discovery of new technologies for protein separation, mass spectrometry technology, labeling techniques for protein quantification, and large-scale bioinformatics tool [1].
Mass spectrometry study has emerged as a multipurpose and
comprehensive tool in analyzing large-scale proteomics due to its
rapid advancement in resolution, sensitivity, mass accuracy, and
protein analysis scan rate [1]. The system can distinguish different
protein species by high precision measurements. The structural
information is obtained from isolated and fragmented molecular
ions using tandem mass spectrometry approach (MS/MS) [2].
Mass to charge ratio (m/z) of a molecule is applied to calculate
the mass of a protein following an ionization process in the instrument. Ionization of proteins and peptides were achieved by methods such as electrospray ionization (ESI) and matrix-assisted laser
desorption ionization (MALDI) [3, 4]. ESI produced ions from a
high voltage electrospray to transform liquid analytes into aerosol
while MALDI uses laser energy absorbing matrix to create ions
from large molecules. ESI ion sources were originally applied on
ion-trap or triple-quadrupole (QQQ) MS/MS, whereas MALDI
was most often coupled with time-of-flight (TOF) analyzers. However, the availability of hybrid-quadrupole TOF (Q-TOF) MS/MS
spectrometers have allowed and used frequently with ESI
[2]. These instruments conduct isolation of specific ions based on
their m/z ratio and the fragmentation of the ions within the gas
phase and allow the MS/MS spectra to be recorded. The MS/MS
spectrum of a peptide is then used as a basis to determine the amino
acid sequence of a specific protein. Availability of sequence databases has greatly improved protein identification from mass spectrometric analysis by using algorithms that complement MS/MS
spectra to the database [5, 6].
Protein characterization can be accomplished by different
approaches such as the bottom-up or top-down proteomics.
Bottom-up protein analysis or shotgun proteomics refers to the
characterization of different proteins based on the analysis of peptides that were digested from the protein prior to analysis [1]. In
this approach, proteins were typically digested with enzyme such as
trypsin, fractionated, and subjected to LC-MS/MS analysis. The
amino acid sequence and peptide identification can be achieved by
comparing the MS/MS spectra of the digested peptides with theoretical MS/MS spectra from a protein database.
Unique or shared peptides of a particular protein will determine the confidence score and grouped according to their specific
protein families [1]. In contrast, top-down proteomics is commonly applied for intact protein characterization. The method is
advantageous in the determination of post-translational modifications and isoforms [7]; however, difficulties with protein fragmentation and ionization has made shotgun proteomics a much
preferred method for protein analysis [1].
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Syafiq Asnawi Zainal Abidin et al.
Mass spectrometry study has emerged as a multipurpose and
comprehensive tool in analyzing large-scale proteomics due to its
rapid advancement in resolution, sensitivity, mass accuracy, and
protein analysis scan rate [1]. The system can distinguish different
protein species by high precision measurements. The structural
information is obtained from isolated and fragmented molecular
ions using tandem mass spectrometry approach (MS/MS) [2].
Mass to charge ratio (m/z) of a molecule is applied to calculate
the mass of a protein following an ionization process in the instrument. Ionization of proteins and peptides were achieved by methods such as electrospray ionization (ESI) and matrix-assisted laser
desorption ionization (MALDI) [3, 4]. ESI produced ions from a
high voltage electrospray to transform liquid analytes into aerosol
while MALDI uses laser energy absorbing matrix to create ions
from large molecules. ESI ion sources were originally applied on
ion-trap or triple-quadrupole (QQQ) MS/MS, whereas MALDI
was most often coupled with time-of-flight (TOF) analyzers. However, the availability of hybrid-quadrupole TOF (Q-TOF) MS/MS
spectrometers have allowed and used frequently with ESI
[2]. These instruments conduct isolation of specific ions based on
their m/z ratio and the fragmentation of the ions within the gas
phase and allow the MS/MS spectra to be recorded. The MS/MS
spectrum of a peptide is then used as a basis to determine the amino
acid sequence of a specific protein. Availability of sequence databases has greatly improved protein identification from mass spectrometric analysis by using algorithms that complement MS/MS
spectra to the database [5, 6].
Protein characterization can be accomplished by different
approaches such as the bottom-up or top-down proteomics.
Bottom-up protein analysis or shotgun proteomics refers to the
characterization of different proteins based on the analysis of peptides that were digested from the protein prior to analysis [1]. In
this approach, proteins were typically digested with enzyme such as
trypsin, fractionated, and subjected to LC-MS/MS analysis. The
amino acid sequence and peptide identification can be achieved by
comparing the MS/MS spectra of the digested peptides with theoretical MS/MS spectra from a protein database.
Unique or shared peptides of a particular protein will determine the confidence score and grouped according to their specific
protein families [1]. In contrast, top-down proteomics is commonly applied for intact protein characterization. The method is
advantageous in the determination of post-translational modifications and isoforms [7]; however, difficulties with protein fragmentation and ionization has made shotgun proteomics a much
preferred method for protein analysis [1].
234
Syafiq Asnawi Zainal Abidin et al.