Protein Mass
Spectrometry: Novel Approaches in Pharmaceutical Biotechnology
Dibyajyoti Saha* and Ankit Tamrakar
School of Pharmacy, Chouksey Engineering College, Lal
Khadan, Masturi Road,
Bilaspur-495004,C.G.
ABSTRACT:
Protein
mass spectrometry refers to the application of mass spectrometry to study the
proteins and nowadays it is introduced in proteomies
science as well as pharmaceutical biotechnology. Mass spectrometry is an
important emerging method for ionization of whole proteins are electrospray ionization (ESI) and matrix assisted laser
desorption/ionization (MALDI). In keeping with the performance and mass range
of available mass spectrometers, two approaches are used for characterizing
proteins. In the first, intact proteins are ionized by either of the two
techniques described above, and then introduced to a mass analyzer. This
approach is referred to as “top-down” strategy of protein analysis. In the
second, proteins are enzymatically digested into
smaller peptides using a protease such as trypsin.
Subsequently these peptides are introduced into the mass spectrometer and
identified by peptide mass fingerprinting or tandem mass spectrometry. Hence
this latter approach (also called “bottom up” proteomies)
uses identification at the peptide level to infere
the existence of proteins.
KEYWORDS: Mass spectrometry, Proteins, Peptides, High
performance liquid chromatography, quantization.
INTRODUCTION:
Whole
protein mass analysis is primarily conducted using either time-of-flight (TOF)
MS, or fourier transform ion
cyclotron resonance (FT-ICR). These two types of instrument are preferable here
because of their wide range, and in the case of FT-ICR, its high mass accuracy.
Mass analysis of proteolytic peptides is a much more
popular method of protein characterization, as cheaper instrument designs can
be used for characterization. Additionally, sample preparation is easier once
whole proteins have been digested into smaller peptide fragments. The most
widely used instrument for peptide mass analysis are
the MALDI time-of-flight instruments as they permit the acquisition of PMFs at
high pace. Multiple stage quadruple –time-of-flights and the quadruple ion trap
also find use in the application1-3.
PROTEIN AND PEPTIDE FRACTIONATION COUPLED
WITH MASS SPECTROMETRY:
Proteins
of interest to biological researches are usually part of a very complex mixture
of other proteins and molecules that co-exist in the biological medium.
This
presents two significant problems. First, the two ionization techniques used
for large molecules only work well when the mixture contains roughly equal
amounts of constituents, while in biological samples, different proteins tend
to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a
tendency to “drown” or suppress signals from less abundant ones. The second
problem is that the mass spectrum from a complex mixture is very difficult to
interpret because of the overwhelming number of mixture components. This is
exacerbated by the fact that enzymatic digestion of a protein gives rise to a
large number of peptide products4-5.
To
contend with this problem, two methods are widely used to fractionate proteins,
or their peptide products from an enzymatic digestion. The first method
fractionates whole proteins and is called two-dimensional gel electrophoresis.
The second method, high performance liquid chromatography is used to
fractionate peptides after enzymatic digestion. In some situations, it may be
necessary to combine both of these techniques6.
PROTEIN IDENTIFICATION:
There
are two main ways MS is used to identify proteins. Peptide mass fingerprinting
(mentioned in the previous section) uses the masses of proteolytic
peptides as input to a search of a database of predicted masses that would
arise from digestion of a list of known proteins. If a protein sequence in the
reference list gives rise to a significant number of predicted masses that
match the experimental values there is some evidence that this protein was
present in the original sample7.
Tandem
MS is becoming a more popular experimental method for identifying proteins.
Collision-induced dissociation is used in mainstream applications to generate a
set of fragments from a specific peptide ion. The fragmentation process
primarily gives rise to cleavage products that break along peptide bonds.
Because of this simplicity in fragmentation, it is possible to use the observed
fragment masses to match with a database of predicted masses for one of many
given peptide sequences. Tandem MS of whole protein ions has been investigated
recently using electron capture dissociation and has demonstrated extensive
sequence information in principle but is not in common practice. This is
sometimes referred to as the “top-down” approach in that it involves starting
with the whole mass and then pulling it apart rather than starting with pieces
(proteolytic fragments) and piecing the protein back
together using De novo repeat detection (bottom-up)8.
PROTEIN QUANTIZATION:
Several
recent methods allow for the quantization of protein by mass spectrometry
(quantitative proteomics). Typically, stable (e.g. non-radioactive) heavier
isotopes of carbon (13C) or nitrogen (15N) are incorporated into one sample
while the other one is labeled with corresponding light isotopes (e.g. 12C and
14N). The two samples are mixed before the analysis9. Peptides
derived from the different samples can be distinguished due to their mass
difference. The ratio of their peak intensities corresponds to the relative
abundance ratio of the peptides (and proteins). The most popular methods for
isotope labeling are SILAC (stable isotope labeling by amino acids in cell
culture), trypsin-catalyzed 180 labeling, ICAT
(isotope coded affinity tagging), TRAQ (isobaric tags for relative and absolute
quantization)10.
“Semi-quantitative” mass spectrometry can be performed without labeling of
samples. Typically, this is done with MALDI analysis (in linear mode). The peak
intensity, or the peak area, from individual molecules (typically protein) is
here correlated to the amount of protein in the sample. However, the individual
signal depends on the primary structure of the protein, on the complexity of
the sample, and on the settings of the instrument. Other types of “labels-free”
quantitative mass spectrometry, uses the spectral counts (or peptide counts) of
digested proteins as a means for determining relative protein amounts11,12.
CONCLUSION:
Characteristics
indicative of the 3 dimensional structures of proteins can be probed with mass
spectrometry in various ways. By using chemical cross linking to couple parts
of the protein that are close in space, but far apart in sequence, information
about the overall structure can be inferred. By following the exchange of amide
protons with deuterium from the solvent, it is possible to probe the solvent
accessibility of various parts of the proteins.
REFERENCES:
1.
Siddiqui AS. A
Database of Protein Structural Domains. Bioinformatics. 17; 2001: 200-201.
2.
Tomlinson E and Livingston C. Pharm J. 1989.
243, 646-648.
3.
Schulz GE and Shirmin RH. Principles of
Protein Structure. Springer-Verlag, New York. 1979:
222-224
4.
Marshak D and Liu D. Therapeutic Peptides and Proteins
Formulation, Delivery, Targeting. Cold Spring Harbor Laboratory. Cold Spring
Harbor. New York. 1989: 198-200
5.
Lee VHL. Peptide and Protein Drug Delivery. Marcel Dekker. New York.
1991: 789-790
6.
Davis SS. et.al.
Delivery Systems for Peptide Drugs
Plenum Press, New York. 1986: 345-350
7.
Langer R. Pharm. Tech. 13; 1989: 18-30.
8.
Johnson KA. Adv. Drug Deliv. Rev. 26; 1997:
3-15.
9.
Bornstein P and Traub W. The Proteins.
Academic Press. New York. Vol.4; 1979. 567-568
10. http://en.wikipedia.org.wiki/Mass_spectrometry
11. http://masspec.seripps.edu/redirect.html
12. http://science.widener.edu/sub/masspec/massspec.pdf
Received on 23.02.2011 Accepted
on 10.05.2011
© Asian Pharma Press All
Right Reserved
Asian J.
Pharm. Ana.
1(2): April-June 2011; Page 25-26