Introduction to Proteomics - Human Proteomics Program - University ...

Introduction to Mass SpectrometryTable of Contents• What is mass spectrometry?• Where can mass spectrometry be used?• How did mass spectrometry originate?• A history of mass spectrometry• What is a mass spectrometer?• Concepts of mass analysis, ions and charges• What’s inside a mass spectrometer?• What are ionization techniques?• How does mass analyzer work?• How does mass spectra look like?• What is tandem mass spectrometry?• How to use mass spectrometry for quantitation?• What are accuracy and precision?Ying Ge, Ph.D.Human Proteomics ProgramSchool of Medicine and Public HealthUniversity of Wisconsin-Madison* This presentation is solely used for public education purpose.

What Is Mass SpectrometryMass Spectrometrist Definition:Mass spectrometry is a powerful analytical technique that is used toidentify unknown compounds, to quantify known materials, and toelucidate the structure and chemical properties of molecules.Simple Definition:Mass spectrometry is a technologythat helps scientists:1. identify molecules present insolids, liquids and gases.2. determine the quantity of each typeof molecule.3. determine which atoms comprise amolecule and how they arearranged.© Donald H. Chace, 2005 Pediatrix Medical Group

Where Can Mass Spectrometry Be Used?Mass spectrometry provides valuable information to a widerange of professionals: physicists, chemists, biologists,physicians, astronomers, and more.Mass spectrometry is used to...• Detect and identify the use of steroids in athletes• Monitor the breath of patients by anesthesiologists during surgery• Detect heart attack from a blood test• Determine the composition of molecular species found in space• Locate oil deposits by measuring petroleum precursors in rock• Monitor fermentation processes for the biotechnology industry• Detect dioxins in contaminated fish• Determine how drugs are used by the body• Determine gene damage from environmental causes• Sequencing proteins, nucleic acid and oligosaccharides• Determine the age and origins of specimens in geochemistry andarchaeology• … …

How Did Mass Spectrometry Originate?The technique of mass spectrometry begins in J.J. Thomson'svacuum tube in 1889 – demonstrate the existence of electrons and"positive rays“.©2003 AMERICAN CHEMICAL SOCIETY

A History of Mass Spectrometry©2005 Scripps Center for Mass Spectrometry

Nobel Prize in Mass Spectrometry• In 1899, J.J. Thompson built the first mass spectrometerawarded Physics Nobel Prize in 1906• In 1919, Francis Anston observed isotopes using massspectrometry, awarded Chemistry Nobel Prize in 1922• In 1953, Wolfgang Paul invented ion quadrupole and ion trapmass spectrometers, awarded Physics Nobel Prize in 1989• In 1988-9, John Fenn and Koich Tanaka developed ESI andMALDI for ionizing large molecules, awarded Chemistry NobelPrize in 2002

What Is A Mass SpectrometerMass Spectrometrist Definition:A mass spectrometer is an instrument that measures the masses ofindividual molecules that have been converted to ions, i.e. moleculesthat have been electrically charged.Simple Definition:A machine used to weighmolecules.A molecular scale.© Donald H. Chace, 2005 Pediatrix Medical Group

Concept of Mass AnalysisSorting/counting coins analogyMixture of moleculesMolecules of different weight, sizeSeparation by “mass”spectrumquantityPocket change (mixture of coins)Penny, dime, nickel, quarter, half $Sorting change by value or sizeConcept of visual interpretation83542© Donald H. Chace, 2005 Pediatrix Medical Groupdime cent nickel quarter half $value

Concept of Ions and ChargesA mass spectrometer measures mass-to-charge ratio (m/z)instead of mass. Therefore, in order to be measured, moleculesneed to be charged (or ionized).1. An ion is an electrically chargedmolecule.3. A mass spectrometer “weighs”molecules electronically byattracting and repelling ions.Consider magnets. Oppositesattract. Like charges repel.2. An ion can be positively (+) charged ornegatively (-)charged. Consider the poleson a battery.© Donald H. Chace, 2005 Pediatrix Medical Group

How Does A Modern Mass Spectrometer Look LikeAn actual mass spectrometer ranges in size from about the size of amicrowave oven to large research instruments that dominate entire rooms.Agilent MSD(Quadrupole)Bruker Apex III FTMS( Fourier Transform IonCyclotron Resonance)Thermo LTQ(Ion Trap)

What’s Inside A Mass SpectrometerA mass spectrometer is composed of five essential parts:SampleInletHigh Vacuum (10 -6 –10 -9 Torr)MarathonAnalogyIon transferIon SourceMassAnalyzer DetectorRecorder1. Inlet: introducing samples from ambient room pressure into ion source2. Ion source: converting sample molecules to ions3. Mass analyzer: separating ions according to their mass4. Detector: detecting ions and amplifying the signal5. Recorder: receiving signal from detector, further amplifying, recording,creating mass spectrum

How Is The Sample Introduced Into Mass Spectrometer?An inlet system is needed to transfer the sample from theatmospheric pressure (760 Torr) into the source as massspectrometers are operated in vacuum (~10 -6 -10 -9 Torr).Common Inlet Systems:1. Chromatography inlets:Gas chromatography (GC)Liquid chromatography (LC)Capillary electrophoresis (CE)Agilent HPLC2. Syringe for direct infusion3. Probe or plateStandard InfusionSyringe Pump fromHarvard Apparatus

What Are Ionization Techniques?Ionization is a process of charging a molecule. Molecules must becharged in order to measure them using a mass spectrometer.“It makes a molecule fly in a mass spectrometer.”Common Ionization TechnologiesBasic TypeGas Phase(for volatilecompounds)Condensed PhaseDesorption(for nonvolatilecompounds)Name and AcronymElectron Ionization (EI)Chemical Ionization (CI)Fast atom bombardment (FAB)Matrix-assisted desorption/ionization (MALDI)Atmospheric pressure chemical ionization (APCI)Electrospray ionization (ESI)

ESI and MALDIESI and MALDI are the two ionization techniques that makemolecule elephant (i.e. Proteins) fly.ESI (Electrospray):formation of charged liquiddroplets from which ionsare desolvated or desorbed.ESIMALDI+++ +MALDI (matrix assisted laserdesorption ionization)impact of high energyphotons on a sample imbeddedin a solid organic matrix.++++© Donald H. Chace, 2005Pediatrix Medical Group

How Does Mass Analyzer Work?The analyzer uses dispersion or filtering to sort ions according totheir mass-to-charge ratios or a related property.The most common analyzers:1. Magnetic sectors:Magnetic field affect radius of curvature of ions -> m/z2. Quadrupole mass filtersScan radio frequency field-> m/z3. Quadrupole ion traps (QIT)Scan radio frequency field-> m/z4. Fourier transform ion cyclotron resonance (FT ICR)Image current – ion cyclotron frequency -> m/z5. Time-of-flight mass (TOF)Flight time - correlated directly with ion’s m/z

How Does Time-Of-Flight Mass Analyzer Work?SampleIon beamLaser pulseField-free regionFlight Tubedm/z = 2Vt 2 /d 2DetectorTime-of-flightm/z = 2Vt 2 /d 2m/z determined fromion’s time of arrivalSmaller ions - higher Vreach the detectorearlier than larger ionsRabbit runsfaster than Sheepand faster thanElephant.

How Does A Mass Spectrum look like?- A Bar Graph FormatMolecular ion: An ion formed by the removal of one or more electrons to form apositive ion or the addition of one or more electrons to form anegative ion, also called parent ion or precursor ion.Fragment ion: A product ion (or daughter ion) resulting from the dissociationof a precursor ion.Relative abundance10050fragment ion(or daughter ion)molecular ion(or parent ion)13C isotope peak13C 2isotope peak0m/z (mass to charge ratio)

How Does Mass Spectra look like?- Isotope EffectIsotope: one of two or more forms of a single element; the atoms of eachisotope have the same number of protons but different numbersof neutrons in the nuclei of an atom.Element M, % M+1, % M+2, %Mass SpectrumH 1, 100 2, 0.015Cl 35, 10037, 32135 37

+What Is Tandem Mass Spectrometry?A sample is measured (weighed) in the first mass spectrometer, then a certain ionis selected and broken into pieces in the collision cell, and the fragments (production/daughter ion) are weighed in the second mass spectrometer.Coupling two stages of mass analysis (MS/MS) to identify compounds in complexmixtures and in determining structures of unknown substances.++Gas/Heat/Light/ElectronFragmentationchamber+++MS2Product ion/Daughter ionMS1+Ion Selection(parent ion/precursor ion)+

What Is Tandem Mass Spectrometry?Simple Definition: Two mass spectrometers joined by a chamberthan breaks apart molecules. **This definition is appropriate for tandem-in-space but not for tandem-in-time.Puzzle analogyMS #1SortingmoleculesFragmentationChamberBreakingmoleculesMS #2SortingPieces2 21111© Donald H. Chace, 2005 Pediatrix Medical Group

What Is Tandem Mass Spectrometry? (Cont’d)MoleculesComprised of atomsArrangement of atomsgives molecules function.Word AnalogyWordsComprised of lettersArrangement of lettersgives words meaning.Special groups of atomsmake functional groups.Common side chains = acidsH 2NCH 2COOC 4H 9H 2NCH(CH 2)COOC 4H 9H 2NCH(C 6H 5)COOC 4H 9Detect all molecules containing abutyl formate functional group from anα-amino acid – Product ion scan.Use a neutrol loss scan function todetect only molecules that lose abutylformate function group weighing102 Da. Neutral loss scan© Donald H. Chace, 2005 Pediatrix Medical GroupSpecial groups of lettersmake syllables.Common endings = suffixesTalkingWritingPlayingList all words containing “ing” in thebook of abstracts.Use a computer to search for thestring “ing” and it displays all wordscontaining “ing.”

How to Use Mass Spectrometry for Quantification?- Isotope Dilution Mass SpectrometrySimple Definition:It is a method that measures how much compound X is present in a liquid, solid orgas. This method uses non-radioactive elements called stable isotopes to make acomparison of compound X with the standard that contains the stable isotope.Since the amount of stable isotope standard is known we can calculate how muchcompound X is present.Jelly Bean AnalogyHow much phenylalanine isHow many Cherry Jelly Beans arepresent in Bloodin the jar?1. Add a marker orstandard10 picomolesd 5Phenylalanine10 BlueberryJelly Beans10 uL (1 drop)2. Obtain a sample.1 oz© Donald H. Chace, 2005 Pediatrix Medical Group

How to Use Mass Spectrometry for Quantification?- Isotope Dilution Mass Spectrometry (Cont’d)Jelly Bean AnalogyHow many Cherry Jelly Beans arein the jar?How much phenylalanine ispresent in BloodIon countSort Phenylalanine by MS/MSCount how many.200010002,000 Phe1,000 d 5 -Phe3. AnalysisSort jelly beans in thecup by flavor/color Counthow many.232234. Calculationsa.) 3 Cherry Red, 2 Blueberry in 1 oza.) 2,000 Phe, 1,000 i.s.b.) 3/2 ratio of Cherry to Blueberryb.) 2/1 ratio of Phe to I.S.c.) 10 Blueberry added to 1 oz.c.) 10 pmol i.s. added to 1 mL bloodd.) 10 x 3/2 = 15d.) 10 x 2/1 = 20 pmole.) 15 Cherry Red Jelly Beans in Jare.) 20pmol Phe per mL of blood© Donald H. Chace, 2005 Pediatrix Medical Group

What Are Accuracy and Precision?Mass Spectrometrists always say:Mass Spectrometry is very accurate and precise.Dart Board AnalogyAccurate & PrecisePrecise, Not AccurateAccurate, Not PreciseNot Accurate, Not Precise© Donald H. Chace, 2005 Pediatrix Medical Group

References1. Donald H. Chace , “Mass what?”, © Donald H. Chace, 2005Pediatrix Medical Group.2. Stu Borman, Hailey Russel, and Gary Siuzdak, “A Mass SpecTimeline”, SEPTEMBER 2003 TODAY’S CHEMIST AT WORK,©2003 AMERICAN CHEMICAL SOCIETY3. Chia M. Chiu and David C. Muddiman “What is mass spectrometry”,American Society for Mass Spectrometry: Educationhttp://www.asms.org/whatisms/index.html4. Gary Siuzdak, http://masspec.scripps.edu/MSHistory/ ©2005 ScrippsCenter for Mass Spectrometry

Acknowledgements• The author would like to thank Dr. Donald H. Chace for kindlygranting the permission to use the materials in his poster, “Masswhat?”, © Donald H. Chace, 2005 Pediatrix Medical Group.• The author would like to thank Dr. Gary Siuzdak for kindlygranting the permission to use the materials from his websitehttp://masspec.scripps.edu/MSHistory/ ©2005 Scripps Centerfor Mass Spectrometry.• The financial support was provided by WisconsinPartnership Fund for a Healthy Future.