with significantly different expression proportions of their isoforms were found. The proportions of isoforms were as follows: isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%). Deamidation of asparagine to aspartate was identified with matrix-assisted laser desorption/ionization-time of flight MS. Tandem mass spectrometry data demonstrated that hGH is a phosphoprotein (spot 6); phosphorylation was found at Ser-77 in the tryptic peptide (68)YSFLQNPQTSLCFSESIPTPSNR(90), at Ser-176 in the tryptic peptide (172)FDTNSHNDDALLK(184), and at Ser-132 in the peptide (126)SLVYGASDSNVYDLLK(141). The phosphorylation sites at Ser-77 and Ser-176 were consistent with computer-program predictions (NetPhos). These results provide novel clues for further studies of the functions, and mechanisms of action, of hGH in the human pituitary and in growth hormone-related diseases.

Alternative Splicing↗

PubMed2005

Two-dimensional gel electrophoresis characterization of the mouse leukocyte proteome, using a tri-reagent for protein extraction.

The use of "tri-reagents" allows the concomitant isolation of DNA, RNA, and proteins for a complete molecular characterization of biological samples. The aim of the current study was to perform the comparative evaluation of two-dimensional gel proteomes isolated with or without a tri-reagent protein extraction step before dissolving the samples in the first-dimension electrophoresis buffer. We conclude that the use of tri-reagents increases the amount of protein extracted from the sample. Furthermore, an average of 301 +/- 3.6 spots were found in gels from both sample preparation methods, whereas 71.7 +/- 8.1 and 49.7 +/- 2.3 spots were uniquely seen in tri-reagent and non-tri-reagent samples, respectively.

Animals↗

PubMed2004

The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry.

Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched to four different proteins: synaptosomal-associated protein, actin, immunoglobulin alpha Fc receptor, and cGMP-dependent protein kinase 2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility.

Amino Acid Sequence↗

PubMed2004

LC-MS/MS analysis of peptides with methanol as organic modifier: improved limits of detection.

With the advent of soft ionization methods such as MALDI and ESI, mass spectrometry has become the most important technique for the analysis of proteins and peptides. ESI-MS is often preceded by separation of the peptide sample by reversed-phase liquid chromatography (LC). Acetonitrile (ACN) is the most commonly employed organic solvent in LC-ESI-MS analysis of peptides. In this report, we demonstrate that the use of methanol (MeOH) as the organic modifier improves the detection limits for analysis of peptide mixtures such as those found in tryptic digests of proteins. A nanoLC-ESI-quadrupole ion trap instrument (LCQ Deca, ThermoFinnigan) was used to analyze peptide standards, protein digests of known concentrations, and tryptic digests of 2-DGE-separated proteins. MeOH displayed excellent chromatographic performance (separation and sensitivity), and shorter gradient times were possible for chromatographic separation with MeOH versus ACN. Sensitivity levels of a few hundred attomoles were achieved with MeOH; those levels could not be achieved with ACN. In addition, MeOH-based nanoLC-MS/MS yielded superior results for the analysis of digests of 2-DGE-separated proteins. For the 14 protein spots analyzed, the success rate of protein identification with MeOH-based nanoLC-ESI-MS/MS was 100%, with multiple proteins identified in several of the spots. In contrast, ACN-based procedure failed to identify any proteins in 21% of the spots and overall identified 33% fewer proteins than the MeOH-based procedure. In summary, higher sensitivity and shorter gradient times make MeOH an excellent organic modifier for the use in nanoLC-ESI-MS/MS analysis of peptides.

Chromatography, Liquid↗

PubMed2004

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry.

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.

Animals↗

PubMed2004

Identification and characterization of phosphorylated proteins in the human pituitary.

Post-translational modifications of proteins from the human pituitary gland play an important role in the regulation of different body functions. We report on the application of a liquid chromatography-tandem mass spectrometry (MS/MS) based approach to detect and characterize phosphorylated proteins in a whole human pituitary digest. By combining an immobilized metal affinity column-based enrichment method with MS/MS conditions that favor the neutral loss of phosphoric acid from a phosphorylated precursor ion, we identified several previously undescribed phosphorylated peptides. The identified peptides were matched to the sequences of six pituitary proteins: the human growth hormone, chromogranin A, secretogranin I, 60S ribosomal protein P1 and/or P2, DnaJ homolog subfamily C member 5, and galanin. The phosphorylation sites of these important regulatory proteins were determined by MS/MS and MS(3) analysis.

Amino Acid Sequence↗

PubMed2004

Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests.

Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.

Mass Spectrometry↗

PubMed2003

Spot volume vs. amount of protein loaded onto a gel: a detailed, statistical comparison of two gel electrophoresis systems.

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome.

Electronic Data Processing↗

PubMed2003

Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systems.

Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume.

Electrophoresis, Gel, Two-Dimensional↗

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↗