Mass spectrometry basic principles

(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 .

1. RANIA MOHAMED EL-SHARKAWYRANIA MOHAMED EL-SHARKAWY
Lecturer of clinical chemistryLecturer of clinical chemistry
Medical Research InstituteMedical Research Institute , ,
Alexandria UniversityAlexandria University
MEDICAL RESEARCH INSTITUTE– ALEXANDRIA UNIVERSITYMEDICAL RESEARCH INSTITUTE– ALEXANDRIA UNIVERSITY
15
th
conference (3
rd
international)
ADVANCES IN MEDICAL RESEARCHADVANCES IN MEDICAL RESEARCH
` ` From molecular medicine-to clinical From molecular medicine-to clinical
applicationapplication``
Mass spectrometric techniquesMass spectrometric techniques

2. Mass Spectrometry

3. 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 the
structure and chemical properties of
molecules
A mass spectrometer A mass spectrometer is a device that
measures the mass-to-charge (m/z) ratio of ions.

5. Mass spectrometry

6. I. Sample Ionization

7. Simple Definition:
Ionization is a process of charging
a molecule.
Molecules must be charged in
order to
measure them using a mass
spectrometer

8. TypesTypes
Of ionizing radiationOf ionizing radiation
Important to determine the target Important to determine the target
from analyte investigatedfrom analyte investigated

9. 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.

10. Mass spectrometry

11. 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

12. 2-Electrospray
Ionization (ESI(

13. Electro spray ionization

14. 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

15. 3-Matrix Assisted 3-Matrix Assisted
Laser Desorption Laser Desorption
Ionization (MALDI)Ionization (MALDI)

16. 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

18. II. Analysis and Separation of
Sample Ions

19. 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.

20. Time Of Flight Mass AnalyzerTime Of Flight Mass Analyzer
))TOFTOF((

21. Quadrupole Mass AnalyzerQuadrupole Mass Analyzer

22. III. Detection of sample ions.

23. •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

24. m/z
Mass spectruM

25. Mass spectrum of CO
2

26. 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 ??

27. 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

28. Mass spectrometry

29. TechniquesTechniques
Of 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

30. TechniquesTechniques
Of 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

31. MALDI

32. SELDI-TOF

33. 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.

34. MS/MS uses 2 stages of mass analysis:
1. Selection of an ion
2. Analysis of ion fragments produced by collision with inert gas.

35. Phe
Subs B

36. Proteomics and mass Proteomics and mass
spectrometryspectrometry
APPLICATIONS OF mass spectrometry
PharmacokineticsPharmacokinetics
New born screeningNew born screening

37. 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

38. Newborn screening (NBS)Newborn screening (NBS)

39. MS/MS allows the screening of a numerous
array of metabolic disorders in a single
analytical run:
•Amino Acid Disorders
PKU, MSUD, Tyr, Cit, etc
•Fatty Acid Oxidation Disorders
MCAD, VLCAD, LCHAD, etc.
• Organic Acid Disorders
PA, MMA, IVA, GA-1, etc

40. %

in
t
e
n
s
it
y

41. 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

42. 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

43. 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

44. 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..

45. 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)

46. 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)