MASS SPECTROMETRY FOR

PROTEOMICS

Presented by – Navneet Kumar BBT 1516 | Aditi Majumdar BBT 1513 | Gauravaaditya Kulkarni BBT 1510
 INTRODUCTION
• Mass spectrometry (MS) is a technique for creating gas phase ions
from the molecules or atoms in a sample, separating the ions
according to their mass-to-charge ratio, m/z, and measuring the
abundance of the ions formed.

• MS is an analytical technique that provides qualitative and

quantitative information , including the mass of molecules and atoms
in samples as well as the molecular structure of organic and inorganic
compounds.
 PRINCIPLE AND TERMS
• The mass spectrometer is an instrument that separates gas phase
ionized atoms, molecules , and fragments of molecules by the
difference in their mass-to-charge ratios.
• The mass to-charge ratio is symbolized by m/z, where the mass m is
expressed in unified atomic mass units and z is the number of charges
on the ion.
• The mass of an ion is given in unified atomic mass units, u. One
unified atomic mass unit is equal to 1/12 of the mass of the most
abundant, stable, naturally occurring isotope of carbon, 12C. The
mass of 12C is defined as exactly 12 u.
• A synonym for the unified atomic mass unit is the Dalton (Da); 1 u =
1 Da. In the SI unit of mass, 1 u = 1.665402 x 10−27 kg.
• The term z symbolizes the number of charges on the ion; this number
may be positive or negative, such as +1, -1, +2, +10, and so on. The
number of charges is not the same as the total charge of the ion in
coulombs. The total charge q = ze, where e is the magnitude of the
charge on the electron, 1.6 X 10−19 C.
• The resolving power of a mass spectrometer is defined as its ability
to separate ions of two different m/z values. Numerically, the
resolution is equal to the mass of one singly charged ion, M, divided
by the difference in mass between M and the next m/z value that can
be separated.
 Instrumentation

Reference: http://www.premierbiosoft.com/tech_notes/mass-spectrometry.html
SAMPLE RESULT
TYPES OF MASS SPECTROMETRY
 COMPONENTS & EXPERIMENTS

Schematic of a proteomic experiments. Protein is digested

using trypsin and pre-fractionated using HPLC. MS device
Fig: The basic components of mass spectrometer. makes use of a combination of ion source and one
or two mass analyzers.
 IONIZATION SOURCE

Fig: Different ionization sources: gas phase, solution phase & solid phase.
MALDI (Matrix Assisted Laser Desorption-
Ionization)

Fig: A schematic diagram of ionization using MALDI

ESI (Electrospray ionization)

Fig: A schematic diagram of ionization using ESI.

 MASS ANALYZER

Fig: Different types of mass analyzers used for proteomics applications.

 TIME OF FLIGHT

The time required by a peptide to

move across the entire flight tube is
given by:

A time of flight (TOF) mass analyzer and its working principle:

Ions are accelerated at different velocities depending on their
m/z ratios. Ions of lower masses are accelerated to higher
velocities and reach the detector first.
 TANDEM MASS SPECTROMETRY

Tandem MS/MS - One of the peaks obtained is chosen and that particular peptide is allowed to move into the
next mass analyzer where it is further fragmented and detected. Ideally, the different daughter peptides so
obtained differ by one or more amino acid and hence from there, the sequence of the peptide can be
determined.
ADVANTAGES
As an analytical technique it possesses distinct advantages such as:
i. Increased sensitivity over most other analytical techniques because
the analyzer, as a mass-charge filter, reduces background
interference
ii. Excellent specificity from characteristic fragmentation patterns to
identify unknowns or confirm the presence of suspected
compounds
iii. Gives information about molecular weight
iv. Provides information about the isotopic abundance of elements
v. Temporally resolved chemical data is obtained
DISADVANTAGES
A few of the disadvantages of the method are as follows –
i. It often fails to distinguish between optical and geometrical
isomers and the positions of substituent in o-, m- and p- positions
in an aromatic ring.
ii. Its scope is limited in identifying hydrocarbons that produce similar
fragmented ions.
APPLICATIONS
• Proteomics: Characterization of proteins and protein complexes,
sequencing of peptides, and identification of posttranslational
modifications.
• Metabolomics: Cancer screening and diagnosis, global metabolic
fingerprinting analysis, biomarker discovery and profiling, biofuels
generation and use, lipidomics studies, and metabolic disorder
profiling.
• Environmental analysis: Drinking water testing, pesticide screening
and quantitation, soil contamination assessment, carbon dioxide and
pollution monitoring, and trace elemental analysis of heavy metals
leaching. Also can be used in space exploration.
• Pharmaceutical analysis: Drug discovery and absorption, distribution,
metabolism, and elimination (ADME) studies, pharmacokinetic and
pharmacodynamic analyses, metabolite screening, and preclinical
development.
• Forensic analysis: Analysis of trace evidence (e.g., fibers in carpet,
polymers in paint), arson investigation (e.g., fire accelerant),
confirmation of drug abuse, and identification of explosive residues
(bombing investigation).
• Clinical applications: Clinical drug development, Phase 0 studies,
clinical tests, disease screening, drug therapy monitoring, analysis of
peptides used for diagnostic testing, and identification of infectious
agents for targeted therapies.
THANK YOU