Proteomics & Genomics Dr.
Vikash Kumar Dubey
Lecture 15: Introduction to mass spectrometry-I
Mass spectrometry (MS) is an analytical technique that measures the mass/charge ratio of
charged particles in vacuum. Mass spectrometry can determine masse/charge ratio with high
accuracy. Molecules in a test sample are converted to gaseous ions that are subsequently
separated according to mass/charge ratio. Several types of experiments can be performed with
mass spectrometry. We shall see a few examples in coming lectures.
A typical mass spectrometry instrument has three components as shown in Fig. 1.
1. Ion source
2. Analyzer
3. Detector: The detector records the current produced when an ion passes by or hits a surface.
Several types of detector are used like electron multiplier, Faraday cups and ion to photon
detectors.
Figure 1: Basic components of mass spectrometry
All mass spectrometry operate under vacuum (10-6 torr pressure). Without high vacuum, the
ions produced in the source will not reach the detector. At atmospheric pressure, the mean
free path of a typical ion is around 52 nm; at 1mtorr, it is 40 mm; and at 10-6 torr, it is 40 m.
Sample inlet may be coupled to a liquid chromatography system (call LC-MS), gas
chromatography system (GC-MS) or capillary electrophoresis system.
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We shall study components of mass spectrometry (MS) in detail. A simplified scheme of mass
spectrometry (MS) and MS-MS (tandem mass spectrometry) is shown in Fig 2)
A simplified scheme of mass spectrometry (MS)
A simplified scheme of MS-MS
Figure 2: A simplified scheme of mass spectrometry and Tandem mass spectrometry (also
called MS MS). In MS-MS, after first analyzer, analytes are fragmented and fragments are
analyzed in second analyzer.
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Ion source:
There are several types of ionization methods in mass spectrometry. The physical basis of
ionization methods are very complex and outside the scope of the course. Most common
methods are:
(a) Matrix-assisted laser desorption/ionization (MALDI)
This method of ionization is a soft ionization method and results in minimum fragmentation
of sample. This method is used for non-volatile, and thermally labile compounds such as
proteins, oligonucleotides, synthetic polymers. Sample is mixed with 1000 times molar excess
of sample and spotted onto a metal plate and dried. Matrix plays a key role in this technique
by absorbing the laser light energy and causing a small part of the target substrate to vaporize.
Although, the process of forming analyte ions is unclear, it is believed that matrix which has
labile protons, such as carboxylic acids, protonates neutral analyte molecules after absorbing
laser light energy. Scheme of MALDI is shown in Fig. 3 and some common matrices are
listed in Table 1
Figure 3: Scheme of Matrix-assisted laser desorption/ionization (MALDI)
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Proteomics & Genomics Dr. Vikash Kumar Dubey
Table 1: Few common matrices used in MALDI
Matrix Application
3,5-Dimethoxy-4- Higher mass biopolymer
hydroxycinnamic acid
-cyano-4-hydroxycinnamic acid Protein, peptides, organic compounds
2,5 dihydroxy benzoic acid Oligonucleotide
Trihydroxyacetophenone Peptides, Oligonucleotide
(b) Electrospray Ionisation (ESI)
Electrospray Ionisation (ESI) is a preferred method of ionization when the sample is in liquid
form. This is also a soft method of ionization and results in less fragmentation. ESI is a very
valuable method for analysis of biological samples. The method was developed by John Fenn
and he shared 2002 Nobel prize in chemistry for this work. The analyte is introduced either
from a syringe pump or as the eluent flow from liquid chromatography with a flow rate 1µl
min-1. The analyte solution passes through the electrospray needle (Stainless steel capillary
with 75-150 µm internal diameters) that has a high potential difference (with respect to the
counter electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the
spraying of charged droplets from the needle with a surface charge of the same polarity to the
charge on the needle. As droplet moves towards counter electrode cone (which passes it to
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Proteomics & Genomics Dr. Vikash Kumar Dubey
analyzer), solvent evaporation occurs and droplet shrinks until it reaches the point that the
surface tension can no longer sustain the charge (the Rayleigh limit) and at that point droplets
break. This produces smaller droplets and the process is repeated. Finally after all solvent
evaporated, charge is passed on to analyte. These charged analyte molecules can have single
or multiple charges (Fig. 4)
Figure 4: A schematic of the mechanism of ion formation in ESI.
(c) Electron ionization
Electron Ionization (EI) works well for many gas phase molecules, but it results in extensive
fragmentation and molecular ions are not observed for many compounds. Fragmentation mass
spectra are sometime useful because it provides structural information of a molecule.
The electron beam is produced by a filament of rhenium or tungsten wire by thermionic
emission. When cathode filament of rhenium or tungsten is heated at temperature over 1000
K, electrons are emitted. The generated electrons are accelerated to 70 eV which results in
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Proteomics & Genomics Dr. Vikash Kumar Dubey
electron beam. The volatile sample or sample in gaseous phase containing neutral molecules
is introduced to the ion source in a perpendicular direction to the electron beam. Electron
impact on the analyte results in either loss of electron (to produce cation) or gain of electron
(to produce anion). Chemical bonds in organic molecules are formed by pairing of electrons.
Electron impact may knock out one of the electron. This leaves the bond with a single
unpaired electron. This is radical as well as being cation written as M+., where (+) indicates
ionic state while (.) indicates radical. Electron impact may result in electron capture (extra
unpaired electron). This generates a radical as well as being anion written as M-., where (-)
indicates ionic state while (.) indicates radical (Fig. 5)
Figure 5: A schematic of the mechanism of ion formation in electron ionization
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(a) Chemical Ionization
Set-up for chemical Ionization is similar to electron impact ionization. However,
in this method, a reagent gas like CH4 is injected in the ion chamber. Due to
electron impact, the reagent gas in the chemical ionization source gets ionized.
This follows injection of analyte molecule. Analyte molecules undergo many
collisions with the reagent gas. The reagent gas ions in this cloud react and
produce adduct ions as shown in figure, which are excellent proton donors for
analyte.
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