advances-in-protein-chemistry

Figure 1: Biochemical and biophysical properties of proteins can form basis for their structural and functional characterization.
different molecular species. Precisely it involves separation of molecules according to mass/size ratio of ionized species in
an electric field. Mass spectrometry instruments are fundamentally composed of three components: an ionization source
that converts particles into ions, a mass analyzer that sorts ions according to m/z and an ion detector that measures m/z [2].
In these instruments positive ions of analyte are generated in short source region in presence of an electrical field E, which
imparts kinetic energy K= ZeEs to each ion. In this equation Ze is the charge, E the electrical field and s the length of source
region. The ions generated in this manner into the field-free drift region of length D have the same kinetic energy regardless
of their size. The velocity with which these ionized particles move is defined by K = mv 2 /2. Equating the above two equations
leads to ZeEs= mv2/2 from which velocity of the particle can be deduced as
1/2
⎛2ZeEs
⎞
v = ⎜ ⎟
⎝ m ⎠
The time that each ion will take to traverse the drift region is given by:
1/2
D ⎛ m ⎞
t = = ⎜ ⎟ D
v ⎝2ZeEs
⎠
Combination of equations (1) and (2) allows expressing mass/charge ratio in terms of t/D:
m ⎛ t ⎞
= 2ZeEs⎜ ⎟
Z ⎝D⎠
2
Based on method of ionization used, mass spectroscopy can be categorized into two types: Matrix-Assisted Laser
Desorption-Ionization (MALDI) and Electrospray Ionization (ESI). Briefly, in MALDI, protein containing samples are
embedded into specific matrix molecules that absorb the ionization laser beam and transfer energy to analyte. On the other
hand in ESI analyte samples are directly injected into the ionizing chamber that converts peptides into smaller ions. Ionized
analytes in both methods are directed via a mass analyzer towards a detector that generates MS spectra with each peak of
the spectra representing a characteristic m/z ratio.
(3)
(1)
(2)
Application: Role of mass spectrometry in protein characterization has been far beyond merely identifying masses
and forms a very formidable technique in ‘proteomics’. Thus role for mass spectroscopy in protein characterization can be
envisioned in protein quantification, protein profiling, protein interaction analysis and studying modifications like posttranslation
modifications [3,4]. Protein mass estimation involves by mass spectrometry involves use of single-stage mass
spectrometers that act like balances [5]. Proteome analysis on other hand involves use of tandem mass spectrometry (MS/
MS) which is accompanied by second stage fragmentation of specific ions after their mass has been determined [5]. MALDI
is usually coupled to time of flight (TOF) analyzers that directly measure mass of intact peptides whereas ESI is coupled to
ion traps and triple quadrupole analyzers and are used to generate fragment ion spectra (collision-induced (CID) spectra)
of selected precursor ions [3,6,7].
Studying proteins through mass spectroscopy is a multi-step process. First step involves isolation of proteins from their
biological sources to be followed by digestion and fractionation. While as peptide- mass mapping by MALDI-TOF is
useful in peptide identification (peptide mass fingerprinting-PMF), peptide sequencing can be accomplished by ESI-MS/
MS [3,8]. There are two approaches that can be employed during protein sequencing. The top-down approach does not
involve enzymatic digestion of protein sample but relies on transfer of intact protein to gas phase. The main advantage
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