Improvement of an "In-Gel" Digestion Procedure for the Micropreparation of Internal Protein Fragments for Amino Acid Sequencing

Hellman, Ulf; Wernstedt, Christer; Gonez, Jorge; Heldin, Carl‐Henrik

doi:10.1006/abio.1995.1070

Cited by 731 publications

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“…Protein identification was carried out by peptide mass fingerprinting on an Ettan matrix-assisted laser desorption/ ionization (MALDI) time of flight (TOF) Pro mass spectrometer (Amersham Biosciences), as previously described [35,36]. Electrophoretic spots, visualized by a colloidal Coomassie staining protocol, were manually excised, destained, and acetonitrile-dehydratated.…”

Section: Protein Identification By Mass Spectrometrymentioning

confidence: 99%

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

et al. 2010

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We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.

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Section: Protein Identification By Mass Spectrometrymentioning

confidence: 99%

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

et al. 2010

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“…The resolved protein was visualized by colloidal Coomassie Blue staining and the gel piece containing the stained band corresponding to gp70 was excised. The proteins in the gel were digested with trypsin using a protocol modified by Hellman et al [37], and the tryptic peptides were analyzed by MALDI-TOF MS spectrometry. The gel pieces were rapidly and completely dried in a vacuum centrifuge, rehydrated in a trypsincontaining solution and incubated on ice (45 min).…”

Section: Cytokine Studiesmentioning

confidence: 99%

“…The method developed by Hellman et al [37] was used to extract the tryptic peptides. Aliquots (0.5 mL) were eluted from a C 18 ZipTip, mixed with a-cyano-4-hydroxycinnamic acid (10 mg/mL) in acetonitrile/water (50%) containing trifluoroacetic acid (0.1%) and then applied directly onto a target.…”

Section: Maldi-tof/tof Analysis and Peptide Sequencingmentioning

confidence: 99%

Passive immunization with monoclonal antibody against a 70‐kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

et al. 2008

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Cell-mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that specific antibodies show different degrees of protection against mycosis. In a previous study, antigens secreted by Sporothrix schenckii induced a specific humoral response in infected animals, mainly against the 70-kDa molecule, indicating a possible participation of antibodies to this antigen in infection control. In the present study, an IgG1 mAb was produced against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. Moreover, in a second schedule treatment, the mAb was injected after infection was established, and again we observed a significant reduction in CFU associated with an increase of IFN-cproduction. Also, the 70-kDa antigen is shown to be a putative adhesin present on the surface of this fungus. In conclusion, we report for the first time the protective effect of a specific antibody against S. schenckii.Key words: Adhesins . Mab . Passive immunization . Sporothrix schenckii . Sporotrichosis IntroductionSporotrichosis is a chronic subcutaneous mycosis that occurs worldwide. However, this disease is commonly seen in tropical and subtropical regions [1]. It is caused by traumatic inoculation of the dimorphic fungus Sporothrix schenckii, which is an ubiquitous environmental saprophyte that can be isolated from soil and plant debris [2]. In the past, infections were limited to the cutaneous forms of the disease.Recently, occurrences of more severe clinical forms of this mycosis were described, especially among immunocompromised individuals [3,4].The immunological mechanisms involved in prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting The host against S. schenckii [5]. In experimental infections, athymic nude mice are more susceptible to sporotrichosis, and acquired immunity against S. schenckii is mainly mediated by T-cell-activated macrophages [6,7].In contrast, the role of the humoral immune response in protection against this fungus has not been studied in detail. In a previous study, we showed that antigens secreted by S. 3080induce a specific humoral response in infected animals, mainly against the 70-kDa molecule, indicating a possible participation of specific antibodies to this molecule in infection control [8].Cell-mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that antibodies with defined specificity show different degrees of protection against mycosis [9]. Administration of mAb-mediated protection against Paracoccidioides brasiliensis and Candida albicans and modif...

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“…The gel pieces were then treated essentially as described in Hellman et al, (1995). Brie¯y, the gel pieces were washed with 0.1 M Tris-HCl bu er, pH 9.2, containing 50% acetonitrile, and then dried thoroughly.…”

Section: Immobilization Of Peptides On Agarose Beadsmentioning

confidence: 99%

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

et al. 1998

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Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) a-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, a nity puri®cation using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the puri®ed proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF a-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was speci®cally inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF a-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8-and 1.3-fold, respectively, upon ligand stimulation of the wild-type areceptor. In contrast, the Y762F mutant PDGF areceptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF a-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF a-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF b-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the b-receptor, which is localized at an analogous position to Tyr-762 in the a-receptor, binds RasGAP. RasGAP is not bound to the a-receptor.

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Improvement of an "In-Gel" Digestion Procedure for the Micropreparation of Internal Protein Fragments for Amino Acid Sequencing

Cited by 731 publications

References 0 publications

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

Passive immunization with monoclonal antibody against a 70‐kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

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