Mass spectrometry is an analytical technique that identifies chemicals in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. It produces a mass spectrum plot showing ion abundance versus mass-to-charge ratio. Mass spectrometry is used to quantify known materials, identify unknown compounds, and elucidate molecular structure and properties. The technique involves converting the sample to gaseous ions, optionally with fragmentation, and then separating and detecting the ions.

1. Presented by,
Erin Davis

2.  Mass spectrometry (MS) is an powerful
analytical chemistry technique
 It helps to identify the amount and type of
chemicals present in a sample by
measuring
 mass-to-charge ratio
 abundance of gas-phase ions
 A mass spectrum is a plot of the ion signal
as a function of the mass-to-charge ratio
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3.  It is a used to:
 quantify known materials
 identify unknown compounds within a
sample
 to elucidate the structure and chemical
properties of different molecules
 The complete process involves the
conversion of the sample into gaseous ions,
with or without fragmentation
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5.  A mass spectrometer generates multiple ions from
the sample
 it separates them according to their specific
mass-to-charge ratio (m/z)
 records the relative abundance of each ion
type
 The production of gas phase ions of the
compound, basically by electron ionization.
 This molecular ion undergoes fragmentation.
 Each primary product ion derived from the
molecular ion, in turn, undergoes fragmentation,
and so on.
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6.  A mass spectrum of the molecule is thus produced
 It displays the result in the form of a plot of ion
abundance versus mass-to-charge ratio
 Ions provide information concerning
 the nature and the structure of their precursor
molecule
 In the spectrum of a pure compound,
 molecular ion appears at the highest value of
m/z
 followed by ions containing heavier isotopes
 Thus provides the molecular mass of the compound
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8.  The instrument consists of three major
components:-
 Ion Source
For producing gaseous ions from the substance
being studied
 Analyzer
For resolving the ions into their characteristics
mass components according to their mass-to-
charge ratio
 Detector System
For detecting the ions and recording the relative
abundance of each of the resolved ionic species22-11-2015Mass Spectrometry 8

9. In addition,
 sample introduction system is necessary to admit
the samples to be studied to the ion source
 maintain the high vacuum requirements (~10-6
to 10-8 mm of mercury)
 computer is required
 to control the instrument
 acquire and manipulate data
 compare spectra to reference libraries
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10.  Produce ions from the sample in the ionization
source
 Separate these ions according to their mass-to-
charge ratio in the mass analyzer
 Eventually, fragment the selected ions and
analyze the fragments in a second analyzer
 Detect the ions emerging from the last analyzer
and measure their abundance with the detector
that converts the ions into electrical signals
 Process the signals transmitted to the computer
and control the instrument using feedback
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WORKING

11. Analysis of Biomolecules
 Till the1970s, the analytical techniques were
electrophoresis, chromatography or ultracentrifugation
methods
 The results were based on characteristics other than the
molecular weight
 Exact molecular weight of a macromolecule was found
by calculation based on its chemical structure
 The development of desorption ionization methods
based on the emission of pre-existing ions such as:-
 plasma desorption(PD)
 fast atom bombardment(FAB)
 laser desorption (LD)
allowed the application of mass spectrometry for
analyzing complex biomolecules
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12.  The determination of the complete structure of
oligosaccharides is more complex than that of proteins.
 It involves the determination of isomeric nature of
monosaccharides and their capacity to form linear or
branched oligosaccharides.
 Knowing the structure of an oligosaccharide requires
determination of monosaccharide sequence
 its branching pattern
 isomer position
 anomeric configuration
 Advances in glycobiology involves
comprehensive study of structure, bio-synthesis,
biology of sugars and saccharides.
 Mass spectrometry is emerging as an enabling
technology in the field of glycomics and glycobiology
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13.  Lipids are made up of many classes of different
molecules which are soluble in organic solvents
 Lipidomics constitutes the detailed analysis and
global characterization, both spatial and temporal, of
the structure and function of lipids within a living
system
 Many new strategies for mass-spectrometry-based
analyses of lipids have been developed. The most
popular lipidomics methodologies involves
 electrospray ionization (ESI) sources
 triple quadrupole analyzers
 It determines the mol.wt, elemental composition, the
position of branching and nature of substituents in
the lipid structure 22-11-2015Mass Spectrometry 13

14.  Proteins and peptides are linear polymers made up of
combinations of the 20 amino acids linked by peptide bonds
 Proteins undergo several post translational modifications,
extending the range of their function via such modifications
 The term Proteomics is the analysis of complete protein content
in a living system, including co- and post-translation modified
proteins and alternatively spliced variants
 Mass Spectrometry allows the determination of the mol.mass of
peptides
 It is used for protein identification, de novo sequencing,
identification of post-translational modifications
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15.  Oligonucleotides (DNA orRNA), are linear polymers
of nucleotides.
 It is composed of a nitrogenous base, a ribose
sugar and a phosphate group.
 Oligonucleotides may undergo several natural
covalent modifications in tRNA and rRNA resulting
from reactions with exogenous compounds
 Mass spectrometry plays an important role in the
determination of
 structural modifications
 Positions and mol.wt of oligonucleotide
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16. SimGlycan®
 Predicts the structure of glycans and glycopeptides from the
MS/MS data acquired by mass spectrometry, facilitates
glycosylation and post translational modification studies
 It accepts the experimental MS profiles, of both glycopeptides
and released glycans, matches them with its own database
and generates a list of probable structures
 It also supports multi stage mass spectrometry data analysis
which enables structural elucidation and identification of
fragmentation pathways
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17. SimLipid
 Innovative lipid characterization tool which enables
structural elucidation of unknown lipids using
MS/MS data
 This software analyzes lipid mass spectrometric
data for characterizing and profiling lipids
 Annotate mass spectra with the lipid structures
identified using abbreviations
 Studies the effect of ionizing energy on molecules.
It depends upon chemical reactions in the gas
phase in which sample molecules are consumed
during the formation of ionic and neutral species
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18.  Digest the protein to peptides (in gel or solution).
MS gets limited sequence data from whole proteins,
but easily analyze peptides
 Trypsin is used for digestion. Majority of peptides
are ideal size for analysis, and peptides behave
nicely in mass spectrometer
 Separate peptides, usually on reverse phase column
with acetonitrile gradient(column dia 75 µm) We
use acetic acid in the solvent.
 Place ionized peptides in vapour phase by passing
the column elute, containing peptides and solvent,
to form tiny droplets. After evaporation of solvent,
peptides are left in the vapour phase. Charged
surfaces move the ionized peptide into the MS22-11-2015Mass Spectrometry 18

19.  Using chromatography to introduce molecules into a
mass spectrometer is LC-MS or HPLC-MS
 Measure mass of peptides
 Fragment peptides -- A collision with gas molecules
fragments peptides at peptide bonds. This is CID
(collision induced dissociation) or CAD (collisionally
activated dissociation)
 Measure mass of fragments from peptides. Because
there are two steps of mass spectrometry (mass of
peptide, mass of fragment of peptide), this is called
MS/MS, or MSn because there can be 2 or more
fragmentation steps. A two step process is also called
tandem mass spectrometry
 Use fragment mass data to determine the sequence
of the peptide by seeing which combinations of
amino acids gives the observed masses of peptide
fragments
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