Mass Spectrometry

Where is MS used? Biotechnology: Pharmaceutical: Clinical: analysis of proteins, peptides, oligonucleotides Pharmaceutical: drugs discovery and metabolism, combinatorial chemistry, pharmacokinetics, Clinical: neonatal screening, haemoglobin analysis, drug testing Environmental: water, food, air quality (PCBs etc) Forensic analysis identification of drugs and blood

Mass Spec Principles General operation of MS Sample Ion Source + _ Mass Analyzer Detector General operation of MS create gas-phase ions separate ions in time or space by their mz ratio measure the quantity of ions

Schematic mass spectrometer MS Source MALDI ElectroSpray EI/CI Under vacuum MS Analyzer Quadrupole Time of Flight Ion Trap

Informazioni analitiche dalla MS Informazioni qualitative Struttura di specie molecolari complesse Composizione di una miscela Purezza di un picco cromatografico Informazioni quantitative Concentrazione dei componenti di una miscela

Maggior contenuto di Informazioni (Tandem mass spectrometry) Studio della struttura Informazioni addizionali per determinare la struttura di un composto Rivelazione selettiva di uno ione Drastica riduzione delle interferenze Studio delle reazioni ione-molecola Esperimenti SRM, PIS e Neutral loss

MS vs NMR Absorbance aspirin EI-MS NMR

MS vs. NMR MS peaks are narrower than NMR peaks MS is much more (104 x) more sensitive than NMR (among most sensitive tools) MS allows one to analyze much larger molecules (>50 kD) than NMR MS samples are more difficult to prepare MS is not particularly quantitative MS instruments cost a little less than NMR

Calcolo delle masse • Unità di massa: Dalton ==>1 u = 1 Da = 1.660540 x 10-27 kg • Massa nominale: Massa Intera (e quindi approssimata) degli isotopi più abbondanti: CH3Br: 12 + 3 x 1 + 80 = 95 Da • Massa monoisotopica : Massa Esatta degli isotopi più abbondanti: CH3Br: (12.00 + 3x 1.007825 + 78.918336) = 93.941011 Da • Massa media (average mass, av): calcolata tenendo in considerazione l’abbondanza relativa dei diversi isotopi: CH3Br: 12.01115 + 3 x 1.00797 + 79.904 = 94.93906 Da

Monoisotopic mass When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement

Average Mass Average mass corresponds to the centroid of the unresolved peak cluster When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass

Isotopic profiles Mass Spectrometry “Monoisotopic mass” One 13C atom Two 13C atoms No 13C atoms (all 12C) The isotopic distribution for a C60 fullerene shows a significant M+1 ion peak as well as easily measurable M+2 and M+3 peaks

(a + b)n Isotope Peaks for High Molecular Weight Species C1 0.99 12C C3 0.97 12C12C12C 0.01 13C 0.03 13C12C12C (3 forms) 0.0004 13C13C12C C2 0.98 12C v. small 13C13C13C 0.02 12C 13C or 13C12C 0.0001 13C 13C BINOMIAL EXPANSION (Yergey, Anal. Chem. 1983, 55, 353) (a + b)n # of atoms Abundance heavy isotope Abundance light isotope

C60 C100 Rel. Abund. Rel. Abund. 12C60 100 12C100 100 12C5913C1 66 12C9913C1 110 12C5813C2 21 12C9813C2 60 12C5713C3 4.6 12C9713C3 22 C60 C100 *isotope peaks from other element not included H, O, N, S, ...!! large MW ® many isotope peaks What is molecular weight?

Isotopic profiles

Mass spectrum of insulin 2 x 13C 13C 12C : 5730.61 Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used.

Important performance factors Mass accuracy: How accurate is the mass measurement? Resolution: How well separated are the peaks from each other? Sensitivity: How small an amount can be analyzed?

Risoluzione La capacità di uno spettrometro di massa di differenziare le masse è generalmente espressa dalla risoluzione R definita come: R = m/Δm dove Δm è la differenza di massa tra due picchi adiacenti risolti e m è la massa nominale del primo picco (o la media delle masse dei due picchi). Due picchi sono considerati risolti se l’altezza della valle tra di essi è inferiore ad una certa percentuale dell’altezza del picco meno intenso (di solito il 10%). Uno spettrometro con una risoluzione di 4 000 risolverà due picchi con valori di m/z 400,0 e 400,1 (o di 40,00 e 40,01). Gli spettrometri commerciali hanno R che variano circa tra 500 e 500 000.

Risoluzione Definizione alternativa Picchi risolti al 10% della valle Intensità Definizione alternativa La risoluzione di un picco isolato si può anche definire come larghezza δm del picco al x% dell’altezza. Spesso si prende x = 50%, e δm è la larghezza a metà altezza.

Effect of Resolution Mass spectrum of a 50 carbon atom compound Elemental Composition: C H N O R = 1000 R = 10000 R = 100000

Mass Accuracy Mass accuracy indicates the accuracy of the mass information provided by the mass spectrometer Mass accuracy is the difference between the theoretical mass and the measured mass: Mass accuracy = mreal – mmeasured in ppm ppm = ∆m / mmeasured * 106 i.e.: theoretical mass: 1000, measured mass: 999.9 error: 100 ppm

High resolution means better mass accuracy Mass accuracy depends on resolution High resolution means better mass accuracy 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Resolution = 14200 Resolution = 4500 Resolution =18100 15 ppm error 24 ppm error 55 ppm error

Ionization Methods

Sample Introduction

Different Ionization Methods Electron Impact (EI - Hard method) Chemical Ionization (CI - Hard method) small volatile molecules, 1-1000 Daltons, structure Electrospray Ionization (ESI - Soft) ionization in condensed phases peptides, proteins, up to 200,000 Daltons Matrix Assisted Laser Desorption (Soft) ionization occurs from solid phase peptides, proteins, DNA, up to 500 kD

Choice of Ionization Methods Physical state of the sample Volatility and thermal stability Type of information needed molecular mass molecular structure sequence analysis

Electron Ionization Sample is evaporated into the ion source Gas phase sample is bombarded with electrons (energy = 70 eV or variable) Sample is ionised according to the mechanisms: M + e-  M•+ + 2e- M + e-  M•- Molecular ions are “shattered” into fragments Fragments sent to mass analyzer

Electron Ionization

Fragment Ions

Electron Impact MS of CH3OH Molecular ion EI Breaks up Molecules in Predictable Ways

Libraries of Mass Spectra Application Oriented Pfleger/Maurer/Weber:toxicology Zamecnik (TX)): toxicology Adams: terpenes and fragrances Ockels: pesticides Spiteller: steroids General Wiley NIST

Different fragmentation There will be different degrees of fragmentation depending on the stability of the sample molecule For a stable aromatic compound the primary peak is the parent ion For a less stable cyclic compound fragmentation is predominant

Isomeric Structures

Different energy spectra 70 eV 12 eV

Chemical Ionization (CI)

Chemical Ionization Sample is evaporated into the ion source Reagent gas (methane, isobutane, ammonia) is ionized and reacts with sample to produce ions Ionization occurs according to the mechanisms: CH4 + e-  CH4•+ + 2e- CH4+• + CH4  CH5+ + CH3• CH4+ + CH4  C2H5+ + H2 M + CH5+  MH+ + CH4 M + C2H5+  MH+ + C2H4 -advantages - molecular weight determination -disadvantages - no fragmentation, less informative

Control of fragmentation by different CI reagent gas Chemical Ionization Control of fragmentation by different CI reagent gas Methane Isobutane IE = 15.8 eV soft reagent hard reagent

EI CI versus EI gives both a molecular ion and a fragmentation pattern CI produces one or the other depending on the reagent gas chosen

Soft Ionization Methods 370 nm UV laser MALDI cyano-hydroxy cinnamic acid Gold tip needle Fluid (no salt) ES + _

ESMS

Sorgente elettrospray Gas di trasporto Gocce di circa 1 µm Gas di desolvatazione Liquido di trasporto Campione Cono di Taylor Capillare riscaldato + - 2-6 kV

Meccanismo di deplezione degli ioni Soluzione del campione 4000 V Pressione atmosferica Calore o gas secco Il calore e/o il gas secco causano la riduzione delle dimensioni delle gocce Vapori del solvente Livelli successivi di vuoto (2 torr – 0.1 torr – 0.01 torr) Spettrometro di massa Esplosione Coulombiana Limite di Rayleigh: Limite di dimensioni della goccia al quale si prevede che le molecole cariche vengano espulse.

Ionization Source Tandem MS 12/17/2017 However, back to more serious things…… This is a schematic representation of the ZSPRAY™ ionization source that we use in our instruments. The sample, in the flowing solvent stream, is introduced into the source through a 125µm stainless steel capillary and passed into the ionization/source region. The sample is introduced at a flow rate in the range of 10 – 100µL per minute and we have to generate a fine spray in order to generate the ions from the sample being analyzed. The fine spray is generated by the action of two phenomena: nebulization and electrospray. The sample passing through the probe is nebulized using a stream of nitrogen gas—similar to what you would see in a perfume atomizer. The stainless steel capillary is held at a high voltage potential—typically 2 – 4kV—while the rest of the instrument is held at about ground potential. This means that we have a very strong electric field between the end of the probe and the entrance into the mass spectrometer and this causes considerable polarization in the droplets formed in the nebulization, leading to the formation of ions—this is the electrospray phenomenon that we saw a couple of slides ago. The ions are generated at atmospheric pressure and then pass through two orthogonal orifices in order to get into the vacuum system of the mass spectrometer. This is what gives ZSPRAY its name. Because we have a right angled turn, all the non-volatile material in the sample passes straight on into a baffle plate, and does not clog up the orifice into the instrument—but we are not interested in that material—only the ions that have been formed. The ions are guided through these small holes (they are only 400µm in diameter) by a combination of gas dynamics (first hole) and electrostatic lensing (second hole). Dr. Edward Randell

Ionization Source Sample Cone Spraying Needle Vacuum Isolation Valve Tandem MS 12/17/2017 Ionization Source Spraying Needle Sample Cone Orifice = 400µm Vacuum Isolation Valve …and a close up photo showing some of the detail of the source. Dr. Edward Randell

ElectroSpray Ionisation Le condizioni applicate sono sufficienti a nebulizzare la soluzione in una miriade di goccioline cariche, contenenti il campione

Multiply Charged Ions

Electrospray ionization M + nH+ = (MHn)n+ m/z(1) = M + nH+ n m/z(2) = M + (n+1)H+ n(+1) = 1211.8 = 1131.1 n = 10

ESI mass spectrum of bovine Blg B m/z after data system transform processing Expected MW = 18276.2 Da, Calculated MW = 18276.6 Da Mass (Da)

Heterocycle Heterocycles and Cyclodextrines by ESMS Rotaxane

Accurate Mass Determination IS Theoretical MW 829.1522 Observed MW 829.1514 Accuracy 1 ppm

MALDI

MALDI - Matrix Assisted Laser Desorption Ionisation

Schematic of MALDI ionization

The principle of matrix assistance Isolate the analyte molecules from one another such that the incident laser radiation primarily heats the matrix, rather than the analyte. Source of protons (H+ ions) to ionize the analyte molecules. Absorbing medium for the ultraviolet light, thus converting the incident laser energy into molecular electronic energy, which may be used both for desorption and ionization.

Analisi Oligosaccaridi MALDI ionization matrices Analisi Peptidi Analisi Proteine Analisi Oligosaccaridi -Cyano-4-hydroxycinnamic Acid Sinapinic Acid 2,5-dihydroxybenzoic Acid Matrix properties Organic acids that absorb in the region of the laser wavelength Used in very high ratios to minimize analyte interactions More acidic matrices tend to cause greater fragmentation

MALDI ionization Two peaks 107Ag and 109Ag Depending on the nature of the matrix we can obtain different molecular ions Cationized species Protonated species Deprotonated species Two peaks 107Ag and 109Ag

Mixture Analysis m/z Relative abundance (%) 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 50 100 1776.79 1531.75 1177.65 1100.59 990.48 902.50 1288.69 1844.04 2607.15 2479.08 2191.95 m/z Relative abundance (%)

MALDI MS Lipids MALDI mass spectra of pospholipids in the (A) cerebellar cortex and (B) cerebellar peduncle regions in rat brain with DHA matrix in positive-ion mode

MALDI Spectrum of PNIPAM (poly-N-isopropylacrylamide) polymer MALDI MS of Polymers MALDI Spectrum of PNIPAM (poly-N-isopropylacrylamide) polymer

MALDI MS of Me-complexes

Ionization techniques Ionization Method Typical Analytes Sample Introduction Mass Range Method Highlights Electron Impact (EI) Relatively small. Volatile. GC or liquid or solid probe To 1000 Daltons Hard method. Provides structural info Chemical Ionization (CI) Soft method. Molecular ion peak [M+H]+ Electrospray (ESI) Peptides/proteins. Non-volatile. Liquid Chromatography To 200,000 Soft method. Ions often multiply charged. Matrix Assisted Laser Desorption (MALDI) Sample mixed in solid matrix To 500,000 Soft method. Very high mass range. Fast Atom Bombardment (FAB) Carbs/peptides. viscous matrix To 6000 Soft method, but harder than ESI or MALDI