21. After thawing cell lysates, always centrifuge at 16,000 Â g for
20 min to remove aggregates.
22. 96-well plate could be stored at À20
C until further use.
23. The last three steps could be repeated if blue staining remains.
24. Any other mass spectrometers system could be used but chromatographic systems in front of the mass spectrometer should
be robust and reproducible enough to allow runs alignments
and semi quantitative analysis. Mass spectrometer should be
high performant and sensitive enough to cover the analyzed
interactome. The more performant the system, the more
robust and deep the analysis.
25. MaxQuant and Perseus versions 1.6.6.0 have been used to
describe the following steps of the bioinformatics analysis.
26. For example data analysis can be performed on Intel
® Xeon
®
CPU ES-2620 v4 @ 2.10 GHz (2 processors) with 46.9 GB of
installed memory (RAM) and Windows Server 2016 (Microsoft), 64 bit operating system. Sufficient space on the hard disk
is important first to copy all raw files and secondly for the
output files and temporary files. For the latter files an SSD
hard disk is desirable.
27. Alternatively the load folder option can be used to download all
raw files.
28. Other quantification methods can be used for affinity proteomics; however, Label-free quantification is mainly used. For
example, isobaric labeling [19] or metabolic labeling can also
be used [20] and set the “LFQ min. ratio count” to 1.
29. The LFQ min. ratio count can be left at the default value;
however, we prefer to use 1 at this step and then if necessary
to filter proteins quantified using 1 value during the
statistical step.
30. For example, Human protein database can be downloaded as a
fasta file from Uniprot (Click on the “Proteomes” type the
organism under study for example “Homo sapiens,” record the
organism ID 9606 and click the proteome ID link
“UP000005640”). Then download the reviewed (around
20,416 entries), the unreviewed (around 54,000 entries) or
the reference proteome (around 74,416 entries).
31. This option allows for the transfer of identification to
non-identified MS features in other LC-MS runs and so
decreases the missing values.
32. The iBAQ value can be useful because it yields an approximation of the protein abundance.
38
Avais M. Daulat et al.
20 min to remove aggregates.
22. 96-well plate could be stored at À20
C until further use.
23. The last three steps could be repeated if blue staining remains.
24. Any other mass spectrometers system could be used but chromatographic systems in front of the mass spectrometer should
be robust and reproducible enough to allow runs alignments
and semi quantitative analysis. Mass spectrometer should be
high performant and sensitive enough to cover the analyzed
interactome. The more performant the system, the more
robust and deep the analysis.
25. MaxQuant and Perseus versions 1.6.6.0 have been used to
describe the following steps of the bioinformatics analysis.
26. For example data analysis can be performed on Intel
® Xeon
®
CPU ES-2620 v4 @ 2.10 GHz (2 processors) with 46.9 GB of
installed memory (RAM) and Windows Server 2016 (Microsoft), 64 bit operating system. Sufficient space on the hard disk
is important first to copy all raw files and secondly for the
output files and temporary files. For the latter files an SSD
hard disk is desirable.
27. Alternatively the load folder option can be used to download all
raw files.
28. Other quantification methods can be used for affinity proteomics; however, Label-free quantification is mainly used. For
example, isobaric labeling [19] or metabolic labeling can also
be used [20] and set the “LFQ min. ratio count” to 1.
29. The LFQ min. ratio count can be left at the default value;
however, we prefer to use 1 at this step and then if necessary
to filter proteins quantified using 1 value during the
statistical step.
30. For example, Human protein database can be downloaded as a
fasta file from Uniprot (Click on the “Proteomes” type the
organism under study for example “Homo sapiens,” record the
organism ID 9606 and click the proteome ID link
“UP000005640”). Then download the reviewed (around
20,416 entries), the unreviewed (around 54,000 entries) or
the reference proteome (around 74,416 entries).
31. This option allows for the transfer of identification to
non-identified MS features in other LC-MS runs and so
decreases the missing values.
32. The iBAQ value can be useful because it yields an approximation of the protein abundance.
38
Avais M. Daulat et al.