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Mass Spectrometry of Macromolecules API 4000, API Q-Tof ultima API, Micromass HCT plus, Bruker History • In 1886, Eugen Goldstein observed rays in gas discharges under low pressure that traveled away from the anode and through channels in a perforated cathode, opposite to the direction of negatively charged cathode rays (which travel from cathode to anode). • Goldstein called these positively charged anode rays "Kanalstrahlen"; the standard translation of this term into English is "canal rays". • Wilhelm Wien found that strong electric or magnetic fields deflected the canal rays and, in 1899, constructed a device with parallel electric and magnetic fields that separated the positive rays according to their charge-to-mass ratio (Q/m). • Wien found that the charge-to-mass ratio depended on the nature of the gas in the discharge tube. • English scientist J.J. Thomson later improved on the work of Wien by reducing the pressure to create the mass spectrograph. History • The first application of mass spectrometry to the analysis of amino acids and peptides was reported in 1958. • Carl-Ove Andersson highlighted the main fragment ions observed in the ionization of methyl esters. History • Some of the modern techniques of mass spectrometry were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively. • In 1989, half of the Nobel Prize in Physics was awarded to Hans Dehmelt and Wolfgang Paul for the development of the ion trap technique in the 1950s and 1960s. • In 2002, the Nobel Prize in Chemistry was awarded to John Bennett Fenn for the development of electrospray ionization (ESI) and Koichi Tanaka for the development of soft laser desorption (SLD) and their application to the ionization of biological macromolecules, especially proteins. In a typical MS procedure 1. A sample is loaded onto the mass spectrometer, and undergoes vaporization 2. The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions) 3. The ions are separated according to their mass-tocharge ratio in an analyzer by electromagnetic fields 4. The ions are detected, usually by a quantitative method 5. The ion signal is processed into mass spectra MS instruments consist of three modules 1. An ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase) 2. A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields 3. A detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present GENERAL PRINCIPLES: THE PROBLEM • The ability to determine accurate molecular masses, including those of fragments of large molecules such as proteins and nucleic acids, now allows us to identify molecules from a cellular mixture, monitor their changes, and even provide detailed information on their structures (from the primary sequence to potentially the tertiary fold and quaternary association of subunits) and mechanisms of folding. The three primary components of a mass spectrometer. The molecule(s) of interest as a charged ion are placed into the gas phase, then the molecular species must be separated (or resolved) according to their masses, and, finally, the molecular ions must be detected. mass-to-charge (m/Z) ratio • Mass spectrometry uses an electrostatic potential to accelerate molecular ions and, thus, the method does not measure the mass of a molecule per se, but rather the mass-to-charge (m/Z) ratio of the molecular ion. • We will see that molecular ions with smaller masses or larger numbers of charges will have a higher velocity, and this can be used as a basis to resolve various molecular ions according to differences in their respective masses. Matrix-assisted laser desportionionization (MALDI) Ionization of the analyte results from exchange of electrons and/or protons (shown here as a proton transition) with the matrix compound. In MALDI-TOF-MS, the macromolecule to be analyzed (the analyte) is first embedded in a crystal of a matrix compound, typically a weak organic acid. The power density required to generate a significant ion current corresponds to an energy flux of ~20 mJ/cm2. Matrix for MALDI-TOF 1. 2,5-Dihydroxybenzoic acid (DHB) Peptides, proteins, lipids, and oligosaccharides 2. 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) Peptides, proteins, and glycoproteins 3. a-Cyano-4-hydroxycinnamic acid (CHCA) Peptides, proteins, lipids, and oligonucleotides A mass spectrum of myoglobin Both singly and doubly charged ions are present. Electrospray ionization (ESI) mass spectrometry. RESOLVING MOLECULAR WEIGHTS BY MASS SPECTROMETRY Schematic drawing of a time-of-flight mass spectrometer (TOF-MS) mass-to-charge (m/Z) ratio The units of the mass itself may be in atomic mass units (amu) or, for biological molecules, in daltons (Da, equivalent to 1 gm/mol) or kilodaltons (kDa equivalent to 1 kg/mol), units that are familiar to biochemists. Principle of a reflectron in TOF-MS Magnetic sector mass spectrometry Only those ions whose angular momentum (as defined by their mlZ ratios) defines an unimpeded pathway through the magnetic field will reach the detector. Quadrupole mass filter constant electric potential (dc potential) alternating potential (ac) or a radio frequency (rf) field The potential at any point (x and y) within the quadrupole Any ion coming into the quadrupole will experience a static potential (with magnitude U) and alternating potential (with a magnitude of V). Quadrupole mass filter Notice that the field applies a force of F = 0 along the z-axis Equations 15.10 and 15.11 can be rewritten in the form of Mathieu‘s differential equation (which was derived in 1866 to describe the propagation of waves in membranes) to provide a quantitative prediction of the stability of ions within a quadrupole field. Quadrupole filter low mass high mass very heavy ions The x-z plane acts as a low mass filter while the y-z plane acts as a high pass filter, leaving only those masses that overlap between the two to be detected. DETERMINING MOLECULAR WEIGHTS OF BIOMOLECULES ESI-MS spectrum of leucineenkephalin C13 Na+ ESI-MS of lysozyme The m/Z ratios of the (M + nH)+n ions are labeled. ESI-MS Solving the two simultaneous equations results in n = 9.0, or that the m/Z = 1590.6 species has 9 protons added. MW = 14,306.4 Da IDENTIFICATION OF BIOMOLECULES BY MOLECULAR WEIGHTS Strategy for a proteomic experiment using mass spectrometry and MS fingerprinting Two-dimensional (2-D) gel electrophoresis separation of the human prostrate proteome The proteins identified by mass spectrometric fingerprinting of the tryptic peptides are labeled. Protein proteolytic fingerprinting by MS Trypsin fingerprint of sera transferrin SEQUENCING BY MASS SPECTROMETRY How do we determine the sequence of a molecule such as a polypeptide by mass analysis? Residue Masses (Within a Polypeptide Chain) of the 20 Common Amino Acids Residue Masses (Within a Polypeptide Chain) of the 20 Common Amino Acids Strategy for assembling a complete polypeptide sequence from the sequences of proteolytic fragments. Peptide sequencing by tandem mass spectrometry (MS/MS). The fragmentation is accomplished in a collision chamber, which is filled with a neutral gas (argon or xenon) that breaks the peptide backbone of the peptide-this is called collision-induced dissociation (CID) and results in a set of product or daughter ions. Characteristics of the CID Daughter Ions Daughter ions produced by collisioninduced dispersion (CID) Proposed mechanism for generation of B- and Y-ions from CID fragmentation MS/MS spectrum of the peptide NFESGK Nomenclature for CID fragments CID Mass Spectrum Note: Not all b or y ions will present in the spectrum PROBING THREE-DIMENSIONAL STRUCTURE BY MASS SPECTROMETRY Comparison of the hydrogen/deuterium exchange rates for the protein thioredoxin Mapping the Core of hPAP Fibrils by H/D Exchange 15N-HSQC No D2O With D2O Spectra of hPAP in 90% DMSO Finding Disorder in Order When a Crystal Structure Is Not Enough Mechanisms for hydrogen/deuterium exchange in proteins EXERCISES EXERCISES