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(MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the atoms or molecules comprising a sample of material. The spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds,so it is considered one f the very important diagnostic analytical techniques .
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RANIA MOHAMED EL-SHARKAWYRANIA MOHAMED EL-SHARKAWYLecturer of clinical chemistryLecturer of clinical chemistry
Medical Research InstituteMedical Research Institute , , Alexandria UniversityAlexandria University
MEDICAL RESEARCH INSTITUTE– ALEXANDRIA UNIVERSITYMEDICAL RESEARCH INSTITUTE– ALEXANDRIA UNIVERSITY
15th conference (3rd international)
ADVANCES IN MEDICAL RESEARCHADVANCES IN MEDICAL RESEARCH
` ` From molecular medicine-to From molecular medicine-to clinical applicationclinical application`̀
Mass spectrometric techniquesMass spectrometric techniques
Mass Spectrometry
Mass spectrometry (MS)Mass spectrometry (MS)
Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known materials,
and to elucidate thestructure and chemical properties of
moleculesA mass spectrometer A mass spectrometer is a device that measures the mass-to-charge (m/z) ratio of ions.
Mass spectrometry
I. Sample Ionization
Simple Definition:Ionization is a process of charging
a molecule. Molecules must be charged in
order tomeasure them using a mass
spectrometer
TypesTypesOf ionizing radiationOf ionizing radiation
Important to determine the target Important to determine the target from analyte investigatedfrom analyte investigated
Hard ionization: the resulting unique fragmentation patterns can be used as a fingerprint for the
identification of the sample. Fragment ions also provide important information about the primary
structure (i.e. sequence) of the sample molecules.
Soft ionization is applied to a stable molecule, the exact m/z value of its stable molecular
thus, allows for the compositional analysis of the sample of unknown structure under
study.
Mass spectrometry
A proton is transferred to or abstracted from ,a gas phase analyte by a reagent
gas molecule
Chemical ionization
Uses: analyte molecular mass determination & quantification
Little or no fragmentation as the protonated molecule is not highly excited in the chemical
ionization
2-Electrospray
Ionization (ESI)
Electro spray ionization
The 2002 Nobel Prize in
Chemistry was awarded to
two mass spectrometrists
(J. Fenn and K. Tanaka) for
their development of
ionization techniques,
which include electrospray
ionization
3-Matrix Assisted 3-Matrix Assisted Laser Desorption Laser Desorption Ionization (MALDI)Ionization (MALDI)
SELDI
-Modification of MALDI surface a type of affinity capture property
-Sample of interest is exposed to affinity surface , certain analyte will preferentially bind
-Washing to remove excess-Matrix is added to asset ionization
Uses: analysis of protein mixture with low sample size (purification&analysis occur on the same
surface
II. Analysis and Separation of Sample Ions
The mass analyzer separates the ions formed
in the ionization source according to their
mass-to-charge (m/z) ratios using some
physical property e.g. electric or magnetic
fields.
Time Of Flight Mass AnalyzerTime Of Flight Mass Analyzer((TOFTOF))
Quadrupole Mass AnalyzerQuadrupole Mass Analyzer
III. Detection of sample ions .
• A tiny current is produced when the ion reaches the detector.
• The detector amplifies the signals which are then transmitted to the data system to be represented as peaks on a mass spectrum
Mass spectrum: graphic representation of ions separated according to their m/z ratio
m/z
Mass Spectrum
Mass spectrum of CO2
Mass spectrumMass spectrum
Questions needs to be asked??1.Negative or positive mode
2.Types of ion source result in different arrays of fragments produced from the original molecules.
3.origin of a sample
4. How the sample was prepared ??
Interpretation of mass spectra
• compare its experimental mass spectrum against a compare its experimental mass spectrum against a library of mass spectralibrary of mass spectra
• If the search comes up empty, then manual If the search comes up empty, then manual interpretation or interpretation or software assisted interpretation of mass software assisted interpretation of mass spectraspectra are performed are performed
•A recent technique for structure elucidation in mass A recent technique for structure elucidation in mass spectrometry, called spectrometry, called precursor ion fingerprintingprecursor ion fingerprinting identifies individual pieces of structural information by identifies individual pieces of structural information by conducting a search of the tandem spectra of the conducting a search of the tandem spectra of the molecule under investigation against a library of the molecule under investigation against a library of the product-ion spectra of structurally characterized product-ion spectra of structurally characterized precursorprecursor
Mass spectrometry
TechniquesTechniquesOf mass spectrometryOf mass spectrometry
• GC –MS : GC –MS :
1.1.Definitive method to quantify Definitive method to quantify standard reference materialsstandard reference materials
2.2.Identifying trace contaminants Identifying trace contaminants or toxinsor toxins
3.3.Identification of drugsIdentification of drugs
TechniquesTechniquesOf mass spectrometryOf mass spectrometry
• HPLC –MS : HPLC –MS :
1.1.Screening & confirmation in Screening & confirmation in inborn errors of metabolisminborn errors of metabolism
2.2.Analysis of amino acidsAnalysis of amino acids
MALDI
SELDI-TOF
Tandem Mass Spectrometry
A tandem mass spectrometer is a mass spectrometer that has more than one analyser, usually two, with a collision chamber in between.
MS/MS uses 2 stages of mass analysis: 1. Selection of an ion2. Analysis of ion fragments produced by collision with inert gas.
Phe
Subs B
Proteomics and mass Proteomics and mass spectrometryspectrometry
APPLICATIONS OF mass spectrometry
PharmacokineticsPharmacokinetics
New born screeningNew born screening
applications of mass spectrometry applications of mass spectrometry
to study to study proteinsproteins
Best two techniques: Best two techniques: •ESI ESI •MALDIMALDI
Using two approaches: Using two approaches: •Top-DownTop-Down•Bottom-UpBottom-Up
Newborn screening (NBS)Newborn screening (NBS)
MS/MS allows the screening of a numerous array of metabolic disorders in a single analytical run:
• Amino Acid DisordersPKU, MSUD, Tyr, Cit, etc
• Fatty Acid Oxidation DisordersMCAD, VLCAD, LCHAD, etc.
• Organic Acid DisordersPA, MMA, IVA, GA-1, etc
%in
tens
ity
Advantages of MS/MS as a screening tool in NBS Sensitive Specific Accurate Quantitation
Internal standards: gold standard for accuracy
High impact Multiple Metabolite, Multiple Disease Screening
cost effective
High throughput
applications of mass spectrometry applications of mass spectrometry
to study to study proteinsproteins
Best techniques: Best techniques: •ESI - QuadrupoleESI - Quadrupole•MALDI-TOFMALDI-TOF•SELDI-TOFSELDI-TOF
TOP-DOWN approachTOP-DOWN approach
intact proteins are ionized intact proteins are ionized by either of the three by either of the three techniques described techniques described
above, and then introduced above, and then introduced to a mass analyzerto a mass analyzer
BOTTOM-UP approachBOTTOM-UP approach
Bottom-up proteomicsBottom-up proteomics is a common is a common method to identify proteins and method to identify proteins and characterize their amino acid characterize their amino acid
sequences and sequences and post-translational post-translational modificationsmodifications by proteolytic digestion by proteolytic digestion of proteins prior to analysis by mass of proteins prior to analysis by mass
spectrometryspectrometry..
applications of mass spectrometry applications of mass spectrometry
to study to study proteinsproteins
Identifying unknown proteinsIdentifying unknown proteins
Protein sequencing ) peptides are Protein sequencing ) peptides are sequenced by generating multiple sets sequenced by generating multiple sets of peptides(of peptides(
applications of mass spectrometry applications of mass spectrometry
to study to study proteinsproteins
Identification of chemical modification Identification of chemical modification ( post-translational modification in ( post-translational modification in proteins after synthesis)proteins after synthesis)
Identification of organisms Identification of organisms (identifying bacteria by finger printing (identifying bacteria by finger printing proteins) proteins)
