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Papers by Jan Havlis
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometr... more In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
Molecular biology of the cell, 2008
The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Gol... more The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic...
Biotechnology and applied biochemistry, 2005
Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxid... more Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxidases (PSAO and GPAO respectively) with BCD (beta-cyclodextrin), performed to improve the thermostability of the enzymes, are described in the present study. Periodate-oxidized BCD reacted with the enzyme proteins via free primary amino groups in a buffered solution containing cyanoborohydride as a reductant. Although the specific activities of PSAO and GPAO partially decreased after modification, Km values determined for the best diamine substrates remained almost unchanged. Both the BCD conjugates could be incubated at 65 degrees C for 30 min without considerable inactivation, and the residual activity remained detectable even after incubation at 75 degrees C. The conjugates contained approx. 30% of neutral sugars. Molecular masses of BCD-PSAO and BCD-GPAO (180 kDa), as estimated by gel-permeation chromatography, were higher compared with the value of 145 kDa for the native enzymes. Thi...
Nature Protocols, 2007
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometr... more In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
1. Úvod − proteolytické enzymy a jejich úloha v proteomice 2. Reakce katalyzovaná proteolytickými... more 1. Úvod − proteolytické enzymy a jejich úloha v proteomice 2. Reakce katalyzovaná proteolytickými enzymy 3. Zdroje a klasifikace proteolytických enzymů 4. Stručný přehled vlastností vybraných enzymů 5. Trypsin a jeho výjimečné postavení v proteomice 6. Měření proteolytické aktivity 7. Počítačová predikce štěpných peptidů 8. Varianty procesu proteolýzy používané v proteomice 9. Modifikace proteolytických enzymů (modulace funkčnosti a termostability) 10. Závěr − perspektivy proteolýzy v moderní proteomice
The influence of bound copper(II) on binding capabilities of tryptophan (Trp) and tyrosine (Tyr) ... more The influence of bound copper(II) on binding capabilities of tryptophan (Trp) and tyrosine (Tyr) to bovine serum albumin (BSA) was studied by means of equilibrium dialysis and ultrafiltration. The number of bound tryptophan increased in the presence of copper(II), coordinated to serum albumin, from 6.87 to 17.16 mols of Trp pro mole of bovine serum albumin in equilibrium studies and from 0.17 to 1.50 in ultrafiltration studies. Tyrosine was found inactive with BSA and BSA-Cu(II) complex.
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometr... more In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
Molecular biology of the cell, 2008
The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Gol... more The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic...
Biotechnology and applied biochemistry, 2005
Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxid... more Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxidases (PSAO and GPAO respectively) with BCD (beta-cyclodextrin), performed to improve the thermostability of the enzymes, are described in the present study. Periodate-oxidized BCD reacted with the enzyme proteins via free primary amino groups in a buffered solution containing cyanoborohydride as a reductant. Although the specific activities of PSAO and GPAO partially decreased after modification, Km values determined for the best diamine substrates remained almost unchanged. Both the BCD conjugates could be incubated at 65 degrees C for 30 min without considerable inactivation, and the residual activity remained detectable even after incubation at 75 degrees C. The conjugates contained approx. 30% of neutral sugars. Molecular masses of BCD-PSAO and BCD-GPAO (180 kDa), as estimated by gel-permeation chromatography, were higher compared with the value of 145 kDa for the native enzymes. Thi...
Nature Protocols, 2007
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometr... more In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
1. Úvod − proteolytické enzymy a jejich úloha v proteomice 2. Reakce katalyzovaná proteolytickými... more 1. Úvod − proteolytické enzymy a jejich úloha v proteomice 2. Reakce katalyzovaná proteolytickými enzymy 3. Zdroje a klasifikace proteolytických enzymů 4. Stručný přehled vlastností vybraných enzymů 5. Trypsin a jeho výjimečné postavení v proteomice 6. Měření proteolytické aktivity 7. Počítačová predikce štěpných peptidů 8. Varianty procesu proteolýzy používané v proteomice 9. Modifikace proteolytických enzymů (modulace funkčnosti a termostability) 10. Závěr − perspektivy proteolýzy v moderní proteomice
The influence of bound copper(II) on binding capabilities of tryptophan (Trp) and tyrosine (Tyr) ... more The influence of bound copper(II) on binding capabilities of tryptophan (Trp) and tyrosine (Tyr) to bovine serum albumin (BSA) was studied by means of equilibrium dialysis and ultrafiltration. The number of bound tryptophan increased in the presence of copper(II), coordinated to serum albumin, from 6.87 to 17.16 mols of Trp pro mole of bovine serum albumin in equilibrium studies and from 0.17 to 1.50 in ultrafiltration studies. Tyrosine was found inactive with BSA and BSA-Cu(II) complex.