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REGULATION AND MECHANISM.PPT Pre-steady State Enzyme Kinetics - Michaelis-Menten kinetics is a ‘steady state’ analysis. It can give us macroscopic parameters for the reaction, but not microscopic ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill PULSED HYDROGEN EXCHANGE AND ELECTROSPRAY CHARGE-STATE 30 íM bradykinin at 10 íL/min] are mixed near the end of the inner capillary to trigger refolding at pH 10.0. Pulse labeling is initiated after a variable time period t1 by mixing with MeOD/D2O (50:50, v/v) from S3, resulting in a total flow rate of 100 íL/min at pD 10.0 and a final protein concentration of 10 íM.Subsequently, the reactionmixture
WILSON LAB @ YORK UNIVERSITYLAKE LOUISE 2016YORKU HOSTS GAIRDNER NEUROSCIENCEHAPPY HOLIDAYSCONGRATS ESTHER Wilson Lab @ York University. Current Projects and Research Opportunities >. A look at the current members of the Wilson Lab >. Tools for analysis - a closer look at our instruments >. Development of mass spectrometry coupled microfluidic devices >. METABOLIC DISEASES AND POISONS Diabetes - Probably the most common metabolic disease is the inability to control sugar metabolism - At the center of the problem: Insulin – another peptide hormone WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
REGULATION AND MECHANISM.PPT Pre-steady State Enzyme Kinetics - Michaelis-Menten kinetics is a ‘steady state’ analysis. It can give us macroscopic parameters for the reaction, but not microscopic ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill PULSED HYDROGEN EXCHANGE AND ELECTROSPRAY CHARGE-STATE 30 íM bradykinin at 10 íL/min] are mixed near the end of the inner capillary to trigger refolding at pH 10.0. Pulse labeling is initiated after a variable time period t1 by mixing with MeOD/D2O (50:50, v/v) from S3, resulting in a total flow rate of 100 íL/min at pD 10.0 and a final protein concentration of 10 íM.Subsequently, the reactionmixture
WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND METABOLISM Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic IRON AND CALCIUM METABOLISM The Other Side of the Enterocyte - On the other side of the Enterocyte is an Iron specific transporter Ferroportin, but it only takes Fe3+! - To re-oxidize we use the oxidoreductase Hephaestin, which is WEEK 9: MS IN SPACE AND PROTEOMICS More Smackdown! 18 •That was followed by this: The remarkable fact of these measurements and their interpretation is that the lowest level of detection at either temperature is 1 μg/g (1 ppm), i.e., a 1,000-fold poorer sensitivity than the 1 ng/g sample (1 ppb) demonstrated with the Viking e ngineering breadboard instrument (seeabove and Fig.
PRINCIPLES AND INSTRUMENTATION IN TIME-OF-FLIGHT MASS 1520 M. GUILHAUS to time Figure 1. Mass-dispersed space-time trajectories in one time (t) and two spatial (x, y) dimensions.For isobaric ions starting at to but originally spread in y, the trajectories are focusing when projected onto the y-t plane.Projections of the trajectories onto the x ENZYMES AND PROTEIN STRUCTURE Enzymes Allow-Milder reaction conditions (same as any catalyst, but better)-We won’t have to heat the reaction up to a gazillion degreesto
WEEK 4: COLLISIONAL ACTIVATION, MASS ANALYZERS: TOF The Triple Quadrupole 7 •The tripple quadrupole was the first, and still most widely used instrument specifically for CAD experiments + + - - + + - - + + - - Q1 Collision (Q2) Q3 •This allowed (initially) 3 types of scan: •Product ion: Select in Q1, Scan Q3 characterize molecular structure of known mass ion based on observed fragments. WEEK 7: ION MOBILITY What is ion mobility? 3 •Ion mobility is a bit like electrophoresis in the gas phase •If we have a packet of ions traveling down a drift tube across which there is a potential, the progress of those ions will be hampered by collisions with background gas. •And here’s the key: The extent to which progress is hampered depends on the sizeof the ion.
ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 WEEK 10: PROTEOMICS AND PROTEIN FUNCTION Quantitative Proteomics with Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) 5 •The study is designed to map phosphorylation on all proteins regulated by ephrin-B1 →ephrin-B2 interaction •To do this, they use a quatitative proteomics technique called ULTRARAPID DESALTING OF PROTEIN SOLUTIONS FOR ELECTROSPRAY ESI.10 Alternatively, using an emitter that is surrounded by a collateral supersonic jet of nebulizing gas provides an increased salt tolerance.13 In fused-droplet ESI, the ion source region is filled with a nebulized methanol/acetic acid mixture. Droplets generated WILSON LAB @ YORK UNIVERSITYHAPPY HOLIDAYSYORKU HOSTS GAIRDNER NEUROSCIENCECONGRATS ESTHER Wilson Lab @ York University. Current Projects and Research Opportunities >. A look at the current members of the Wilson Lab >. Tools for analysis - a closer look at our instruments >. Development of mass spectrometry coupled microfluidic devices >. Want to knowmore?
METABOLIC DISEASES AND POISONS Diabetes - Probably the most common metabolic disease is the inability to control sugar metabolism - At the center of the problem: Insulin – another peptide hormone WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 IRON AND CALCIUM METABOLISM The Other Side of the Enterocyte - On the other side of the Enterocyte is an Iron specific transporter Ferroportin, but it only takes Fe3+! - To re-oxidize we use the oxidoreductase Hephaestin, which is WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she WEEK 10: PROTEOMICS AND PROTEIN FUNCTION Quantitative Proteomics with Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) 5 •The study is designed to map phosphorylation on all proteins regulated by ephrin-B1 →ephrin-B2 interaction •To do this, they use a quatitative proteomics technique called VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill A CAPILLARY MIXER WITH ADJUSTABLE REACTION CHAMBER VOLUME flow experiments, the reaction is initiated by rapid mixing of the reactants, followed by mixing with a quenching agent, such as acid, base, or organic solvent, that abruptly stops the reaction after WILSON LAB @ YORK UNIVERSITYHAPPY HOLIDAYSYORKU HOSTS GAIRDNER NEUROSCIENCECONGRATS ESTHER Wilson Lab @ York University. Current Projects and Research Opportunities >. A look at the current members of the Wilson Lab >. Tools for analysis - a closer look at our instruments >. Development of mass spectrometry coupled microfluidic devices >. Want to knowmore?
METABOLIC DISEASES AND POISONS Diabetes - Probably the most common metabolic disease is the inability to control sugar metabolism - At the center of the problem: Insulin – another peptide hormone WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 IRON AND CALCIUM METABOLISM The Other Side of the Enterocyte - On the other side of the Enterocyte is an Iron specific transporter Ferroportin, but it only takes Fe3+! - To re-oxidize we use the oxidoreductase Hephaestin, which is WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she WEEK 10: PROTEOMICS AND PROTEIN FUNCTION Quantitative Proteomics with Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) 5 •The study is designed to map phosphorylation on all proteins regulated by ephrin-B1 →ephrin-B2 interaction •To do this, they use a quatitative proteomics technique called VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill A CAPILLARY MIXER WITH ADJUSTABLE REACTION CHAMBER VOLUME flow experiments, the reaction is initiated by rapid mixing of the reactants, followed by mixing with a quenching agent, such as acid, base, or organic solvent, that abruptly stops the reaction after PRINCIPLES AND INSTRUMENTATION IN TIME-OF-FLIGHT MASS 1520 M. GUILHAUS to time Figure 1. Mass-dispersed space-time trajectories in one time (t) and two spatial (x, y) dimensions.For isobaric ions starting at to but originally spread in y, the trajectories are focusing when projected onto the y-t plane.Projections of the trajectories onto the x WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
IRON AND CALCIUM METABOLISM The Other Side of the Enterocyte - On the other side of the Enterocyte is an Iron specific transporter Ferroportin, but it only takes Fe3+! - To re-oxidize we use the oxidoreductase Hephaestin, which is3050 LECTURE 7
Reduction Potential - Reduction potential Eo is a measure of how much a molecule likes to gain electrons. - In a reaction ΔEo = Eo (acceptor) –E o (donor) - Thus: NAD ++ H + 2e-↔NADH Eo = -0.315 V O 2 + 2H+ + 2e-↔H 2 OE o = 0.815 V NADH + H+ + 2e-+ ½O 2 ↔NAD+ + H 2 O ΔEo = 1.130 V - Converting to ΔG: AMINO ACID METABOLISM Glutamine Synthetase - When we make glutamine, we’ve got to use some precious ATP ADP/ATP Glutamate - Glutamine synthetase is an important metabolic center for WEEK 7: ION MOBILITY What is ion mobility? 3 •Ion mobility is a bit like electrophoresis in the gas phase •If we have a packet of ions traveling down a drift tube across which there is a potential, the progress of those ions will be hampered by collisions with background gas. •And here’s the key: The extent to which progress is hampered depends on the sizeof the ion.
ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 MECHANISTIC STUDIES ON ENZYMATIC REACTIONS BY ELECTROSPRAY , and remain constant, whereas and increase linearly with time. The rate of reaction under these steady-state conditions is given by the Michealis-Menten expression1 where 0 is the total enzyme concentration. Measurements of A STRUCTURAL ROLE FOR TRYPTOPHAN 188 OF INDUCIBLE NITRIC A Structural Role for Tryptophan 188 of Inducible Nitric Oxide Synthase Derek J. Wilson and Steven P. Rafferty1 Chemistry Department, Trent University, 1600 West Bank Drive, Peterborough, Ontario, CanadaK9J
ULTRARAPID DESALTING OF PROTEIN SOLUTIONS FOR ELECTROSPRAY ESI.10 Alternatively, using an emitter that is surrounded by a collateral supersonic jet of nebulizing gas provides an increased salt tolerance.13 In fused-droplet ESI, the ion source region is filled with a nebulized methanol/acetic acid mixture. Droplets generated WILSON LAB @ YORK UNIVERSITYLAKE LOUISE 2016YORKU HOSTS GAIRDNER NEUROSCIENCEHAPPY HOLIDAYSCONGRATS ESTHER Wilson Lab @ York University. Current Projects and Research Opportunities >. A look at the current members of the Wilson Lab >. Tools for analysis - a closer look at our instruments >. Development of mass spectrometry coupled microfluidic devices >. PRINCIPLES AND INSTRUMENTATION IN TIME-OF-FLIGHT MASS 1520 M. GUILHAUS to time Figure 1. Mass-dispersed space-time trajectories in one time (t) and two spatial (x, y) dimensions.For isobaric ions starting at to but originally spread in y, the trajectories are focusing when projected onto the y-t plane.Projections of the trajectories onto the x METABOLIC DISEASES AND POISONS Diabetes - Probably the most common metabolic disease is the inability to control sugar metabolism - At the center of the problem: Insulin – another peptide hormone WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
REGULATION AND MECHANISM.PPT Pre-steady State Enzyme Kinetics - Michaelis-Menten kinetics is a ‘steady state’ analysis. It can give us macroscopic parameters for the reaction, but not microscopic METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she PULSED HYDROGEN EXCHANGE AND ELECTROSPRAY CHARGE-STATE 30 íM bradykinin at 10 íL/min] are mixed near the end of the inner capillary to trigger refolding at pH 10.0. Pulse labeling is initiated after a variable time period t1 by mixing with MeOD/D2O (50:50, v/v) from S3, resulting in a total flow rate of 100 íL/min at pD 10.0 and a final protein concentration of 10 íM.Subsequently, the reactionmixture
VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill WILSON LAB @ YORK UNIVERSITYLAKE LOUISE 2016YORKU HOSTS GAIRDNER NEUROSCIENCEHAPPY HOLIDAYSCONGRATS ESTHER Wilson Lab @ York University. Current Projects and Research Opportunities >. A look at the current members of the Wilson Lab >. Tools for analysis - a closer look at our instruments >. Development of mass spectrometry coupled microfluidic devices >. PRINCIPLES AND INSTRUMENTATION IN TIME-OF-FLIGHT MASS 1520 M. GUILHAUS to time Figure 1. Mass-dispersed space-time trajectories in one time (t) and two spatial (x, y) dimensions.For isobaric ions starting at to but originally spread in y, the trajectories are focusing when projected onto the y-t plane.Projections of the trajectories onto the x METABOLIC DISEASES AND POISONS Diabetes - Probably the most common metabolic disease is the inability to control sugar metabolism - At the center of the problem: Insulin – another peptide hormone WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
REGULATION AND MECHANISM.PPT Pre-steady State Enzyme Kinetics - Michaelis-Menten kinetics is a ‘steady state’ analysis. It can give us macroscopic parameters for the reaction, but not microscopic METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she PULSED HYDROGEN EXCHANGE AND ELECTROSPRAY CHARGE-STATE 30 íM bradykinin at 10 íL/min] are mixed near the end of the inner capillary to trigger refolding at pH 10.0. Pulse labeling is initiated after a variable time period t1 by mixing with MeOD/D2O (50:50, v/v) from S3, resulting in a total flow rate of 100 íL/min at pD 10.0 and a final protein concentration of 10 íM.Subsequently, the reactionmixture
VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill YORK UNIVERSITY DEPARTMENT OF CHEMISTRY AND DEPARTMENT OF York University Department of Chemistry and Department of Biology Advanced Biochemistry, Winter 2020 Biology 3010 3.0 / Chemistry 3050 3.0 / Biochemistry 3010 3.0 IRON AND CALCIUM METABOLISM The Other Side of the Enterocyte - On the other side of the Enterocyte is an Iron specific transporter Ferroportin, but it only takes Fe3+! - To re-oxidize we use the oxidoreductase Hephaestin, which is3050 LECTURE 7
Reduction Potential - Reduction potential Eo is a measure of how much a molecule likes to gain electrons. - In a reaction ΔEo = Eo (acceptor) –E o (donor) - Thus: NAD ++ H + 2e-↔NADH Eo = -0.315 V O 2 + 2H+ + 2e-↔H 2 OE o = 0.815 V NADH + H+ + 2e-+ ½O 2 ↔NAD+ + H 2 O ΔEo = 1.130 V - Converting to ΔG: WEEK 4: COLLISIONAL ACTIVATION, MASS ANALYZERS: TOF The Triple Quadrupole 7 •The tripple quadrupole was the first, and still most widely used instrument specifically for CAD experiments + + - - + + - - + + - - Q1 Collision (Q2) Q3 •This allowed (initially) 3 types of scan: •Product ion: Select in Q1, Scan Q3 characterize molecular structure of known mass ion based on observed fragments. WEEK 9: MS IN SPACE AND PROTEOMICS More Smackdown! 18 •That was followed by this: The remarkable fact of these measurements and their interpretation is that the lowest level of detection at either temperature is 1 μg/g (1 ppm), i.e., a 1,000-fold poorer sensitivity than the 1 ng/g sample (1 ppb) demonstrated with the Viking e ngineering breadboard instrument (seeabove and Fig.
ENZYMES AND PROTEIN STRUCTURE Enzymes Allow-Milder reaction conditions (same as any catalyst, but better)-We won’t have to heat the reaction up to a gazillion degreesto
WEEK 7: ION MOBILITY What is ion mobility? 3 •Ion mobility is a bit like electrophoresis in the gas phase •If we have a packet of ions traveling down a drift tube across which there is a potential, the progress of those ions will be hampered by collisions with background gas. •And here’s the key: The extent to which progress is hampered depends on the sizeof the ion.
NUCLEOTIDE METABOLISM Nucleotide Metabolism - Mostly, we think of nucleotides as being part of RNA/DNA, but now that we’ve studied metabolism, we know they’reall over!
ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 WEEK 10: PROTEOMICS AND PROTEIN FUNCTION Quantitative Proteomics with Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) 5 •The study is designed to map phosphorylation on all proteins regulated by ephrin-B1 →ephrin-B2 interaction •To do this, they use a quatitative proteomics technique called WILSON LAB @ YORK UNIVERSITYHAPPY HOLIDAYSYORKU HOSTS GAIRDNER NEUROSCIENCECONGRATS ESTHER The Wilson Lab wishes all a restful and happy holiday season PRINCIPLES AND INSTRUMENTATION IN TIME-OF-FLIGHT MASS 1520 M. GUILHAUS to time Figure 1. Mass-dispersed space-time trajectories in one time (t) and two spatial (x, y) dimensions.For isobaric ions starting at to but originally spread in y, the trajectories are focusing when projected onto the y-t plane.Projections of the trajectories onto the x WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she ENZYMES AND PROTEIN STRUCTURE Enzymes Allow-Milder reaction conditions (same as any catalyst, but better)-We won’t have to heat the reaction up to a gazillion degreesto
ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
WEEK 10: PROTEOMICS AND PROTEIN FUNCTION Quantitative Proteomics with Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) 5 •The study is designed to map phosphorylation on all proteins regulated by ephrin-B1 →ephrin-B2 interaction •To do this, they use a quatitative proteomics technique called VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill WILSON LAB @ YORK UNIVERSITYHAPPY HOLIDAYSYORKU HOSTS GAIRDNER NEUROSCIENCECONGRATS ESTHER The Wilson Lab wishes all a restful and happy holiday season PRINCIPLES AND INSTRUMENTATION IN TIME-OF-FLIGHT MASS 1520 M. GUILHAUS to time Figure 1. Mass-dispersed space-time trajectories in one time (t) and two spatial (x, y) dimensions.For isobaric ions starting at to but originally spread in y, the trajectories are focusing when projected onto the y-t plane.Projections of the trajectories onto the x WELCOME TO BIOCHEM 3050 / BIOLOGY 3010: ENZYMES AND … Metabolic Pathways - In sum, pathways are almost always exergonic, and very often largely so. - This does not mean that all reactions in the pathway are exergonic e.g. from Glucose to Pyruvate is ≈ -130 kcal/mol e.g. from Glucose to Glyceraldehyde-3-Phosphate is ≈ +20 kcal/mol - Metabolic pathways can be Catabolic (break stuff down - degradation) or Anabolic AMINO ACIDS TO PROTEINS Amino Acids Henri Braconnot 1780 - 1855 Isolated Glycine Karl Ritthausen – 1866 Glutamic and Aspartic Acid Heinrich Hlasiwetz and Josef Habermann - 1867 METABOLIC PATHWAYS AND ENERGY METABOLISM Sugar to Energy Overview - Glucose is the most direct input into the energetics metabolic pathway - As evidenced by the behavior of a typical 7 year old after he/she ENZYMES AND PROTEIN STRUCTURE Enzymes Allow-Milder reaction conditions (same as any catalyst, but better)-We won’t have to heat the reaction up to a gazillion degreesto
ENZYME DYNAMICS AND FUNCTION mM 0 1 23456 k obs 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kac (light) = (3.2 +/- .3) s-1 kac (heavy) = (3.7 +/- .2) s-1 kac (optical) = (3.6 +/- .2) s-1 K d (light) = (1 WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
WEEK 10: PROTEOMICS AND PROTEIN FUNCTION Quantitative Proteomics with Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) 5 •The study is designed to map phosphorylation on all proteins regulated by ephrin-B1 →ephrin-B2 interaction •To do this, they use a quatitative proteomics technique called VAMPIRES, WEREWOLVES AND… METABOLISM!!? The Origins of Werewolf Folklore First surviving written reference in an 10th century french poem. Lyncanthropy – greek for ‘wolf-man-to’ Things used to kill ENZYMES AND PROTEIN STRUCTURE Enzymes Allow-Milder reaction conditions (same as any catalyst, but better)-We won’t have to heat the reaction up to a gazillion degreesto
IRON AND CALCIUM METABOLISM The Other Side of the Enterocyte - On the other side of the Enterocyte is an Iron specific transporter Ferroportin, but it only takes Fe3+! - To re-oxidize we use the oxidoreductase Hephaestin, which is METABOLIC DISEASES AND POISONS Diabetes - Probably the most common metabolic disease is the inability to control sugar metabolism - At the center of the problem: Insulin – another peptide hormone YORK UNIVERSITY DEPARTMENT OF CHEMISTRY AND DEPARTMENT OF York University Department of Chemistry and Department of Biology Advanced Biochemistry, Winter 2020 Biology 3010 3.0 / Chemistry 3050 3.0 / Biochemistry 3010 3.0 WEEK 3: MASS ANALYZERS Mass Analyzer = the ♥of the Mass Spectrometer 3 •The mass analyzer is what makes a mass spectrometer a mass spectrometer. •There are three classes of analyzer (that I made up): •Separating: Ions are separated in space according to their m/z and detected at different times or locations. •Filtering: All ions except for a specific m/zare removed.
WELCOME TO 4081: PRINCIPLES AND APPLICATIONS OF MASS Assessment Midterm exam 1- 25% November 2nd Project Assignment- 35% Due: Dec 9th, Presentations Dec 7th and 9th Final exam- 40% AcademicHonesty:
WEEK 7: ION MOBILITY What is ion mobility? 3 •Ion mobility is a bit like electrophoresis in the gas phase •If we have a packet of ions traveling down a drift tube across which there is a potential, the progress of those ions will be hampered by collisions with background gas. •And here’s the key: The extent to which progress is hampered depends on the sizeof the ion.
WEEK 6: VACUUM SYSTEMS Measuring High Vacuums 9 •These days, MS instrument operate at ‘high vacuum’, i.e. anything lower than 10-7 torr. •The cheapest high vacuum measuring device is a Penning or Cold Cathode gauge. •Electrons are generated via discharge in a cold cathode.Electronsflow to the
WEEK 4: COLLISIONAL ACTIVATION, MASS ANALYZERS: TOF The Triple Quadrupole 7 •The tripple quadrupole was the first, and still most widely used instrument specifically for CAD experiments + + - - + + - - + + - - Q1 Collision (Q2) Q3 •This allowed (initially) 3 types of scan: •Product ion: Select in Q1, Scan Q3 characterize molecular structure of known mass ion based on observed fragments. ULTRARAPID DESALTING OF PROTEIN SOLUTIONS FOR ELECTROSPRAY ESI.10 Alternatively, using an emitter that is surrounded by a collateral supersonic jet of nebulizing gas provides an increased salt tolerance.13 In fused-droplet ESI, the ion source region is filled with a nebulized methanol/acetic acid mixture. Droplets generated Investigating Protein Dynamics, Structure and Function using MassSpectrometry
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Dec 20, 2019
Happy Holidays!
The Wilson Lab wishes all a restful and happy holiday season...Read more
Aug 13, 2018
Feature in Laboratory Focus The Wilson lab has recently been featured in Laboratory Focus magazine- link! ...
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Apr 2, 2018
Congratulations Derek! Congratulations to Derek for receiving a York Research Chair appointment in Molecular Mechanisms of Disease!...Read more
Derek Wilson Lab GroupYork University,
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