Sarka Beranova-Giorgianni

Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitations

Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics are the key components of current proteomics technology. The purpose of this review is to give an overview of the basic concepts of 2DE-based proteomics methodology, to discuss the advantages and limitations of the approach, and to highlight some recent advancements in 2DE-based technology.

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.

Phosphoproteomic analysis of the human pituitary

The pituitary is the central endocrine gland that regulates the functions of various target organs in the human body. Because of the pivotal regulatory role of the pituitary, it is essential to define on a global scale the components of the pituitary protein machinery, including a comprehensive characterization of the post-translational modifications of the pituitary proteins. Of particular interest is the examination of the phosphorylation status of the pituitary in health and disease. Towards the goal of global profiling of pituitary protein phosphorylation, we report here the application of the in-gel IEF-LC-MS/MS approach to the study of the pituitary phosphoproteome. The analytical strategy combined isoelectric focusing in immobilized pH gradient strips with immobilized metal ion affinity chromatography and mass spectrometry. With this method, a total of 50 phosphorylation sites were characterized in 26 proteins. Because the investigation involved primary tissue, the findings provide a direct glimpse into the phosphoprotein machinery operating within the human pituitary tissue microenvironment.

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A1 (PLA1) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA1. Addition of this recombinant PLA1 significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA1; 2) PLA1 generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.

Effects of Muramyl Peptides on Macrophages, Monokines, and Sleep

Muramyl peptides are fragments of peptidoglycan from the cell walls of bacteria. Because of their unique chemistry, the immune system recognizes that muramyl peptides are products of bacteria, and it responds by becoming activated to resist infection. This resistance to infection is nonspecific, and extends to unrelated species of bacteria, fungi, and viruses. A key mechanism of the resistance to infection is activation of macrophages. Macrophage activation results in increased production of microbicidal oxygen radicals like superoxide and peroxide, and in increased secretion of inflammatory cytokines like interleukin-1β and tumor necrosis factor-α. These cytokines, besides activating neutrophils, B lymphocytes, and T lymphocytes, act on the central nervous system to induce physiological responses like fever and sleep. These physiological responses also aid in combating infection. Muramyl peptides also activate macrophages and other cells of the immune system to kill cancer cells. Muramyl peptides and similar agents will become more important as therapeutic agents in the future, due to increasing resistance of microbes to antibiotics, and increasing numbers of patients with immunodeficiencies.

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.

Mass spectrometry of the human pituitary proteome: identification of selected proteins

The field of proteomics involves the combined application of advanced separation techniques, mass spectrometry, and bioinformatics tools to characterize proteins in complex biological mixtures. Here we report the identification of nine proteins from the human pituitary proteome, using the proteomics approach. The pituitary proteins were separated by two-dimensional electrophoresis, and were visualized by silver staining. The proteins of interest were subjected to in-gel digestion with trypsin, and the masses of the resulting peptides were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This tryptic mass map was used to identify the proteins through a search of a protein-sequence database. The identified proteins include important hormones, and enzymes with various catalytic activities. These proteins will be used to construct a two-dimensional reference database of the human pituitary. This database will be employed to study changes in the pituitary proteome that are associated with the formation of pituitary tumors.

Proteome of monocyte priming by lipopolysaccharide, including changes in interleukin-1beta and leukocyte elastase inhibitor

BackgroundMonocytes can be primed in vitro by lipopolysaccharide (LPS) for release of cytokines, for enhanced killing of cancer cells, and for enhanced release of microbicidal oxygen radicals like superoxide and peroxide. We investigated the proteins involved in regulating priming, using 2D gel proteomics.ResultsMonocytes from 4 normal donors were cultured for 16 h in chemically defined medium in Teflon bags ± LPS and ± 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. LPS-primed monocytes released inflammatory cytokines, and produced increased amounts of superoxide. AEBSF blocked priming for enhanced superoxide, but did not affect cytokine release, showing that AEBSF was not toxic. After staining large-format 2D gels with Sypro ruby, we compared the monocyte proteome under the four conditions for each donor. We found 30 protein spots that differed significantly in response to LPS or AEBSF, and these proteins were identified by ion trap mass spectrometry.ConclusionWe identified 19 separate proteins that changed in response to LPS or AEBSF, including ATP synthase, coagulation factor XIII, ferritin, coronin, HN ribonuclear proteins, integrin alpha IIb, pyruvate kinase, ras suppressor protein, superoxide dismutase, transketolase, tropomyosin, vimentin, and others. Interestingly, in response to LPS, precursor proteins for interleukin-1β appeared; and in response to AEBSF, there was an increase in elastase inhibitor. The increase in elastase inhibitor provides support for our hypothesis that priming requires an endogenous serine protease.

Characterization of the phosphoproteome in LNCaP prostate cancer cells by in-gel isoelectric focusing and tandem mass spectrometry

Reversible protein phosphorylation forms the basis of cell signaling networks. Aberrations in protein phosphorylation have been linked to human diseases including cancer. Phosphoproteomics has recently emerged as an approach that focuses on analysis of protein phosphorylation on a global scale. We have recently developed a new methodology, termed in-gel IEF LC-MS/MS, and we have adapted this methodology for phosphoproteome analysis. Here, we report on the application of in-gel IEF LC-MS/MS to the mapping of the phosphoproteome in the LNCaP human prostate cancer cell line. The analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing (IEF), digestion of the proteins with trypsin, enrichment of the digests for phosphopeptides with Immobilized Metal Ion Affinity Chromatography (IMAC), analysis of the enriched digests by LC-MS/MS, and identification of the phosphorylated peptides/proteins through searches of a protein sequence database. With this analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins. This panel of the LNCaP phosphoproteins is 3-fold larger than the panel obtained in our previous work, which attests to the power of the chosen analytical methodology. The characterized phosphoproteins are functionally diverse and include a number of proteins relevant to cancer.

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis.

Background We investigated sera from elderly subjects with and without age-related macular degeneration (AMD) for presence of autoantibodies (AAbs) against human macular antigens and characterized their identity. Methods Sera were collected from participants in the Age-Related Maculopathy Ancillary (ARMA) Study, a cross-sectional investigation ancillary to the Health ABC Study, enriched with participants from the general population. The resulting sample (mean age: 79.2±3.9 years old) included subjects with early to advanced AMD (n = 131) and controls (n = 231). Sera were tested by Western blots for immunoreactive bands against human donor macular tissue homogenates. Immunoreactive bands were identified and graded, and odds ratios (OR) calculated. Based on these findings, sera were immunoprecipitated, and subjected to 2D gel electrophoresis (GE). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities. Results In AMD, 11 immunoreactive bands were significantly more frequent and 13 were significantly stronger than in controls. Nine of the more frequent bands also showed stronger reactivity. OR estimates ranged between 4.06 and 1.93, and all clearly excluded the null value. Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin. ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls. Conclusions Consistent with other evidence supporting the role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression.