5/11/2015 MASS SPECTROMETRY. Mass spectrometry (MS) - overview. Mass spectrometer. Ionization and mass analyzer. Analyzer: QUADRUPOLE

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1 UNIVERSITY OF PÉCS MEDICAL SCHOOL MASS SPECTROMETRY Mass spectrometry (MS) - overview SAMPLE: charged/ionized sample(fluid biological samples, proteins, peptides, oligosaccharides, DNA, serum, blood, urine, liquor, tissue extracts, ) INSTRUMENT: mass spectrometer, measurement of mass: charge ratio RESULTS: mass spectrum: mass: chargeratio distribution(molecularweight, molecular formula/composition, structural features) OPTIONS: separation of components on the basis of mass : charge ratio, tandem MS, imaging MS(MSI: mass spectrometry imaging) BIOPHYSICS th April Dr. Beáta Bugyi Department of Biophysics What are the advantages? HIGHSENSITIVITY: g LOWAMOUNTOFSAMPLE: g(micro picogramm) BROADMASSRANGE: atomicmassunit(da) SPECIFICITY AND REPRODUCIBILITY Mass spectrometer ION SOURCE: productionof of gasphaseions ionsfrom the sample Positiveion mode: removing an electron(+ charged ion) Negativeion mode: adding an electron(- charged ion) 2. MASS ANALYZER: separation, filtering of ions according to their mass: charge ratio 3. DETECTOR: measuringthe relativeabundance of each mass: charge ratio, electric signal 4. VACUUM: minimizing the collisionsbetweenions and air molecules otherwiseions do not reach the detector /atmospheric presure/ Ionization and mass analyzer ION SOURCE ionization ELECTRON IMPACT IONIZATION/EI/ CHEMICAL IONIZATION/CI/ ATMOSPHERIC PRESSURE CHEMICAL IONIZATION/APCI/ ATMOSPHERIC PRESSURE PHOTO IONIZATION/APFI/ FAST ATOM BOMBARDMENT /FAB/ THERMOSPRAY IONIZATION/TPI/ LIQUID SECONDARY IONIZATION/LSI/ ELECTROSPRAY IONIZATION/ESI/ MATRIX-ASSISTED ASSISTED LASER DESORPTION IONIZATION/MALDI/ MASS ANALYZER separation according to massand and charge SECTOR ANALYSER ION TRAP FOURIER TRANSFORM ION CYCLOTRON RESONANCE QUADRUPOLE TIME-OF OF-FLIGHT FLIGHT /TOF/ Ionization: ELECTROSPRAY IONIZATION(ESI) electrospray Analyzer: QUADRUPOLE QUADRUPOLE: 4 parallel cylindrical rods radio frequency voltage spray the sample through a capillary into electric field + vacuum small drops the solvent evaporates quickly in the vacuum thesizeof thedropdecrases, the charge density in the drop increases increasing forces from electrostatic repulsion split the drop into smaller droplets multiply charged species for large biomolecules direct current voltage the electric field of the quadrupole influences the movement of the ions foragiven setofelectric field ions ofoneparticularmass :chargeratiocanpass through the quadrupole and reach the detector, the others collide with the rods different mass: charge ratios can be scanned by changing the electric fields 1

2 Ionization: MATRIX-ASSISTED ASSISTED LASER DESORPTION IONIZATION /MALDI/ MATRIX-ASSISTED ASSISTED: sample is embedded in a matrix on a metal surface Analyzer: TIME-OF-FLIGHT(TOF) electric field: LASER: the matrix-sample is irradiated by laser(uv), the laser energy is absorbed DESORPTION: the matrix-sample dissociates (desorped) from the metal surface into vacuum IONIZATION: the particles of the sample are ionized by the matrix ions: example: Detector: CONVERSION DYNODE, AMPLIFICATION Mass spectrum, mass : charge spectrum more CO 2 conversiondynode ion electron dynode chain secondary emission cascading effect abundance less O small CO mass: charge(m/z) large IMPORTANT: MASS SPECTRUM = MASS : CHARGE RATIO SPECTRUM amplification: 10 6 molecular weight: 400 charge: + 1 m/z = 400 molecular weight: 1200 charge: + 3 m/z = 400 Analysis of mass spectra UNKNOWN SAMPLE mass spectrum measured experimentally collisionchamber SAMPLE IDENTIFICATION octadecanoic acid methyl ester m/z SPECTRA DATABASE MS1: fix precursorion witha givenm/z ratio fragmentation MS2: scan product ion m/z ratio distribution determine structurally significant fragment ions amino acid composition of peptides CID: collisioninduced dissociation 2

3 ütközés indukálta disszociáció MS2: fix production witha givenm/z ratio fragmentation MS1: scan the m/z ratio distribution of precursor ions group of compounds all give the same fragment ion functional groups, phosphate ester, carbohydrate modification MS1: givenm/z ratio of precursorion and MS2:givenm/z ratio of product ion (fix the difference: neutral fragment) scanthem/z ratio distributionof correspondingprecursorand product ions compounds giving a fragment having that specific loss are detected functional groups, phosphorylation-phosphoric acid MS1: fix precursorion witha givenm/z ratio and MS2: fix production witha givenm/z ratio scan the distribution of the corresponding ions identification of components Applications PROTEOMICS molecular mass determination sequence and structural information posttranslational modicifaction/phosphorylation, glycosilation, / indentificationof disulfide linkeage determinationof active/bindingsite protein-ligandinteraction quantitativeanalysis of protein mixtures MEDICAL JURISPRUDENCE screening of drugs, dopping and poisons MICROBIOLOGY PHARMACEUTICAL RESEARCH MEDICAL SCIENCES AGRICULTURE e.g. active components of wine (resveratrole) FOOD-INDUSTRY SPACE SCIENCES Applications medical applications NEONATAL SCREENING FOR CONGENITAL METABOLIC DISORDERS carnitine ester profile determination: primary/secondary carnitine deficiency, fatty acid oxidation disorders, organic acidurias amino acid profile determination: aminoacidopathies guanidine-compounds: disorders of creatine synthesis disorders of bile acid synthesis homocystein: hiperhomocysteinaemias purines, pirimidines galaktosaemia disorders of steroid synthesis, cholesterol synthesis disorders of hormon synthesis (triiodo-thyronine, catechol amines) disorders of carbohydrate metabolism THERAPEUTIC DRUG MONITORING immunosuppressive drugs/sirolimus/, antimycotic drugs, antiepileptic drugs, antiviral drugs ANDROGEN PROFILE CARNITINE ANALYSIS SULFONYLUREA SCREENING FATTY ACID ANALYSIS diagnosis of Refsum s disease, adrenoleukodystrophy CANNABINOID CONFIRMATION TEST METHYLMALONIC ACID IN URINE OR SERUM diagnosis of vitamin B12 deficiency 3

4 Metabolic disorders medium-chain acyl-coa dehydrogenase deficiency(mcad) fattyacidoxidationdisorder, impairs the break down of medium-chain fatty acids into acetyl-coa, hypoglycemia marker metabolite: C6, C8, C10:1-carnitine Metabolic disorders propionic acidemia(pa) fatty acid oxidationdisorder marker metabolite: C3-carnitine propionic acidemia (PA) marker metabolite: C3-karnitin Mass spectrometry imaging visualize the spatial distribution of endogenous biomolecules /small molecules, drugs, biomarkers, metabolites, lipids, peptides or proteins/ by their molecular masses within tissues and organs MSI - worklfow 1. SAMPLE PREPARATION administration of compound and biopsy histological sectioningof specific organ or animal whole body GOALS DIAGNOSTIC diagnostic studies comparingdifferent tissue types (such as tumor versusnormal) to aid in pathological diagnosis PROGNOSTIC prognostic studies to categorize patientswith long- or short-term survival THERAPEUTIC drug response studies to predict a patient sresponse to a certain treatment 2. MASS SPECTROMETRY mass spectrum in each spatial coordinate pair 3. ANALYSIS reconstruction ofthe distribution of detected molecules as an image /software/ analysis, identificationand quantification of molecules MSI detecting cancer specific compounds in tissues MSI detecting anti-cancer drug distribution in tissues tumour major blood vessels concentration HER2+ breastcancer increased m/z 8404 expression marker: m/z 8404 HER2 - breastcancer A gastrointestinal tract B HER2: human epidermal growth factor its overexpressionplays an inportantrole in the developmentof agressivebreast cancers therapy: trastuzumab/herceptin/ interfereswith HER2 receptors it is effective only in HER2 overexpressingcells, side effects in other cells Orally administered anti-cancer drug distribution in whole mouse section. A) unstained transverse whole mouse section B) IMS image The drug is present inhigh concentrations in the tumourand can also be found in lower concentrations in the digestive system and blood vessels. 4

5 Mass spectrometry keywords ESI-quadrupole quadrupole,, MALDI-TOF techniques Mass spectrum Applications 5