Pulldown). Set the s0 and the FDR values defined at the
previous step and press “OK.” Additional columns will be
added in the new matrix (see Note 41).
13. The new matrix contains two supplemental categorical columns and four supplemental numerical columns. The two
categorical columns are named “Student’s t-test Significant
Control_Pulldown” and “Student’s T-test Significant” containing respectively a “+” and “Control_Pulldown” to spot
significant differences. The four numerical columns are
named “Student’s T-test p-value Control_Pulldown”, “Student’s T-test q-value Control_Pulldown”, “Student’s T-test
Difference Control_Pulldown”, and “Student’s t-test Test statistic Control_Pulldown”. These columns show the P value,
the difference and the pFDR-threshold of the t test; and give
the relative enrichment of proteins between Control and Pulldown groups in a log2 scale.
14. Remove imputed value using “Imputation/Replace imputed
values by NaN” and export the resulting matrix to a text file
using the disk-shaped button (e.g., generic matrix export) (see
Note 42).
3.5 Results
3.5.1 Identification
of TANC1 Associated
Protein Complexes
Using the above described method, we purified the protein complexes associated to FLAG-TANC1 and identified bound proteins
by MS analysis (Fig. 3b). We mutated the last three amino-acids
located at the carboxy-terminus of the TANC1 sequence (SNV
changed to AAA) to abrogate PDZ interactions. Using this
approach, we were able to differentially identify proteins specifically
associated through the PDZBM of TANC1. Among the proteins
identified, we found 5 proteins containing PDZ domains (SCRIB,
DLG1, MMP7, CASK, SNX27) which were absent from the purified complex associated to the mutant form of TANC1 (TANC1AAA). Moreover, we also noticed that 41 FLAG-TANC1 partners
were also absent from the FLAG-TANC1-AAA protein complex
suggesting that association of these proteins with TANC1 relies on
the PDZBM.
We also carried out a peptide-based purification using a
TANC1 peptide ( LTAAKPKRSFIESNV ) and its mutant form
lacking the SNV motif to precipitate partners from HEK293T cell
extracts and we identified 25 PDZ domain containing proteins and
14 proteins lacking a PDZ domain including 2 proteins (CTNNA
and CPVL) found with FLAG-TANC1 (Fig. 4b).
Data obtained by the two approaches confirmed that 6 PDZ
domain proteins (SCRIB, DLG1, MMP7, CASK, PTPN13,
SNX27) (Figs. 3b and 4b) are direct interactors of the TANC1
PDZBM. However, using the peptide-based purification strategy,
we identified an additional set of 19 additional PDZ proteins
(Fig. 4b) demonstrating the higher efficiency of this technique to
identify PDZ interactions.
34
Avais M. Daulat et al.
previous step and press “OK.” Additional columns will be
added in the new matrix (see Note 41).
13. The new matrix contains two supplemental categorical columns and four supplemental numerical columns. The two
categorical columns are named “Student’s t-test Significant
Control_Pulldown” and “Student’s T-test Significant” containing respectively a “+” and “Control_Pulldown” to spot
significant differences. The four numerical columns are
named “Student’s T-test p-value Control_Pulldown”, “Student’s T-test q-value Control_Pulldown”, “Student’s T-test
Difference Control_Pulldown”, and “Student’s t-test Test statistic Control_Pulldown”. These columns show the P value,
the difference and the pFDR-threshold of the t test; and give
the relative enrichment of proteins between Control and Pulldown groups in a log2 scale.
14. Remove imputed value using “Imputation/Replace imputed
values by NaN” and export the resulting matrix to a text file
using the disk-shaped button (e.g., generic matrix export) (see
Note 42).
3.5 Results
3.5.1 Identification
of TANC1 Associated
Protein Complexes
Using the above described method, we purified the protein complexes associated to FLAG-TANC1 and identified bound proteins
by MS analysis (Fig. 3b). We mutated the last three amino-acids
located at the carboxy-terminus of the TANC1 sequence (SNV
changed to AAA) to abrogate PDZ interactions. Using this
approach, we were able to differentially identify proteins specifically
associated through the PDZBM of TANC1. Among the proteins
identified, we found 5 proteins containing PDZ domains (SCRIB,
DLG1, MMP7, CASK, SNX27) which were absent from the purified complex associated to the mutant form of TANC1 (TANC1AAA). Moreover, we also noticed that 41 FLAG-TANC1 partners
were also absent from the FLAG-TANC1-AAA protein complex
suggesting that association of these proteins with TANC1 relies on
the PDZBM.
We also carried out a peptide-based purification using a
TANC1 peptide ( LTAAKPKRSFIESNV ) and its mutant form
lacking the SNV motif to precipitate partners from HEK293T cell
extracts and we identified 25 PDZ domain containing proteins and
14 proteins lacking a PDZ domain including 2 proteins (CTNNA
and CPVL) found with FLAG-TANC1 (Fig. 4b).
Data obtained by the two approaches confirmed that 6 PDZ
domain proteins (SCRIB, DLG1, MMP7, CASK, PTPN13,
SNX27) (Figs. 3b and 4b) are direct interactors of the TANC1
PDZBM. However, using the peptide-based purification strategy,
we identified an additional set of 19 additional PDZ proteins
(Fig. 4b) demonstrating the higher efficiency of this technique to
identify PDZ interactions.
34
Avais M. Daulat et al.