5. Normalized protein concentration is needed for sample comparison, and it can be achieved by protein quantitation assay.
Bicinchoninic acid (BCA) assay offers significant advantage
over Bradford assay for protein quantitation as it is extremely
sensitive and compatible with most ionic and nonionic detergents. BCA assay works by the reduction of cupric ions by
protein in alkaline conditions. Interaction of BCA with the
cupric ions produces a colored (purple) water-soluble complex
that can be detected using spectrophotometer at 562 nm.
6. Proteolytic digestion of proteins from heterogeneous lysate are
able to circumvent several challenges related to mass spectrometry analysis on intact proteins such as the separation, ionization, and characterization [12]. Trypsin is a digestion enzyme
that specifically cleaves at the carboxyl site of arginine and
lysine. The tryptic digested peptides are typically analyzed by
LC-MS/MS and allows for the identification and determination of protein expression levels through peptide abundance
(fragment ion intensity) [5]. Typical in-solution trypsin digestion involves the reduction of peptide disulfide bonds by denaturing agent such as dithiothreitol (DTT). This is followed by
alkylation process to prevent the re-formation of disulfide
bonds by introducing a carbamidomethylation modification
on the free cysteine residues. The alkylation step is important
to allow maximum trypsin digestion efficiency.
7. Database searching through search engines are crucial in proteomic analysis as it can translate raw MS/MS spectra obtained
into protein with identification data (protein ID). Examples of
database search engines include Mascot, SEQUEST, PEAKS
Studio, OMSSA, X!Tandem, and Agilent Spectrum Mill. In
this chapter, the methodology is described based on PEAKS
studio search engine as it offers more sensitive and accurate
peptide identification [11].
8. The results table should display information such as the protein
accession number (Protein ID), protein score (confidence,
-10lgP score), protein coverage (%), number of peptides and
unique peptides identified, average mass (MW), and protein
description. This information is crucial to present as it highlights proteins that were detected with high confidence from
the biological samples. Proteins identified with high confidence
are generally determined by protein score of more than
20 and/or with unique peptides identified equal or more
than 2.
Proteomics and Mass Spectrometry
239
Bicinchoninic acid (BCA) assay offers significant advantage
over Bradford assay for protein quantitation as it is extremely
sensitive and compatible with most ionic and nonionic detergents. BCA assay works by the reduction of cupric ions by
protein in alkaline conditions. Interaction of BCA with the
cupric ions produces a colored (purple) water-soluble complex
that can be detected using spectrophotometer at 562 nm.
6. Proteolytic digestion of proteins from heterogeneous lysate are
able to circumvent several challenges related to mass spectrometry analysis on intact proteins such as the separation, ionization, and characterization [12]. Trypsin is a digestion enzyme
that specifically cleaves at the carboxyl site of arginine and
lysine. The tryptic digested peptides are typically analyzed by
LC-MS/MS and allows for the identification and determination of protein expression levels through peptide abundance
(fragment ion intensity) [5]. Typical in-solution trypsin digestion involves the reduction of peptide disulfide bonds by denaturing agent such as dithiothreitol (DTT). This is followed by
alkylation process to prevent the re-formation of disulfide
bonds by introducing a carbamidomethylation modification
on the free cysteine residues. The alkylation step is important
to allow maximum trypsin digestion efficiency.
7. Database searching through search engines are crucial in proteomic analysis as it can translate raw MS/MS spectra obtained
into protein with identification data (protein ID). Examples of
database search engines include Mascot, SEQUEST, PEAKS
Studio, OMSSA, X!Tandem, and Agilent Spectrum Mill. In
this chapter, the methodology is described based on PEAKS
studio search engine as it offers more sensitive and accurate
peptide identification [11].
8. The results table should display information such as the protein
accession number (Protein ID), protein score (confidence,
-10lgP score), protein coverage (%), number of peptides and
unique peptides identified, average mass (MW), and protein
description. This information is crucial to present as it highlights proteins that were detected with high confidence from
the biological samples. Proteins identified with high confidence
are generally determined by protein score of more than
20 and/or with unique peptides identified equal or more
than 2.
Proteomics and Mass Spectrometry
239