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Research paper thumbnail of In-gel digestion for mass spectrometric characterization of proteins and proteomes

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.

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Research paper thumbnail of Electron Transfer to Coenzyme NAD+ on Mercury Reinvestigated: Excimers (NAD)2, Locally Excited Amide Reacting to β-Lactam

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Research paper thumbnail of Biradical Excited State of NADH and Its Reactions on the Mercury Electrode

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Research paper thumbnail of Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth

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...

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Research paper thumbnail of Thermostable beta-cyclodextrin conjugates of two similar plant amine oxidases and their properties

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...

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Research paper thumbnail of In-gel digestion for mass spectrometric characterization of proteins and proteomes

Nature Protocols, 2007

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Research paper thumbnail of Proteolytic Enzymes: Significance for Proteomics

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Research paper thumbnail of Influence of protein-bound copper (II) on properties of bovine serum albumin-tryptophan complex

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.

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Research paper thumbnail of Determination of purins, pyrimidines and their nucleosides in urine by HPLC and MALDI-TOF mass spectrometry

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Research paper thumbnail of Determination of 5-methyl-2'-deoxycytidine 5'-monophosphate and 2'-deoxycytidine 5'-monophosphate in the cell homogenate using HPLC

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Research paper thumbnail of Recent developments in humic acid characterisation by CE and MALDI-TOF mass spectrometry

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Research paper thumbnail of Partial characterisation of a colored metabolite of Acidithiobacillus ferrooxidans grown on elemental sulfur

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Research paper thumbnail of HPLC Separation of Neuroprotective Peptides. General Approach Towards Optimization: Experimental Design Combined with Artificial Neural Network

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Research paper thumbnail of Využití izotopového značení v hmotnostní spektrometrii

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Research paper thumbnail of Praktické aspekty absolutní kvantifikace proteinů a peptidů pomocí MS

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Research paper thumbnail of Indirect quantification of protein modification degree

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Research paper thumbnail of Proteolytické enzymy: význam pro proteomiku

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Research paper thumbnail of Protein Modification Degree-An Indirect MS Quantitation Approach

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Research paper thumbnail of Modification of Trypsin for Improvement of Its Stability in Protein Digestion Process

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Research paper thumbnail of Analýza metaloproteinů pomocí MS-paralelně kombinované techniky GLIM

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Research paper thumbnail of In-gel digestion for mass spectrometric characterization of proteins and proteomes

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.

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Electron Transfer to Coenzyme NAD+ on Mercury Reinvestigated: Excimers (NAD)2, Locally Excited Amide Reacting to β-Lactam

DESCRIPTION

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Research paper thumbnail of Biradical Excited State of NADH and Its Reactions on the Mercury Electrode

DESCRIPTION

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth

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...

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Research paper thumbnail of Thermostable beta-cyclodextrin conjugates of two similar plant amine oxidases and their properties

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...

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Research paper thumbnail of In-gel digestion for mass spectrometric characterization of proteins and proteomes

Nature Protocols, 2007

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Research paper thumbnail of Proteolytic Enzymes: Significance for Proteomics

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Research paper thumbnail of Influence of protein-bound copper (II) on properties of bovine serum albumin-tryptophan complex

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.

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Research paper thumbnail of Determination of purins, pyrimidines and their nucleosides in urine by HPLC and MALDI-TOF mass spectrometry

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Determination of 5-methyl-2'-deoxycytidine 5'-monophosphate and 2'-deoxycytidine 5'-monophosphate in the cell homogenate using HPLC

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Recent developments in humic acid characterisation by CE and MALDI-TOF mass spectrometry

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Partial characterisation of a colored metabolite of Acidithiobacillus ferrooxidans grown on elemental sulfur

Bookmarks Related papers MentionsView impact

Research paper thumbnail of HPLC Separation of Neuroprotective Peptides. General Approach Towards Optimization: Experimental Design Combined with Artificial Neural Network

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Využití izotopového značení v hmotnostní spektrometrii

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Research paper thumbnail of Praktické aspekty absolutní kvantifikace proteinů a peptidů pomocí MS

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Indirect quantification of protein modification degree

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Research paper thumbnail of Proteolytické enzymy: význam pro proteomiku

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Protein Modification Degree-An Indirect MS Quantitation Approach

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Modification of Trypsin for Improvement of Its Stability in Protein Digestion Process

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Analýza metaloproteinů pomocí MS-paralelně kombinované techniky GLIM

Bookmarks Related papers MentionsView impact

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