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Metalloprotein/Metallocofactor Mass Spectrometry Jeffrey N. Agar Brandeis University Diseases Involving Transition Metals • • • • • • • • • • • • • • • Hereditary hemochromatosis Type 1‐4 Aceruloplasminemia Hypotransferrinemia Menkes disease Wilson disease (autosomal recessive) ALS‐like symptoms in mice Antibiotic resistance plasmids in bacteria ALS Fatal Neonatal Lactic Acidosis Fatal deficiency of multiple respiratory functions dehydrogenase enzymes Swedish myopathy with exercize intolerance Fredrich ataxia Sideroblastic anemia Defect mitochondrial heme biosyn and COX def. Neurodegeneration (AD, PD, HD, Prion, etc) HFE, ?, TFR2, Ferroportin, respectively. Ceruloplasmin (CP) Transferrin (TF) MNK (ATP7A) WD (ATP7B) IRP Heavy metal transporters (MERP, etc) SOD1 C12orf62 NFU1 and BOLA3 cause a and 2‐oxoacid ISCU FXN GLRX5 COX10, COX15 (fatal cardiomyopathy) Mitochondrial dysfunction This is not an exhaustive list email [email protected] if you know of more… Goal of Lecture • Learn fundamentals of mass spectrometry (MS) interpretation • Learn fundamentals of mass spectrometry instrumentation in historical perspective • Overview of MS applications • Metalloprotein MS applications Mass Spectrometry at the Interface of Physics, Chemistry, and Biology. Mass Spectrometer Basics I. Ion Source (makes ions out of neutrals). II. Mass Analyzer (separates ions). III. Detector (Black Box For This Class). IV. Computer (Black Box For This Class). Mass Spectrometer Basics I. Ion Sources (the three most popular) ‐ ‐ ‐ Electrospray ionization ‐ THE ONLY METALLOPROTEIN FRIENDLY TECHNIQUE. MALDI ionization Electron Ionization II. Mass Analyzers (the only four) ‐ ‐ ‐ ‐ Magnetic Sector Quadrupole Time‐Of‐Flight Fourier Transform Physical Concepts Basics I. II. III. IV. Only charged particles are analyzed in MS. Negative attracts positive Magnets bend trajectory of MOVING charges Mass spectrometers perform three basic tasks. I. Weigh II. Purify III. Break (react) MS Basics‐ Monoisotopic Mass versus Average Mass. • Average Weight/Mass. – You are use to thinking in bulk terms (lots of molecules) about weight or mass. For example, if you purify a Mol of Cu, it will weigh 63.5463 grams. That’s Cu’s “standard molecular weight” or “molar mass” or “relative atomic mass” or “average mass.” – Obviously, no single atom of Cu weights 63.54 Daltons (Da, or amu)‐ that would require having half of a nucleon. The molecular weight of Cu is the weighted average of ~ 69% 63Cu and ~31% 65Cu. MS Basics‐ Monoisotopic Mass versus Average Mass. • Monoisotopic Mass (not weight!). – In mass spec we can often resolve the isotopomer distribution. If we can, the monoisotopic mass, which is a type of molecular mass (a mass of a particular atom or molecule), is a better (more accurate) description. – The picture to the right illustrates the mass spectrum of Cu. http://www.webelements.com/copper/isotopes.html MS Basics: The mass defect • Carbon is by definition 12.000000. • Using this standard, mass spectrometrist realized that no other atom mass ends in “.000000 • Nuclei that are more stable than carbon weigh a bit less (32S “weighs” 31.97 Da), and less stable weight a bit more (226Rn, with a few millisecond halflife, “weighs” 226.0309). • This is the result of differences in the relativistic mass of the nuclear binding energy (E=mc2)‐ particularly the mass of photons released when a particular element is created. • The nominal mass is the number of nucleons. • We can use the nominal mass in conjunction with the mass defect to infer molecular formula! For example a molecular mass of 720.000000 is C60, not C59H12 (720.084 Da). MS Basics: Mass Defect Mass Defect results from difference in binding energy per nucleon (E=mc2) MS Basics: Mass Defect Mass Defect results from difference in binding energy per nucleon (E=mc2) MS Basics: Resolution Resolving Power (m/z ∕ ∆ m/z) Resolution (∆ m/z) (full width of peak at half height, (FWHH or 10% of valley) High resolution >10,000 resolving power, FTMS > 100,000 MS Basics: Molecular Formula From Mass • One of the most useful things a mass spectrometer can be used for is to determine the formula (and often identity) of a compound using ONLY ITS ACCURATE MASS. • If you measure a compound that weighs 26.0140 Da, what is its molecular formula? • The key is using both the nominal mass (26) and the mass defect. • Remember that C12 is DEFINED as mass 12.00000000000000000000000000000 • What are the mass defects of the two elements present in this formula? • Upper mass limit of roughly ~600 Da (using an FTMS) for normal organic compounds MS Basics: How to interpret a mass spectrum 1. Assign charge state‐ in high resolution MS this is done by determining the distance in m/z between two isotopes of a molecule. The reciprocal of this distance is the charge state. For low resolution data use MAXENT or a charge state ruler. 2. Multiply the charge state by m/z and you have determined the molecular mass of a given peak within the mass spectrometer. 3. Next subtract mass that is an artifact of ionization to determine the actual mass. Charges are added during ionization in the form of proton adducts. MALDI usually adds a single proton and ESI multiple protons. Be careful‐ metals carry charges and are a special case that most mass spectrometrists wouldn’t think to account for. Basics – Calculate Mass ~674.350 m/z MS Basics Applied to Metals • Metals have huge mass defects, 63Cu = 62.9295989, making them easier to detect. Automated data processing programs wont account for metals. • Metals have characteristic isotope abundances‐ whereas only 1% of Carbon is heavy, 31% of Cu is heavy (63Cu). • Metals have characteristic nominal masses. • Metals usually have charges‐ It is critical for data analysis that you manually account for these charges being holes (not protons). Data analysis programs are not equipped to. Failure will result in a 1 Da overestimation in molecular mass per metal charge (so 4 Da on SOD1). A Brief History of MS Technology # of Mass Spectrometry Pubs Per Decade (decade ending in a given year, e.g. 2010 represents years 2000-2010) MS Technology: Discovery of the electron’s e/m (Nobel Prize Thomson) MS Technology: Thomson- Aston’s Parabola Mass Spectrograph. Nobel Prize 1922 MS Technology: Electrospray Ionization (ESI) enabled metalloprotein characterization In the Words of John Fenn: Nobel Prize for ESI 2002 Mass Spectrometry is the art of measuring atoms and molecules to determine their molecular weight. Such mass or weight information is sometimes sufficient, frequently necessary, and always useful in determining the identity of a species… Clearly the sine qua non (essential element) of such a method is the conversion of a neutral analyte molecules into ions.. In recent years, the efforts of many investigators have led to new techniques for producing ions of species too large and complex to be vaporized without substantial, even catastrophic, decomposition. MS Technology: Nanospray‐ more sensitive and gentler than ESI‐ better for metalloproteins ABI’s Lab On A Chip Advion’s Nanomate Matrix Assisted Laser Desorption Ionization (MALDI): Not good for metalloproteins‐ Often OK for small metal clusters (<1000 Da) Tanaka’s 2002 Nobel Prize MS Technology: Nanospray and MALDI in action MALDI Ionization Nanoelectrospray Taylor Cone Typical Sensitivities of Ionization Techniques MS Technology: Nier Sector Mass Spectrograph Nobel Prizes Thomson/Aston 1910, 1922 MS Technology: Cyclotron‐ Lawrence Nobel Prize. Predecessor of first Fourier transform (high res) mass spectrometry. Why is MS Technology so advanced? Preparative MS: The Manhattan Project Calutron magnet was 15 feet in diameter Original cyclotron below with blue background was a couple of inches. http://www.mbe.doe.gov/me70/manhattan/im ages/MassSpectrograph.jpg MS Technology: The Manhattan Project MS Technology: The Manhattan Project Lawrence wondering why all of the quarters in his pockets keep disappearing. Q‐FTMS Hans Dehmelt Nobel Prize 1989 Agar Lab FTMS is a Quad+TOF+ICR MS Applications: -Quantify small molecules including metallocofactors and artificial additives in food and humansoften uses triple quad as with small molecule lab Ben Johnson, Canadian athlete Furabazol, Anabolic steroid MS Applications ‐Identify explosives, biohazards, radioactive materials, toxic chemicals. MS Applications ‐Identify Chemical Composition =>Saturn Mission; Jupiter Mission (1982 Galileo); Comet Mission MS Applications ‐Identify Chemical Composition (Saturn Mission) Science 13 May 2005: Vol. 308. no. 5724, pp. 982 - 986 DOI: 10.1126/science.1110652 Ion Neutral Mass Spectrometer Results from the First Flyby of Titan J. Hunter Waite, Jr., et. al. MS Application: Compound ID Using Electron Ionization Fragmentation Ketone Major fragmentation peaks result from cleavage of the C-C bonds adjacent to the carbonyl. 4-Heptanone C7H14O MW = 114.19 Fifteen Years experience in providing mass spec libraries Over 600,000 measured and identified mass spectra Nearly 4 times the size of the NIST 2002 collection MS Applications: Proteomics using MS/MS Product Ion MS/MS (MS2) The Basis For The Proteomics Experiment 31MonoAverage letter letter isotopic Amino Acid Structure 647 (+2) at all m/z (+3) 1) Scan Q1 to get Intensity mass code code mass = mass spectrum (MS Survey) Alanine Ala A 71.03712 71.08 647 (+2) C3H5NO Amino Acid Masses Intens. x10 6 Intens. x106 648.9 3+ 433.5 3 4 649.4 3 2+ 649.0 2 2) Lock Q1 to Isolate only m/z 647 156.10112 156.19 And trap molecules in Q2 (collision cell) 2 Arginine C6H12N4O Arg R 649.9 1 1 650.4 Asparagine C4H6N2O2 2+ 464.0 Asn N 114.04293 114.10 0 640 642 Angio_tune_cid_000002.d: +MS 667.9 513.4 0 200 400 Aspartic acid C4H5NO3 e n s. x1 0 5 6 Asn or Asp 5 Cysteine C3H5NOS 4 600 800 Asp 1000 D 6 4 7 .4 115.02695 115.09 3) Scan Asx B Q3 to get Intensity at all m/z = MSMS (MS2; Collision Induced Dissociation MS (CID; CAD); Product Ion MS) Cys C 103.00919 103.14 7 8 4 .4 3 Glutamic acid Glu C5H7NO3 E 2 129.04260 129.12 5 3 4 .3 Glutamine C5H8N2O2 4 2 6 .2 1 3 8 2 .2 1 0 2 8 .6 Gln Q 9 1 3 .5 128.05858 128.13 8 7 0 .4 0 500 750 An g i o _ tu n e _ ci d _ 0 0 0 0 0 5 .d : +M S2 (4 3 2 .0 ) M a xRe s 1000 1250 644 646 648 650 652 Angio_tune_cid_000003.d: +MS 654 656 658 m/z MS Applications: 60,000 Year Old Protein MSMS The osteocalcin that became a star was extracted from fossilized bison bone, Bison priscus, which was radiocarbon-dated back almost 60'000 years. Bison priscus, more commonly known as the steppe bison, though now extinct is quite well known thanks to prehistoric paintings in Paleolithic caves and fossils found in permafrost. Automated Proteomics: Mascot ID Good for IP “pulldowns” of interacting proteins Metalloprotein MS applications: Don’t digest metalloproteins if you want to detect metals homogenize Many post-translational modifications, including metals lost following endoproteinase digestion. digest Structural information can be lost during digestion, especially unexpected modifications Metalloprotein MS Applications: High res methods and algorithms for isotopic fine structure detection Substance P, human neuropeptide (C63H98N18O13S) m = E / c2 A A transformative transformative effect effect because because higher higher magnetic magnetic field field increases increases the the upper upper mass of observing mass of observing isotopic isotopic fine fine structure structure and and isotopic isotopic fine fine structure structure is is a a powerful powerful tool tool for for elemental elemental composition composition confirmation confirmation Karabacak, NM et. al. 2010 JASMS . Li., L. 2009 JASMS and 2010 RCM. Metalloprotein MS Applications: Metal Content By Mass AND Charge State Distribution G85R SOD1- Causes ALS (left) WT SOD1 (right) tens. x107 G85R holo in pure w ater_000003.d: +MS, Deconvoluted (MaxEnt) Single metal 8 Intens. x107 4 Deconvoluated spectrum 6 AS holo in pure w ater_000001.d: +MS, Deconvoluted (MaxEnt) Deconvoluated spectrum holo 3 4 2 Single metal+98 Da 2 1 holo apo 0 15825 Single metal apo 0 15850 15875 15900 15925 15950 15975 16000 16025 m/z 15740 15760 15780 15800 15820 15840 15860 15880 15900 15920 m/z Metalloprotein MS Applications: Compare Experimental and Simulated Spectrum Intens. x108 15906.779 1.0 Red trace is experimental A4V spectrum and black trace is simulated spectrum w/ Cu/Zn each as 2+ 0.8 0.6 0.4 0.2 0.0 15890 15895 15900 15905 15910 15915 A4V holo in pure water_000001.d: +MS, Deconvoluted (MaxEnt) A4V holo in pure water_000001.d: C681H1083N203O225S2CuZn, M,15895.76 15920 15925 15930 m/z Metalloprotein MS Applications: Effects of Metals on Protein Structure Using Charge State Distribution Purification of metallated and apo SOD1 (multiple charge states) within the mass spectrometer Quadrupole mass filter to eliminate low m/z As Isolated 0.2 kcal/mol Time-Of-Flight cutoff to eliminate high m/z Metalloprotein MS Applications: Effects of Metals on Protein Structure Using H/D Exchange WT (left panel) versus fALS mutant G85R (right panel) superoxide dismutase showing percentage deuteration levels. Molnar K S et al. J. Biol. Chem. 2009;284:30965-30973 H/D Exchange: 13 fALS SOD1 Variants Intact Protein H/D‐Ex MS: Cu and Zn loss 220 200 G41S Cu+Zn Mixture 160 Δ mass (Da) apo 180 140 120 100 80 60 WT Cu+Zn G41S (+8) holo 40 WT (+8) holo 20 G41S (+12) apo 0 0.0 20.0 40.0 time (min) Loss of metal: enormous structural consequence Apo G41S Apo G41S Apo WT Cu/Zn G41S and WT Cu/Zn WT 0.2 kcal/mol Lose Cu/Zn >> structural consequence than mutation Metalloprotein MS Applications: Metal Oxidation State and Nuclearity Figure 8 ESI-FTICR mass spectrum of [3Fe-4S]ferredoxin from Pf (top) in which the intact cluster can be seen at m/z 1866. Peaks at higher mass-to-charge ratios represent oxygen, sodium, and sulfur adducts, whereas more intense peaks at lower mass-to-charge ratios represent losses of inorganic sulfide from the cluster. The mass spectrum of 34S labeled [3Fe-434S] protein from Pf is also shown (bottom). The degradation of the protein is similar to the wild-type [3Fe-4S] protein. The loss of inorganic sulfide from the iron−sulfur cluster is confirmed by this example, as the mass difference between the sulfur-loss peaks is 34 Da, whereas in the unlabeled form the difference is 32 Da. Published in: Keith A. Johnson; Marc F. J. M. Verhagen; Phillip S. Brereton; Michael W. W. Adams; I. Jonathan Amster; Anal. Chem. 2000, 72, 1410-1418. DOI: 10.1021/ac991183e Copyright © 2000 American Chemical Society