Identification of proteins in laser-microdissected small cell numbers by SELDI-TOF and Tandem MS

BACKGROUND: Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques. RESULTS: Hemalaun stained mouse lung sections were used to isolate 500–2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip(® )Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip(® )Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks. CONCLUSION: Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization

The Human Urine Metabolome

Urine has long been a “favored” biofluid among metabolomics researchers. It is sterile, easy-to-obtain in large volumes, largely free from interfering proteins or lipids and chemically complex. However, this chemical complexity has also made urine a particularly difficult substrate to fully understand. As a biological waste material, urine typically contains metabolic breakdown products from a wide range of foods, drinks, drugs, environmental contaminants, endogenous waste metabolites and bacterial by-products. Many of these compounds are poorly characterized and poorly understood. In an effort to improve our understanding of this biofluid we have undertaken a comprehensive, quantitative, metabolome-wide characterization of human urine. This involved both computer-aided literature mining and comprehensive, quantitative experimental assessment/validation. The experimental portion employed NMR spectroscopy, gas chromatography mass spectrometry (GC-MS), direct flow injection mass spectrometry (DFI/LC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) experiments performed on multiple human urine samples. This multi-platform metabolomic analysis allowed us to identify 445 and quantify 378 unique urine metabolites or metabolite species. The different analytical platforms were able to identify (quantify) a total of: 209 (209) by NMR, 179 (85) by GC-MS, 127 (127) by DFI/LC-MS/MS, 40 (40) by ICP-MS and 10 (10) by HPLC. Our use of multiple metabolomics platforms and technologies allowed us to identify several previously unknown urine metabolites and to substantially enhance the level of metabolome coverage. It also allowed us to critically assess the relative strengths and weaknesses of different platforms or technologies. The literature review led to the identification and annotation of another 2206 urinary compounds and was used to help guide the subsequent experimental studies. An online database containing the complete set of 2651 confirmed human urine metabolite species, their structures (3079 in total), concentrations, related literature references and links to their known disease associations are freely available at http://www.urinemetabolome.ca

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

Evidence for Cu(I) clusters and Zn(II) clusters in neuronal growth-inhibitory factor isolated from bovine brain

etallothionein. By analogy with Cu 8 -metallothionein, we propose the presence of a Cu 4 cluster with similar electronic structure in native GIF. However, the determined Cys/Cu 1 ratio of approximately 2 : 1 in Cu, Zn-GIF is higher than the ratio found in mammalian Cu(I)-metallothionein forms (approximately 1.6 : 1), which implies that the coordination geometry of Cu 1 -binding sites is different in the Cu 4 cluster. The spectroscopic characterization of Zn 21 -substituted and Cd 21 -substituted GIF (627 metal ions/protein) showed CD and MCD features at positions identical to those reported for the well-characterized mammalian Zn 7 -metallothionein and Cd 7 -metallothionein. Therefore, it is inferred that the cluster organization in GIF with divalen

Isolation and characterization of a novel monomeric zinc- and heme-containing protein from bovine brain

AbstractAn acidic zinc- and heme-containing protein was isolated from the soluble fraction of bovine brain and has been purified to homogeneity. The zinc-heme protein is a monomeric globular protein with a molecular mass of 31200 Da as determined by electrospray mass spectrometry. The protein was isolated with 0.90±0.05 zinc per protein and with substoichiometric amounts of heme. Amino acid sequences of four peptides (ca. 20% of the protein) were determined and the comparison of these sequences with those of protein and gene sequence databases revealed no significant correlation with any known protein. Thus, it is concluded that it is a novel protein of currently unknown biological function

Isolation of an alveolar septum by laser-assisted microdissection and manipulation from a hemalaun stained frozen lung section (magnification 200×)

<p><b>Copyright information:</b></p><p>Taken from "Identification of proteins in laser-microdissected small cell numbers by SELDI-TOF and Tandem MS"</p><p>BMC Biotechnology 2004;4():30-30.</p><p>Published online 3 Dec 2004</p><p>PMCID:PMC539305.</p><p>Copyright © 2004 Kwapiszewska et al; licensee BioMed Central Ltd.</p> A) Alveolar septum is selected for isolation. B) Laser photolysis is used to disconnect the cells from adjacent ones. C) Septum cells adhere tightly to the approximated sterile needle and can be transferred into a reaction tube