3.4 Mass
Spectrometry–Based
Quantification
The bioinformatics analysis can be done according to the following
references using the free software’s MaxQuant and Perseus
described below [8–11]. Alternatively, other software programs
can be used as for example the free academic software Proline
[12] (http://www.profiproteomics.fr/proline/) or a commercial
one, Progenesis QI for proteomics (Nonlinear dynamics a Waters
company). Here, a summary of the bioinformatics analysis of raw
files from MS is given. Only changes from the default parameters
are mentioned (see Note 25).
3.4.1 Protein
Identification
and Quantification Using
MaxQuant
1. Download MaxQuant from https://www.maxquant.org/
website.
2. Install the software on a compatible computer (see Note 26).
3. Load all LC-MS/MS files using “Raw data/Load” (see Note
27).
4. Fulfil your experimental setup in the “Experiment” column.
Select LC-MS/MS file and then click “raw data/Set experiment” and name the line using an unique name for each
technical replicate and biological replicate (e.g., Control1,
Control2, Control3, etc. and Pulldown1, Pulldown2, Pulldown3, etc.).
5. Under “Group-specific parameters/Label-free quantification”
select LFQ for label-free quantification (see Note 28) and set
the “LFQ min. ratio count” to 1 (see Note 29).
6. Download a protein database corresponding to your studied
species from the Uniprot website (https://www.uniprot.org/)
(see Note 30).
7. Load the fasta file in the MaxQuant software using “Global
parameters/Sequences” and do not forget to enter the organism ID using the “Taxonomy ID” button (e.g., 9606 for
Human) and set the parse rule using “Identifier rule” (e.g.,
For Uniprot database use the Uniprot identifier).
8. Under the “Group-specific parameters/Identification” set on
the Match between run option (see Note 31).
9. Under the “Group-specific parameters/Label-free quantification” set on the iBAQ calculation (see Note 32).
10. Under the “Group-specific parameters/Folder locations” set
on the location of the temporary folder and the Combined
folder locations (see Note 33).
11. Set the number of processors according to your computer at
the bottom left of the MaxQuant window. Press “Start” to run
the program and save the parameters files (mqpar.xml) using
the menu “File/Save parameters” in the combined folder (see
Note 34).
32
Avais M. Daulat et al.
Spectrometry–Based
Quantification
The bioinformatics analysis can be done according to the following
references using the free software’s MaxQuant and Perseus
described below [8–11]. Alternatively, other software programs
can be used as for example the free academic software Proline
[12] (http://www.profiproteomics.fr/proline/) or a commercial
one, Progenesis QI for proteomics (Nonlinear dynamics a Waters
company). Here, a summary of the bioinformatics analysis of raw
files from MS is given. Only changes from the default parameters
are mentioned (see Note 25).
3.4.1 Protein
Identification
and Quantification Using
MaxQuant
1. Download MaxQuant from https://www.maxquant.org/
website.
2. Install the software on a compatible computer (see Note 26).
3. Load all LC-MS/MS files using “Raw data/Load” (see Note
27).
4. Fulfil your experimental setup in the “Experiment” column.
Select LC-MS/MS file and then click “raw data/Set experiment” and name the line using an unique name for each
technical replicate and biological replicate (e.g., Control1,
Control2, Control3, etc. and Pulldown1, Pulldown2, Pulldown3, etc.).
5. Under “Group-specific parameters/Label-free quantification”
select LFQ for label-free quantification (see Note 28) and set
the “LFQ min. ratio count” to 1 (see Note 29).
6. Download a protein database corresponding to your studied
species from the Uniprot website (https://www.uniprot.org/)
(see Note 30).
7. Load the fasta file in the MaxQuant software using “Global
parameters/Sequences” and do not forget to enter the organism ID using the “Taxonomy ID” button (e.g., 9606 for
Human) and set the parse rule using “Identifier rule” (e.g.,
For Uniprot database use the Uniprot identifier).
8. Under the “Group-specific parameters/Identification” set on
the Match between run option (see Note 31).
9. Under the “Group-specific parameters/Label-free quantification” set on the iBAQ calculation (see Note 32).
10. Under the “Group-specific parameters/Folder locations” set
on the location of the temporary folder and the Combined
folder locations (see Note 33).
11. Set the number of processors according to your computer at
the bottom left of the MaxQuant window. Press “Start” to run
the program and save the parameters files (mqpar.xml) using
the menu “File/Save parameters” in the combined folder (see
Note 34).
32
Avais M. Daulat et al.