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Mass Spectrometry

Basic principles Components Types Applications of MS

Basic principles
Qualitative & Quantitative analytical technique. Basic principle: Charged molecule moves through electric or magnetic field can be separated according to m/z ratio. Essential features: Production ions in the gas phase. Acceleration of the ions to a specific velocity in an electric field. Separation of ions in a mass analyzer. Detection of ions of particular m/z ratio. Ionization Separation Detection.

Mass spectrum: Intensity of each ion is plotted against the m/z ratio of each ion. Base peak: Max intensity in the spectrum 100% relative abundance

Mass Spectrum

Components of MS
High Vacuum system (10-5 torr - 10-7 torr)
To create & maintain the high vacuum Sample inlet: sample or target plate. To introduce the sample in to ion source

Ionization source:
To convert molecules into gas phase ions.

Mass filter (or) analyzer:


separate the ions according to m/z ratio

Detector:
For collection, amplification, detection of ions. Computer system: For operate, acquisition, display, manipulation,& interpretation of data

Vacuum system
MS operates under high vacuum to minimize collision At atmospheric pressure ions will not reach the detector. Mean free path of ion is At atmospheric pressure -52 nm 1mtorr-40 mm 1torr-40 m Optimize detection, resolution, transmission of ions Types: Rotary vane pumps, Turbomolecular pumps, Diffusion pumps. Rotary vane pumps-provide initial vacuum(1 torr) Turbomolecular or diffusion pump provide high vacuum(1mtorr-1ntorr) Maintain the ion source pressure 10-5-10-7 torr

Ionization source:
Molecules molecular Ions Types Electron ionization(EI) gaseous sample molecule Chemical ionization(CI) Electrospray ionization(ESI) Sonic spray ionization(SSI) Atmospheric pressure Chemical ionization(APCI) Atmospheric pressure photo ionization(APPI) Inductively coupled plasma(ICP) Matrix assisted laser desorption ionization(MALDI) Atmospheric pressure Matrix assisted laser desorption ionization(APMALDI) Fast atom bombardment(FAB) Surface enhanced laser desorption ionization (SELDI)

Electron Impact Ionization(EI)


Gas phase sample molecules Electron beam(70eV) molecules ionization molecular ions fragmentation Fragment ions repel Mass analyzer M+ e M+ M++2eF1+F2+F3

Chemical ionization
Samples similar to EI Electron beam ionization Reagent gas Reagent gas ions Molecules ionization Molecular ions. reagent gas - Methane, Ammonia, isobutane, water CH4 + 2e- > CH4+ + CH3+ +H.+4e CH4+ + CH4 > CH5+ + CH3. CH3+ + CH4 > C2H5+ + H2 M + C2H5+ --> MH+ + C2H4 M + CH5+ --> MH+ + CH4

Chemical Ionization
High sensitivity, specificity Soft ionization Less fragmentation much clear spectrum, min overlap Determinations of molecular mass Eliminates false positive results Detect high m/z molecule Accurate identification Molecular mass determination & quantification Detects femtogram level

Spectrum of Cocaine in EI

CI spectrum
Isobutane

Methane

Fast atom bombardment


Ionize protein & smaller molecules Soft ionization technique Formation ions with low internal energies Little fragmentation. Electron beam bombardment Sample + viscous solution (glycerol,thioglycerol,m-nitrobenzyl alcohol) vaporization & ionization Molecular ions Mass analyzer

Fast atom bombardment

Electrospray Ionization
Soft ionization Sample - HPLC effluent

Sample +solvent spray Electric Field warm gas (nitrogen) Evaporation & ionization molecular ions solvent: methanol,isopropanol, acetonitrile
Produce multiple charged ions Less resolution

ELETROSPRAY IONIZATION

ELETROSPRAY IONIZATION

Matrix Assisted LASER Desorption/ Ionization(MALDI)


Matrix+sample
drying

Cocrystallise ionization & Evaporation


pulse of laser beam

Molecular ions Matrix analyte ratio: 1000:1 High sensitivity, large mass ions Soft ionization, less fragmentation

MALDI

MALDI

MALDI

Mass analyzer
Separate the ions according to m/z ratio & allows to reach the detector Separation can be electronically & magnetically Types: Quadrupole Quadrupole Ion Trap Magnetic sector Time of flight EI,CI,& FAB combined with magnetic sector analyser ESI with QI,QIT MALDI with TOF

Quadrupole analyzer
Consists four parallel rods DC & RF are applied Creates electric field along the length of rods In particular electric field particular m/z charge ions are separated & accelerated towards detector. Other m/z ratio ions impact the quadrupole rods Analyze upto m/z 3000(protein & biological molecules)

Quadrupole ion trap


combine function of ion source and mass analyzer Traps & stores the ions of interest. Trapped ions ejected to the detector Trapping & storage causes concentration of ions of interest. High sensitivity Analysis of peptides & small molecules Gives Structural information

Magnetic sector analyzer


m/z =H2r2/2V H= magnetic field r = radius V =accelerating voltage High resolution, mass accuracy More complex & expensive Resolution= m/m M-mass of ion m difference between mass of ion & adjacent ion High resolution > 10000 Low resolution < 1000

Time of flight
t=L(m/2E)1/2
Flight time: The time taken to traverse the flight tube Lighter ions travel faster than heavier High mass accuracy, speed, sensitive High resolution, exact mass measurement, Confirm molecular formula Unlimited m/z range Reasonable cost Used for protein and peptide identification

MALDI-TOF

Insulin : 5733.5 -Lactoglobulin:18,388

Detector
Types: Conversion diode Electron multiplier-discrete, continuous Microchannel plate electron multiplier,array detector Faraday cup

Ions from analyzer strike surface of detector Collection, amplification, detection

Tandem MS
2 or more MS arranged in sequentially (tandem) Common type triple quadrupole MS. Separation faster than GC/LC Analysis of several analytes take 1-3 min In GC/LC/MS take 30 min. Rapid analysis ,high throughput (100-1000) GC/LC/MS one test one disorder MS/MS one test- multiple disorder Less cost per sample Improved detection limits in complex mixture Rapid, sensitive,& selective analysis of complex mixture with minimal or no sample cleanup.

Tandem MS

Tandem MS

Parent ion Ion source

Daughter ion

MS1

Collision cell

MS2

Detector

Applications
Biotechnology: the analysis of proteins, peptides, oligonucleotides Pharmaceutical: drug discovery, pharmacokinetics, drug metabolism Clinical: neonatal screening, haemoglobulin analysis, drug testing Environmental: water quality, food contamination Geological: oil composition

Accurate molecular weight measurements: sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-translational modifications, to calculate the number of disulphide bridges Reaction monitoring: to monitor enzyme reactions, chemical modification, protein digestion Amino acid sequencing: sequence confirmation, de novo characterization of peptides, identification of proteins by database searching with a sequence "tag" from a proteolytic fragment Oligonucleotide sequencing: the characterization or quality control of oligonucleotides Protein structure: protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination

Applications
GC/MS ng or pg sample amount required Analysis biological compounds Drug screening for clinical & toxicological (nonpolar,volatile,small molecular weight). To detect drugs of abuse in forensic toxicology. Screening is done by immuno assay technique. positive samples are confirmed by GC/MS. Analysis of anabolic steroids ,pesticides pollutants ,TCA

Xenobiotic compound analysis

Applications
HPLC /MS : Polar, nonpolar,large molecules of protein compound Drug screening Anlysis of all immunosuppressants,antiretroviral, homocystiene, bigenic amines ,methyl melonic acid, haemoglobulin varients. Identyfy new taxanes Therapeutic approach: .Recognition of target protein receptor associated with selected disease -used for drug design.

MALDI-TOF
Identification of proteins,peptides Identification of organism- bacteria Identification of particular compounds Identification of phosphatidyl choline Very rapid 2min/sample >100 samples deposited on a single MALDI plate

SELDI-MS Analysis biomarker (tumor marker) for disease Eg : Breast cancer marker

ICP-MS
Determinations of toxic trace elements Al ,Hg,Ar,Cad,Pb.Co,Pt,Se

Tandem MS
Screening & determination of genetic disorders & inborn errors of metabolism. Analysis of multiple compound in single run. Aminoacid metabolism disorders: Argininemia (Argininosuccinic Acid(ASA) lyase deficiency) Citrullinemia (ASA synthetase deficiency) Homocystinuria (HCU) Hyperphenylalaninemias(PKU, HPhe) Classical PKU, Hyperphenylalaninemia, Biopterin cofactor defects Maple syrup disease

Fatty acid oxidation disorders


Carnitine/acylcarnitine translocase deficiency (CAT)

Carnitine palmitoyl transfereasedeficiency, type I (CPT-I)


Carnitine palmitoyl transferasedeficiency, type II (CPT-II) Carnitine transport defect Long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD)deficiency/Trifunctional protein deficiency (TPF) Medium chain acyl-CoA dehydrogenase (MCAD) deficiency Multiple acyl-CoA dehydrogenase deficiency(MADD or GAII, Glutaric acidemia, type II) Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency

Organic aciduria
2-methylbutyryl-CoAdehydrogenase deficiency (2MBCD or SBCAD) 2-methyl-3-OH butyric aciduria 3-methylcrotonyl-CoA carboxylase deficiency (3MCC) 3-methylgluta-conyl-CoA hydratase deficiency 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG) Glutaric acidemia, type I (GA-I) Isovaleric acidemia (IVA) Malonic acidemia Methylmalonic acidemias (MMA) Mitochondrial acetoacetyl-CoA thiolase deficiency (SKAT, BKT, 3-ketothiolase deficiency, ketothiolase) Multiple carboxylase deficiency (MCD) Propionic acidemia (PA)

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