Mass Spectrometry Basics, Ionization Methods, and Mass Analyzers PDF

Biological Applications of Mass Spectrometry (MS)  Applications of MS in Biology In general, MS can be used to study structural features of many different types of molecules (biological molecules, environmental pollutants, inorganic complexes, toxins, drugs, etc.) MS is most powerfu...

Biological Applications of Mass Spectrometry (MS)  Applications of MS in Biology In general, MS can be used to study structural features of many different types of molecules (biological molecules, environmental pollutants, inorganic complexes, toxins, drugs, etc.) MS is most powerful (in my opinion) as a tool for biomolecular structure elucidation. Biomolecules such as proteins and nucleic acids are large polymers with complex structures. MS is ideally suited to handle this complexity. Soft ionization has lead to biomolecule analys Many of the recent advances in MS instrumentation have been fueled by a need for better methods of biomolecular analysis. We will look at some of the many different ways that mass spec has been used to study fundamental biological processes such as protein folding, enzyme function, biosynthesis, and protein-protein interactions.  Biology Primer – The Central Dogma DNA Transcription There’s a correlation RNA (DNA to mRNA) between DNA (gene) polymerase sequence and protein amino acid sequence mRNA Translation Ribosome (mRNA to protein) tRNA protein  Biology Primer - Protein Function Catalysis – enzymes Structural – keratin Transport – hemoglobin Trans-membrane transport – Na+/K+ ATPases Toxins – rattle snake venom, ricin Contractile function – actin, myosin Hormones – insulin Storage Proteins – seeds and eggs Defensive proteins – antibodies Biology Primer – Protein Structure here the fun begins Interaction of local structures Protein function is related to protein structure MS can characterize all structures 4 levels of protein structure Sequencing Proteinabundance H Dexch nativest primary – the linear sequence of the polymer, composed of the 20 amino probing acids secondary – certain amino acid sequences H-bond to form helices and sheet tertiary – secondary structural elements fold up in 3-dimensions to form native structure quaternary – non-covalent interactions between multiple proteins Studying Protein Structure with MS MS shines w̅ large Dyn proteins bc harder to crystalize or ge NMR No spacial resolution Buthavingan idea ofhow things move maybe en Complex mixtures Curr. Opin. Struct. Biol. 2011, 21, 641  Why are proteins good models for learning about MS? The structural and functional diversity of proteins is vast Proteins have inspired the development of many different types of mass spectral analyses. Almost all of the modern mass analyzers, and the two most common ionization modes (ESI and MALDI) are routinely used to study proteins and biological systems Proteins are excellent model systems for understanding the many different ways that mass spectrometry can be used to study and discriminate molecular structures What do mass spectrometers do? Mass spectrometry (MS) is a suite of analytical techniques whereby chemical species are ionized, transferred to the gas phase, and then sorted based on their mass to charge ratios (m/z) Requiresgas phase ions i must ionize sample b c mass MS is a gas phase technique. works w̅ charge acceleration spec We are dealing with ions. Molecules to be analyzed must be ionized in order to respond to the electric fields used in the mass spectrum. An ion in an electric field has potential energy that is proportional to charge (ez) and the potential difference (U) of the field. As the field accelerates the ion, the potential energy is converted into kinetic energy. Basic Components of All Mass Specs High Vacuum System Introduce sample HPLC GC Ion Mass Data Inlet source Analyzer Detector System Photomultiplier lifted EYE p etc Getsignal electrode detector Vacuum – needed to prevent ion annihilation via collision with other gas molecules Inlet – how samples get into ion source (GC, LC, direct infusion) Ion Source – hardware that ionizes sample (ESI, MALDI) Mass analyzer – hardware that separates/manipulates ions (TOF, quadrupole, ion trap, FT-ICR, orbitrap, etc.)  The Mass Spectrum The output of an MS experiment is a mass spectrum. The mass spectrum is a plot of relative intensity (on the y-axis) vs. m/z (on the x-axis) The mass spectrum is a histogram of which ions are detected by the mass analyzer The resolving power of the mass analyzer can greatly affect the appearance of this histogram. Mass spectra can contain all sorts of information on a compound (or a mixture of compounds) depending on how the ions were generated and analyzed/manipulated once in the gas phase  Examples of Mass Spectra EI mass spectrum of phenol (C6H6O, monoisotopic mass = 94.04132 g/mol) Intact ion The Parent ion, in this case, is also d the base peak (i.e. the most intense ion to which all others are normalized) Daughter ions (formed by fragmentation of parent ion by the EI source)  Examples of Mass Spectra MALDI TOF spectrum of IgG (a large protein with MW near 150,000 g/mol) For biomolecules, MWs are often given in units of Dalton (Da), which are equivalent to g/mol M Not sticks broad distribe MALDI typically generates MH + b c resolution w̅ 40000 much larger sample Relative Abundance It ion 30000 (M+2H)2+ Note the difference in m/z 20000 scale from last spectrum. Mass spectrometers cover 10000 a vast range of m/z values (M+3H)3+ 0 50000 100000 150000 200000 m/z  Examples of Mass Spectra ESI TOF spectrum of Horse Heart Myoglobin (MW = 16951.48 Da) ESI gives many charge states Some mass spectrometers generate multiply charged ions Relative Abundance +13 charge state +12 charge state +11 charge state m/z  Important Concepts - Monoisotopic Mass Monoisotopic mass is the mass of a compound calculated using the isotopic mass of the most abundant isotope of each atom type. Most abundant isotope for each element can be readily looked up Most abundant isotopes relevant for organic compounds: 1H, 12C, 14N, 16O, 32S, 31P So, to calculate monoisotopic mass for glucagon (C153H224N42O50S): 12C 1H 14N [M]mono = 153 x (12.000000) + 224 x (1.007825) + 42 x (14.003074) + 16O 32S 50 x (15.994915) + 1 x (31.972071) isotopic masses = 3481.5997 Monoisotopic mass uses most common isotope mass  Important Concepts - Average Mass Average mass (molecular weight) is the mass of a compound calculated using the atomic masses of each of the constituent atoms. Average mass is a weighted average calculated using the atomic masses read off of the periodic table So, to calculate average mass for glucagon (C153H224N42O50S): atomic masses from periodic table [M]avg= 153 x (12.011) + 224 x (1.008) + 42 x (14.007) + 50 x (15.999) + 1 x (32.066) Average mass uses average mass molecular weight of = 3483.785 all isotopes for atoms  Identifying masses on a mass spectrum Theoretical mass spectrum of glucagon (a peptide hormone) monoisotopic mass - if isotope distribution is asymmetric, the lowest m/z peak will be the monoisotopic mass average mass – the weighted average of the isotope peaks most abundant mass Most MS systems now have 30 40k resolution i Can see isotopalogues Not all sample will be I isotope Asymetric distribution typically means monoisotopic mass is smallest Not an actual peak bc based on weighted average  Monoisotopic mass is not always observable in a mass spectrum Spectrum of [glucagon + H]1+ ion at a resolving power of 10,000 Spectrum of [glucagon + H]1+ ion at Monoisotopic a resolving power of 1,000 mass In order to observe the monoisotopic mass, the mass spec must be able to resolve individual isotopes in the relevant m/z range  Monoisotopic mass is not always observable will be within monoisotopic If compound is very large (MW > 10,000), go monosiotopicnoise mass may not be visible, even if resolution of mass spectrometer is very high. Why? V large molecule monoisotopic bc more atoms chance that the whole sample is all common isotope Hypothetical C100 compound Hypothetical C1000 compound (resolution = 10,000) (resolution = 100,000) Monoisotopic Monoisotopic Mass Mass W gausian 1K Important MS Concepts - Resolving Power Defined as RP = m/m m = m/z of ion m = full width at half max (FWHM) m/z = This spectrum for glucagon was 3481.6 simulated m = 0.34816 at m/z = 3481.60 (RP = 10,000). Must know what Mz range to sample so you know expect w̅ 1/2 max m = 0.34816 what kind of resolving power you nee  What Resolving Power do you need? [M]mono = 3481.5 RP = 3500 (RP/[M] ~ 1) – can only see envelope RP = 7000 (RP/[M] ~ 2) – isotopic resolution RP = 35,000 (RP/[M] ~ 10) – baseline resolution (RP ~ 30-60k easily obtained with modern TOF analyzer) RP should be 2x MW of molecule As a general rule, you will need a resolving power of approximately 2x the molecular weight of your molecule in order to get isotopic resolution  Resolving Power of Modern Mass Analyzers Analyzer Resolving Power Cost of new instrument ($$) (FWHM) Quadrupole (Q) ~ 1,000 ~ 50 – 200 k Ion Trap ~ 1,000 ~ 50 – 200 k Time of Flight (TOF) ~ 10,000 ~ 200 – 500 k High resolution TOF ~ 50,000 ~ 500 – 700 k Oribitrap ~ 100,000 ~ 1000 k FT ion cyclotron ~ 1,000,000 > 1000 k resonance (FT-ICR) A variety of mass spectrometers with a range of RPs are commercially available Type of analyzer to use depends largely on budget and the types of samples you need to run  Important Concepts - Mass Accuracy Mass Accuracy – the degree to which a measured mass value ([M]obs) approaches the true value ([M]calc) Example: The monoisotopic mass of glucagon: [M]calc = 3841.5997 [M]obs = 3481.61 The mass for the monoisotopic peak observed in the mass spectrum: [M]obs = 3841.61 Mass Accuracy = ([M]obs – [M]calc)/[M]calc = 2.7 x 10-6 = 2.7 ppm If mass accuracy is only 5ppm you can only report to 5 SF  Mass Accuracies of Typical Mass Analyzers Analyzer Mass Accuracy Linear Ion trap ~ 50 – 200 ppm Triple Quadrupole ~ 3 – 5 ppm Time of Flight (TOF) ~ 0.5 – 5 ppm Oribitrap ~ 0.5 – 1 ppm FT ion cyclotron resonance (FT-ICR) ~ 0.1 – 1 ppm Mass accuracy of an instrument is usually determined through calibration against a standard sample (NaI is very common for ESI) The mass accuracy of instrument should always be kept in mind when reporting data…. Series of evenly spaced peaks Compare observed to calculated to get mass accuracy  To summarize: monoisotopic mass – calculated using the masses of the most abundant isotope of each type of atom (12C, 14N, 16O, etc.) average mass – calculated using the atomic mass of each type of atom (i.e. the weighted average taking into account the natural abundance of each isotope) For small molecular weight compounds (< 10,000 Da) analyzed by mass spectrometers with good resolving power, the monoisotopic mass is usually visible in the mass spectrum. There are many different types of mass spectrometers with a range of resolving powers How do we pick the right mass spectrometer for the type of analysis that we need to do? We must understand more about the hardware components of common MS instruments: – Inlet – Ion Sources – Mass Analyzers Basic Components of All Mass Spectrometers High Vacuum System Ion Mass Data Inlet source Analyzer Detector System  Why is Vacuum Needed? In a typical MS, ions must travel between 0.1 and 5 m (from the point of ionization to the detector) you want to avoid collision w̅ molecules in air Mean Free Path () – the average distance traveled by a gas phase ion before it collides with a molecule from air:  = 1/N  = collision cross section (Å2) between ion and gas molecule (based roughly on surface areas of the ion and gas) N = gas number density size of your sample size likelyhood of collision  Why is Vacuum Needed? Pressure (Torr) Gas Number Density (molecules/cm3) Mean Free Path ()* 760 (1 atm) 2.7 x 1019 1 m 1 3.5 x 1016 0.05 mm 0.1 3.5 x 1015 0.5 mm 0.01 3.5 x 1014 0.5 cm 0.0001 3.5 x 1013 5 cm 1 x 10-4 3.5 x 1012 50 cm 1 x 10-6 3.5 x 1010 50 m 1 x 10-8 3.5 x 108 5 km *Calculated for  = 50 Å2 (typical of a small peptide) Longer X α resolving power ddd Pressure Without sufficient vacuum, ions would never reach the detector i In some MS experiment, ions are intentionally collided with gas molecules. This leads to ion fragmentation and can be used to structurally characterize the ion. In these fragmentation experiments, sections of the mass spec have higher pressure where collision/fragmentation occur. The other sections remain under vacuum to maximize detection of fragment ions.  Basic Components of All MS systems High Vacuum System Ion Mass Data Inlet source Analyzer Detector System Must convert analytes to ions (separation by MS relies on m/z) – need an ion source Many types of ion sources - each type is suitable for specific types of analytes Common ion sources: Electron Ionization (EI), Chemical Ionization (CI), Inductively Couples Plasma (ICP), Electrospray Ionization (ESI), Matrix-Assisted Laser Desorption/Ionization (MALDI) Common Ion Sources  Ion Sources for Biomolecules Biological molecules like proteins and nucleic acids are large polymers composed of smaller subunits (amino acids and nucleotides, respectively) Ion sources for biological molecules must be “soft” – i.e. the process of ionization cannot lead to fragmentation of the molecular ion. Otherwise, much of the interesting information regarding the structure of the biomolecule will be lost To keep the molecular ion intact, biomolecules are often ionized with: 1. Matrix-Assisted Laser Desorption/Ionization (MALDI) 2. Electrospray Ionization (ESI) Typically more for proteins  Matrix Assisted Laser Desorption/Ionization (MALDI) Laser is used to ionize samples embedded in a solid matrix will lose folding from Analyze is mixed and proteins dryingout crystalized with matrix, usually on a stainless steel plate The plate is placed in a Creates plume vacuum chamber and irradiated, usually with a matrix destroyed but UV-laser analyte stays okay MALDI is very sensitive (can detect fmol quantities) Like ESI, MALDI is a “soft” ionization technique Produces mostly 1+ ions  established w̅ MALDI well MALDI target Proteins are Peptide  Sample Preparation No I size fits all setup will need to adjust matrix solven dried droplet preparation method for each sample crystalization strongly affected by solvent and components within the sample (salts, etc.) H2O solvents lead to slower crystalization and bigger crystals, but poorer sample-to-sample reproducibility Organic solvents like acetonitrile are often included to speed up cyrstalization Even with best methods and careful handling, reproducibility can difficult – parameters for data acquisition often need to be slightly adjusted  Sample Clean-Up is Critical for MALDI Salts and other components of a sample can drastically supress ionization by MALDI Zip-tips are commonly used to rapidly desalt samples prior to MALDI. Tip contains a stationary phase (such as C18 or ion exchange resin) that allows user to separate analyte from other junk prior to spotting sample on MALDI target  MALDI Matrices Most common matrices: -cyano” matrix DHB sinapinic acid (-cyano-4- (2,5-dihydroxy (3,5-dimethoxy-4- hydroxycinnamic acid) benzoic acid) hydroxycinnamic acid) Good w̅ peptides overall w̅ acid supplement formic Important properties of matrices: Low MW (allows easy vaporization) dry/crystalize quickly with analyte Polar, with good aqueous solubility Often acidic, which facilitates protonation/ionization of analyze Strongly absorb light in 250-350 nm (UV range)  Matrix Selection difficult to predict which matrix will work best Combine like polari highly polar analytes work better with highly polar matrices, and analyte matrix nonpolar analytes are preferably combined with nonpolar matrices Matrix acidity data may help to judge its protonating effect Sometimes, mixtures of different matrices work best to predic Hard what todo mixtures migh work too  Ionization Mechanism Matrix absorbs laser light, causing desorption of ionized matrix molecules into a plume Analyte goes along for the ride Within the plume, a series of proton and charge transfers leads to ionization of analyte  MALDI is well-suited for Good for screening biomolecules Fast Easy Sensitive MALDI ESI Cheap Antibody cytochrome C Was first MS technique suitable for mass analysis of large biomolecules proteins > 300,000 Da can be kept intact MALDI usually generates singly charge molecular ions ([M+H]1+) MALDI is fast, cheap, easy, and sensitive Also some very interesting recent uses in MALDI imaging  MALDI Imaging Soft ionization method Used whenintere in space distrib cover it Eachpixelgets its own MS Can get biomarker in real fi for cancer surgery Can study the spatial distribution of many molecules in a complex sample. Rapidly distinguish disease vs. healthy cells/tissue Used to study chemical interactions between microorganisms (i.e. antibiotic production)  Chemical Warfare between bacteria bacteria close together Growing interaction MALDI senses cellcell Can use MALDI imaging to discover new antibiotics that bacteria use to kill each other 2002 Nobel Prize in Chemistry In 1985, Koichi Tanaka was first to show that soft laser desorption could be used to ionize proteins without destroying them. His technique was a little different than MALDI (they used Co nanoparticles in glycerol) MALDI was being developed simultaneously by Hillenkamp and Karas in Germany, but they did not show that it was soft enough to be applied to biomolecules until after Tanaka’s work. Transfer molecules to gas phase Non destructive  Electrospray Ionization (ESI) accomplishes transfer of ions from solution to gas phase at atmospheric pressure very useful for analysis of large, non-volatile, chargeable molecules such as proteins and nucleic acids ionization method of choice for LC-MS Combination w̅ separation makes it powerful very soft ionization, little fragmentation its extensive application in biology has made ESI the most commonly employed ion source  ESI generates charged droplets + ions attracted to counterelectrode Sample is in sprayer Droplet gets in electrolyte Droplet heated so small it breaks i shrinks e- Epplied charge generates spray charges derived from either polarity can be used to electrolytes and redox generate positive or negatively reactions at interface charged gas phase ions e- Electrolytic flow cell 1. electrical potential placed on spray needle by cylindrical electrode 2. charged ions arise from electrolyte additives, as well as from redox reactions that occur at needle/solution interface 3. charges of one polarity type (either + or -) are attracted towards counter electrode, generating the Taylor cone.  Important Solvent Characteristics for ESI Several features of solvent greatly facilitate ESI Only if youdon't care 1. Organic solvents (e.g. MeOH or acetonitrile) decrease surface I tension and help form smaller initial droplets. This facilitates solvent evaporation, droplet shrinking and transfer of analyte ions to gas phase. 2. Low concentrations of a volatile electrolyte – usually formic acid or acetic acid (for positive ion mode) or NH4OH (for negative ion mode). The electrolyte serves several purposes: 1. enables the charge separation necessary to establish the electrolytic flow cell 2. helps to generate more densely charged (and hence, smaller) initial droplets that will desolvate quicker 3. helps to ionize the analyte via acid/base chemistry desolvate Electrolyte must be volatile Cannot complex w̅ agnate must ex if Nat present your signal could be 9kt Natnot I  Ionization mechanism Solvent evaporates until droplet size reaches the Rayleigh limit At this point, electrostatic repulsion within droplet is stronger than the surface tension holding the droplet together This leads to Coulomb fission, where the droplet explodes into many tinier droplets. During fission, droplets lose a small percentage of mass and charge. This process repeats until there is no more solvent to lose, resulting in a gas phase ion. surface tension E ftp.tsifon Rayleigh Limit zR = Rayleigh charge R = radius of droplet  = surface tension 0 = vacuum permittivity Charge state distribution Δ w̅ ord  Ionization Mechanisms Depends on size and shape of molecule: Large molecules (e.g. folded proteins) ionize via charge residue model (CRM) Small molecules, peptides, unfolded proteins ionize via the ion ejection model (IEM) CRM good for large b c E to eject droplet w̅ small unfolded molecules inside interactions stabilizing transfered Charge important for to surface 1 most proteins CRM Model – analyte remains in center of droplet until solvent is evaporated and charge is transferred IEM Model – analyte samples space near edge of droplet and takes a portion of the surface charge as it escapes CRM model for folded proteins Folded myoglobin CRM is experimentally supported by observation ~ 5 nm that native ESI of Foldedstays globular proteins tends to in drop produce ions near the jhargestates Rayleigh charge of a similarly size water droplet (suggests size of droplet from which globular protein is Unfolded electrosprayed is similar myoglobin to the size of the protein itself) a a.net Picks up charge as leaves Rayleigh Limit  ESI produces multiply charged ions Another very useful property of ESI is its propensity to generate multiply charged ions This often allows even very large molecules (with MW > 10 6 Da) to be detected with standard mass analyzers (remember, MS measures m/z) Each charge state is it's own measurement Similar these mass spectra are all drawn on the same m/z scale Detectors ma'sanaly wide range of analyt How does charge state affect MS spectrum? Remember, MS separates species by m/z. We can deduce charge state and molecular weight from the spacing in the isotope peaks For compound with MWmono = 8876.28 Da: Spacing between Spacing 2 isotope peaks = 1/charge Spacing between adjacent isotopes = 1 m/z unit = 1/1 This is a 1+ ion monoisotopic [M+1H]1+ ion (m/z = 8877.29) How does charge state affect MS spectrum? Better resolving power Easier to distinguish overlaping signals Spacing between Charge stat isotope peaks = distinguishes 1/charge Spacing between between adjacent isotopes = analytes Asymetric peaks 0.33 m/z units = 1/3 can indicate monoisotopic In most cases use centroid This is a 3+ ion Subtract charging species monoisotopic MW = [(m/z)*3] – 3*MWH [M+3H]3+ ion = [2959.77*3] – 3*1.00794 (m/z = 2959.77) = 8876.28  ESI reduces fragmentation One of the main benefits to ESI is that it does not lead to extensive ion fragmentation Desolvation process actually cools the ion and ensures that excess thermal energy does not get bundled up into vibrational modes that can trigger fragmentation Contrast with EI, where an electron collides with the analyte and promotes formation of an excited state that relaxes through vibrational motions that lead to bond cleavage. The “soft” ESI ionization mechanism has revolutionized MS of biological molecules and led to a Nobel Prize for Prof. John Fenn, who first developed ESI-MS in the 1980s John Fenn (1918-2010) 2002 Nobel Prize in Chemistry  Mass Analyzers High Vacuum System Ion Mass Data Inlet source Analyzer Detector System MALDI ESI best for biological samples Mass Analyzer – hardware that manipulates, sorts, and characterizes ions Quadrupole TOF FT-ICR Similar to orbitraps morecommon but motion is better understood  Ion Kinetic Energy charge charge kinetic energy Potential These equations are valid for any ion in an electric field and pertain to all mass analyzers Note that kinetic energy depends only on the charge and potential through which the ion is accelerated Control E of ions i you can breaksamples in controlled  Quadrupole Mass Analyzer Quadrupole - also called quadrupole mass filter (QMF), “quad”, or simply abbreviated “Q” (as in the “Q-TOF” hybrid mass spec) Consists of 4 parallel, cylindrical rods arranged around the ion axis (where gas phase ions are directed) Selectively transmit narrow range Easy to coup quads to syste Gets lost filtered out on milliseco scale Opposite pairs rods are connected electrically. Opposing DC voltages (+ and -) and RF voltages (180° out of phase) are placed on each pair of rods. Ions of the appropriate m/z traverse the quadrupole with a stable trajectory. Other ions collide with the quadrupole and are annihilated.  Electric Field Diagram E = 0 Optimal shape but susceptible to defect It Dc Rods are often cylindrical (left), though hyperbolic rods (right) have been shown to be optimal. Typical rod dimensions: 10-20 mm diameter, 15-25 cm length Cylindrical rods are often used because they are cheaper and easier to manufacture and because even slight distortions to rod geometry can lead to severe losses in ion transmission and resolution  How Exactly a Quad Works Ions entering the quad are first accelerated by a 5-10 V potential to give ions of same charge state uniform kinetic energy Rod electrodes have both RF (AC) and DC voltages applied One pair of rods has: +U + Vcos(t) Equal magnitude opposite The other pair has: -U – Vcos(t) vector DC potentials are RF voltages are opposite polarity 180° phase shifted U – DC voltage (typically in the 102-103 V range) V – RF voltage (typically in the 102-103 V range)  – radio frequency (typically 1-4 MHz)  How a Quadrupole Works Consider the effect that an alternating RF voltage would have on a positively charged ion (ignoring DC for the moment). The polarity of the RF voltage oscillates in time... Ions will track the polarity of the RF When amplitude of RF is +, When amplitude of RF is -, positively charged ions are positively charged ions are pushed to center attracted towards electrode Positively + + Cylindrical charged electrode pair ions + + The motion of ions in the field depends on: Ion Charge – more charge = ion feels stronger force from RF electrode Ion Mass – ion momentum affects how quickly ion changes direction in RF field Field Strength – magnitude of force applied by RF Larger mass dRFmoveme Frequency of Oscillation – how quickly RF polarity changes  Now, considering the DC voltage... Ions of same charge with higher m/z will be less sensitive to fluctuating RF voltage. This is because the momentum of ions of equal KE is proportional to (m)1/2. Thus, larger ions are harder to deflect by an alternating RF field. In the presence of a constant +DC potential, larger + ions (more momentum) will be unresponsive to the oscillating RF field and will be focused at the center of the flight path Ions of lower mass will “feel” the RF more strongly and will be accelerated into the electrodes and neutralized The overlaid +DC and RF voltages thus create a high-pass mass filter for positively charged ions. ON DC RF can + unstable low m/z ions have unstable trajectories tradject Constant +DC collide with electrode + of MW potential + high m/z ions have stable trajectories make it through +  Now, add another pair of electrodes... When a second pair of electrodes are added... Once again, positively charge ions with large mass (more momentum) do not respond strongly to the oscillating RF voltage. They are instead drawn towards the – electrode where they are neutralized. Positively charged ions with smaller mass can be more readily deflected away from the – electrodes by the oscillating RF field (because they have less momentum). This creates a low pass filter for positively charge ions ON DC rods - high m/z ions have unstable trajectories Constant -DC and collide with electrode + potential + low m/z ions have stable trajectories make it through -  Quad acts as a mass filter. For + ion…. high pass filter for + ions transmission + Large m/z have more Allowed m/z momentum - hard to + deflect towards electrode with oscillating m/z RF sign of DC potential low pass filter for + ions transmission Allowed -- Large m/z pulled into m/z electrode – hard to deflect towards center with oscillating RF m/z transmission Allowed + m/z Narrow range - - mass filter + m/z Quads are small, cheap, and allow fast scanning rates. Resolution is not fantastic, but they are still the workhorse mass analyzer of biological MS and are components of nearly every type of MS system  Time-of Flight (TOF) TOF mass analyzers literally measure the time that it takes for an ion to move from the ion source to the detector. Ex 10 000 MS S Coupled with fast detectors that allow measurements of ions over large m/z ranges simultaneously. This is especially powerful when combined with ESI sources that generate multiply charged ions Resolution is very good (RP ~ 80,000 on some instruments) which is sufficient for a large number of applications good tradeoff between cost and performance  Time-of Flight (TOF) Following ionization, ions are accelerated to approximately the same translational kinetic energy (KE) by an electric field potential (usually 103-104 V): Charged species into field KE = zeV = ½(mv2) Same charge thesame pat fly z = ion charge but different mass different e = electronic charge (1.6x10-19 C) V = voltage difference m = mass (in kg) v = velocity Note, all ions with the same charge are assumed to have the same KE Since KE is constant, ion velocity is inversely proportional to mass – smaller masses have higher velocities  Time-of-Flight (TOF) Mass Analyzers Ions are accelerated to same KE and transferred into a field-free drift region, which is usually 1-2 m in length In the absence of any external field, the ions will become separated in time as the smaller ions move through the drift region with faster velocities than large ions The time of flight is given by: tf = L/v = L x (m/2zeV)1/2 remember: L = length of flight tube, v = velocity Ion generation Ions of same KE have different velocity no electric field and separate in time (i.e. field free) dust Longer path resolution flyaccording to mass BUT sensitivity less ions hi TOFs have Unlimited Mass Range Theoretically unlimited m/z range Limited by length of flight tube In practice, it is difficult to achieve a high enough vacuum across the entire flight tube TOFs have been widely used for analyzing large biomolecules and biomolecular assemblies (> 100,000 Da) ESI-TOF mass spectra of in tact viral particles  Increasing TOF resolution The assumption that ions of the same charge accelerated by same field have exactly the same KE is not technically correct. This is especially true for MALDI-TOF, where the initial energy spread and spatial distribution of ions within the plume are significant (due to the MALDI process) Upon exposure to the accelerating V, ions actually have a distribution of velocities (both the position and speed of ions differ) which gives rise to a similar distribution of velocities in the flight tube This distribution decreases mass resolution because the time interval over which a given ion arrives at the detector is increased, which allows overlap with the time intervals of other ions of similar mass/velocity Increasing TOF resolution – Delayed Extraction Ions of a given m/z in the plume have distribution of initial velocities. By using a pulsed laser and a time delay before the accelerating potential is applied (by the grid), velocities are allowed to identical analyte equilibrate. Not all When grid is turned on, ions get the same KE closer to the grid in the expanding plume will experience less force than ions farther away. Ions farther away will be accelerated to a greater extent and will catch up Ions of same m/z will arrive simultaneously at detector  Increasing TOF Resolution - Delayed Extraction control a Delay acceleration to allow 4.1 plume to expand Delayed pulse ensures all ions hit detector same time  Increasing TOF resolution - Reflectron Ions of same type enter drift tube with different velocities and enter a repulsive electric field (the reflectron) , where they change direction and are reflected back to the detector. Ions with greater kinetic energy (because of faster velocity) penetrate farther into the repulsive field, slowing them down and allowing ions with lower velocities to catch up. Distance travelled for faster ions i Land same time accelerating Ions with same m/z, but reflectron: series of voltage different initial velocities ring electrodes of upon entering drift tube increasing potential Ion with faster velocity (ion b) penetrates farther into retarding field of reflectron Ion a catches up with ion b and both arrive at detector simultaneously Depends on S N if you need Botopes etc MALDI Tof  Fourier Transform Mass Spectrometers FTMS provide the best S/N ratios, speed, sensitivity, and resolution Resolving powers up to 107 (i.e. can get baseline resolution of peaks at m/z = 1000.0001 and 1000.0002) Ideal for complex biological samples often encountered in proteomics/metabolomics, where there are many ions will overlapping m/z envelopes Require superconducting magnets to generate strong magnetic field Employ ultrahigh vacuum (mean free path of 10s – 100s of km!) Budget g Costly to buy (> $1M) and maintain V.V expensive  Basic Principles of FTMS A constant external magnetic field is used to trap ions in circular orbits between two electrically charged plates FTMS instruments are a type of ion trap (called a Penning trap) Within the trap, ions circulate with well-defined orbits that are a function of m/z Provided that there is sufficient vacuum, these orbits can be maintained for extended periods of time, allowing the unparalleled sensitivity, mass accuracy, and resolution of FTMS instruments electrically charged plates Strong magnetic field AC plates circular orbit circular orbit that depends on m/z  Ion Cyclotron Resonance More specifically, FTMS measures the ion cyclotron resonance frequency of the ions In strong magnetic fields, charged ions adopt circular motion perpendicular to the field. This motion is characterized by the cyclotron frequency: c (rad/s) = v/r = zeB/m w̅ Strong constant magnetic field c = cyclotron frequency orbit α MIZ v = velocity r = radius of circular orbit In a fixed field, c z = ion charge depends only on m/z e = electron charge B = magentic field m = mass Faster movement bigger orbit  Measurement of ICR Signal The trapped ions can absorb energy from an RF electric field, provided that the frequency of the RF field matches c (resonant excitation) The absorbed energy increases the velocity of the ions without altering c. This, in turn, perturbs the radius of the orbit (because c = v/r must remain constant). The velocity (and radius) of the ion will continuously increase until the RF voltage is shut off. Ions with different c will be unaffected by the AC field after RF is shut applied RF perturbs off, ion packet has radius of orbit until circular orbit the RF is shut off Nondestructive 1 1 ions don't hit It Adjust movement measuremotion  Coherent Motion of Ion Packet is Critical After excitation by the RF, the ion packet remains clustered as it travels in a circular orbit. The packet generates a periodic response as it passes the two plate electrodes: As packet of positive e- ions passes the top electrode, electrons move this way over time This creates an oscillating ions slowly current at the detector known spin into centre as an image current of trap As packet of positive ions e- passes the bottom electrode, electrons move image this way current  Measurement of ICR Signal The magnitude of current depends on number of ions in the packet. The more ions in the packet, the more strongly the electrons in the circuit will be affected The frequency of the current is the cyclotron frequency (c) and is directly related to m/z magnitude of image current related to number of ions Frequency = c and is related to m/z The oscillating signal is called a free induction decay (FID). t Over time, ions lose energy (due to collisions) and return to thermal Eq.  Fourrier Transform FT extracts the frequency and amplitude from a periodic signal. In FTMS, the frequency of the signal is the cyclotron frequency of the ion. This Allows easy conversion of  to m/z (remember:  = c) c (rad/s) = v/r = zeB/m Long mean free path b c non destructive high resolution  Converting FID to Mass Spectrum The m/z for each ion that contributes to FID can be extracted by FT The longer the observation period, the sharper the line, the better the resolution  Rest of Course Tandem MS – fragmentation methods to study protein/peptide 1° structure and post-translational modifications Hydrogen/Deuterium Exchange MS – follow H/D exchange into peptide backbone to measurement dynamics of 2° structure fluctuations Native MS – methods to ionize and transfer proteins into the gas phase in a folded state – allows characterization of 3° and 4° structure