John Fenn

Information about John Fenn

Published on October 15, 2007

Author: Marigold

Source: authorstream.com

Content

Slide1:  Biomolecules and Mass Spectroscopy Lecture 11 September 26th 2005 “...wings for molecular elephants” Slide2:  Nobel Prize In Chemistry 2002 “for his development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules” John B. Fenn, Ph.D Yale 1940, Professor, Yale University 1967-1987 “electrospray wings for molecular elephants” Slide3:  Mass Spectroscopy: Some history and highlights. 1897 JJ Thompson: first mass spec (1906 Nobel) Harold Urey: discovers deuterium (1934 Nobel) H Kroto et al: Fullerenes (1996 Nobel) Bieman: first analysis of amino acids (1958) McFarlane: spectrum of insulin 5750 Da (1982) Fenn: First spectra of proteins above 20,000 Da (1988) Siuzdak: Mass spec. of an intact virus (1996) Slide4:  Mass Spec: The Basics 5 basic components: Inlet, Source, Mass Analyzer, Detector, Signal Processor Source: converts a sample to a mixture of ions, Analyzer: resolves those ions based on their m/z Detector: detects those resolved ions. Produce gas phase ions: Slide5:  Best choice for Peptides and Proteins MALDI: Matrix-assisted Laser Desorption ionization ESI: ElectroSpray Ionization Slide6:  Matrix is typically a small organic molecule with a desirable chromophore produce a crystalline lattice in which the analyte is integrated target is placed in the source laser is fired, matrix absorbs photons, gets excited! Excess energy is transferred to analyte in sample, which along with matrix is ejected, ionized into the gas phase produces + and - ions. (peptides tend to pick up a single proton: [M + H]+) Matrix-assisted laser desorption ionization (MALDI) Slide7:  First MALDI was on a protein so solvent was water Common to now use 70 % water/30 % acetonitrile 10 M peptide to 10 mM matrix solution 0.1 % TFA to keep pH less than 4 If using CHCA or SA as matrix : 337 nm N2 Laser Typical MALDI-TOF experiment Slide8:  very efficient at ionizing proteins (intense signals) produces large fragments particularly in the 500-5000 range where tryptic digests appear (Hence use for <10,000 Da proteins Protein mass fingerprinting not strongly ionizing so fewer multiply charged ions detected high affinity for proteins very non-selective best for hydrophobic proteins best for crude biological extracts alpha-cyano-4-hydroxycinnamic acid (CHCA) sinapinic acid (SA) (3,5-dimethoxy-4-hydroxycinnamic acid)    Slide9:  Concentration of sample contaminants tolerated by MALDI of peptides and proteins * * Mock et al., Rapid Commun. Mass Spectrom. 4 (1992) 233 Pros and Cons of MALDI:  Pros and Cons of MALDI User friendly high accuracy very sensitive (down to attomole concs) excess of matrix molecules helps prevent cluster formation matrix means no need to adjust wavelength to match abs. freq. of each analyte Can be difficult to produce good “matrix” admixtures really only for detection of intact peptide ions (so not great for peptide-sequencing) Slide11:  Time-of-Flight (TOF) measures time it takes ions to fly from one end of the analyzer to the other. The greater the m/z the faster they fly (Newton) Original linear TOF detectors gave poor resolution Average time in TOF: 10-7 sec : average speed 1-2 x 105 km/h Slide12:  Linear TOF vs Reflectron Linear = poor resolution due to velocity variation of ions with the same m/z Reflectron = Contact lens for a near sighted machine! Insulin: (a) with reflectron (b) linear TOF (a) (b) Slide13:  an electrospray is produced by applying a strong electric field to a liquid passing through a capillary tube with a weak flux desolvation by gas flow (N2) or heated capillary-apply vacuum best for peptides/proteins at acidic pH produces multiply charged ions (unique characteristic) multiply charged species bring m/z into quadrupole limit range Electrospray Slide14:  (A) “Multi-charge envelope” ESI+ Spectrum of Bovine apomyoglobin (B) After “Charge-deconvolution algorithm” Slide15:  Quadrupole detection (Q1) 4 metal rods arranged in parallel (hence the name!) DC/RF applied  magnetic field causes “corkscrew” trajectory for ions depending on voltage applied, only specific m/z ions will pass through ions of greater or lesser m/z will fail to pass through by sweeping RF voltages, ions of increasing m/z values can be analyzed limited range (max ~ 4 kDA) - Digestion?? Slide16:  Why digestion? greater the mass-the greater the absolute error Not all proteins are amenable to intact mass measurement (e.g.v.large/v.hydrophobic) Modern computers can use algorithms to assist protein identification Programs can take into account specific cleavage sites of agents such as trypsin Digesting can increase ionization or even introduce it Pros and Cons Resolution vs m/z intact mass window Sequencing vs mass fingerprinting Ease of use/sample preparation Solution?? Slide17:  Triple Quadrupole Tandem mass analysis Q1-q2-Q3 Q1-q2-TOF (Q-TOF) Best Resolution/large upper limit m/z/high mass accuracy Tandem HPLC (John R Hayes (Scripps)) Tandem MS (MS-MS) Historically combined with ESI Slide18:  References D. C. Liebler, Introduction to Proteomics, Humana Press 2002. E. De Hoffmann, J. Charette and V. Stroobant, Mass Spectrometry 1st Ed., Wiley Press, 1996. R. M. Twyman, Advanced Molecular Biology, Springer Press, 1998. Proteome Research: Mass Spectrometry; James, P ed.; Springer: New York, 2001 www.nobel.se http://massspec.scrips.edu/ G. Siuzdak Homepage http://fields.scripps.edu/ John R Yates Homepage

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