MASS SPECTROSCOPY

& ITS APPLICATIONS

 INTRODUCTION
 An analytical technique for determination of
elemental composition of a sample or molecule.
 Used to know the structures of molecule like
peptides & other chemical compounds.

 PRINCIPLE
 Consists of ionizing compound to generate
charged molecule & fragments& measure its
mass to charge ratio.
 PROTEIN MASS SPECTROSCOPY
 Application of mass spectroscopy for study of
proteins.

 To know the peptides of interest are present in

a protein.

 To compare unknown with the known using

databases.
 PROCESS
 Fractionating the proteins using 2-Dgel
Electrophoresis & High Performance Liquid
Chromatography(HPLC).
 Protein spot of interest is digested using Trypsin
as enzyme.
 Ionization of molecules using Electrospray
ionization(ESI)/ Matrix-assisted laser
desorption/ionization(MALDI).
 Introducing into mass spectrometry using Peptide
Fingerprinting/Tandem mass spectrometry.
 ESI & MALDI
 These are techniques used for producing ions.
 ESI-Used for macromolecules as it can
overcome the rigidness & complexity.
 For liquid and wet conditions.
 MALDI(TOF)- Soft ionization technique.
 Uses matrix to protect molecule from laser
beam.
 For solid & fragile conditions.
 TOF-Time of flight measures velocity from
which mass-charge ratio can be determined.
ESI

MALDI
 PROTEIN IDENTIFICATION
 Peptide Fingerprinting:-
 Uses mass-charge ratio values from
MALDI/ESI.
 Compares it with database having known
protein sequence or genome.
 Comparison is done by different software
programs.
 Then it is known whether peptide of interest is
present in given sample.
Tandem Mass Spectroscopy
It is a popular method as it uses Collision-induced
dissociation for fragmentation

It gives a notation for the fragments generated

through this technique.
 a, b, c, - charge on N-terminus.

 x, y, z, - charge on C-terminus.
Notation of Tandem mass spectroscopy
De novo sequencing
 Can be performed without any prior
knowledge about the amino acid sequence.
 An incomplete or corrupted database have
problem of identifying the protein, but De
novo helps in catching these missing files.
 False positives in database search are virtually
eliminated.
 Proven successful in confirming and expanding
results from databases.
 SOFTWARE PROGRAMS
 Two types
 Database search programs.
A. SEQUEST
B. MASCOT
C. X!TANDEM
 De novo search programs.
A. DeNoS
B. PEAKS
C. SPIDER(Homology Sequencing)
HL-60 cells
 Human promyelocytic leukemia cells.
 These proliferates continuously with fetal
bovine serum supplemented in a medium.
 Then differentiates into granulocytes like
eosinophil using retinoic acid.
 During HL-60 cell differentiation certain
proteins were newly synthesized.
 These proteins were identified by application
of mass spectroscopy.
 Sources
 Wikipedia

 ExPASy

 Bio Med
central
Thank you

By

FANI