2. Create a new project and select “mass only” as the correct
precursor. Set the error tolerance in De Novo settings at
0.1 Da for both precursor and fragment mass. Select trypsin
as the digestion enzyme and carbamidomethylation as the fixed
post-translational modification (PTM). Set the maximum
allowed variable PTM at 3 per peptide and report up to 5 candidates per spectrum.
3. In the database/PEAKS DB search, set the error tolerance for
precursor mass at 0.1 Da using monoisotopic mass and fragment ion at 0.1 Da. Select trypsin once again as the digestion
enzyme and allow nonspecific cleavage at both ends of the
peptide. Set the maximum number of missed cleavages per
peptide at 3 and carbamidomethylation as fixed PTM. Set the
maximum number of allowed variable PTM pep peptide at 10.
4. Allow PEAKS software to perform additional analysis by selecting the following options:
(a) Estimate FDR with decoy-fusion.
(b) Find unspecified PTM and common mutations with
Peaks PTM.
(c) Find more mutations with SPIDER.
5. Validate the identified proteins with the following filters: Protein -10lgP score ! 20 or FDR 1% and containing at least two
unique peptides for identification.
6. Export the data from the PEAKS Studio software to spreadsheet (Microsoft Excel) and tabulate the protein data based on
the protein family (see Note 8).
4 Notes
1. Lyophilize protein samples and store at À20
C until
further use.
2. Solution: Prepare all solutions with double distilled water
(ddH 2 O).
3. Solutions for liquid chromatography: Sonicate all solutions for
20 min prior usage.
4. Protein extraction from tissues and/or cultured cells is the
initial step in various biochemical techniques such as
SDS-PAGE, western blot, mass spectrometry, and protein purification via liquid chromatography. Rapid and efficient protein
extraction can be achieved by radioimmunoprecipitation assay
(RIPA) buffer. The buffer can be used together with protease
and phosphatase inhibitors to avoid protein degradation and
maintain protein phosphorylation.
238
Syafiq Asnawi Zainal Abidin et al.
precursor. Set the error tolerance in De Novo settings at
0.1 Da for both precursor and fragment mass. Select trypsin
as the digestion enzyme and carbamidomethylation as the fixed
post-translational modification (PTM). Set the maximum
allowed variable PTM at 3 per peptide and report up to 5 candidates per spectrum.
3. In the database/PEAKS DB search, set the error tolerance for
precursor mass at 0.1 Da using monoisotopic mass and fragment ion at 0.1 Da. Select trypsin once again as the digestion
enzyme and allow nonspecific cleavage at both ends of the
peptide. Set the maximum number of missed cleavages per
peptide at 3 and carbamidomethylation as fixed PTM. Set the
maximum number of allowed variable PTM pep peptide at 10.
4. Allow PEAKS software to perform additional analysis by selecting the following options:
(a) Estimate FDR with decoy-fusion.
(b) Find unspecified PTM and common mutations with
Peaks PTM.
(c) Find more mutations with SPIDER.
5. Validate the identified proteins with the following filters: Protein -10lgP score ! 20 or FDR 1% and containing at least two
unique peptides for identification.
6. Export the data from the PEAKS Studio software to spreadsheet (Microsoft Excel) and tabulate the protein data based on
the protein family (see Note 8).
4 Notes
1. Lyophilize protein samples and store at À20
C until
further use.
2. Solution: Prepare all solutions with double distilled water
(ddH 2 O).
3. Solutions for liquid chromatography: Sonicate all solutions for
20 min prior usage.
4. Protein extraction from tissues and/or cultured cells is the
initial step in various biochemical techniques such as
SDS-PAGE, western blot, mass spectrometry, and protein purification via liquid chromatography. Rapid and efficient protein
extraction can be achieved by radioimmunoprecipitation assay
(RIPA) buffer. The buffer can be used together with protease
and phosphatase inhibitors to avoid protein degradation and
maintain protein phosphorylation.
238
Syafiq Asnawi Zainal Abidin et al.