1. User manual of IMSTIQ 1.0 1

2. Once your computer install the MATLAB R2010a or above, you can install and use the software 2

3. 1.The installation process: 1.1 Release the software in root directory in D disk, As shown in the figure. Figure 1 3

4. 1.2 Open Matlab, Click File button. Then click set path, add in back folder, as shown in the figure, then set the Current Folder to. Now,you can use the software. Figure 2 4 2. Command: matlab_filter (Tpp_Pep_File1, Tpp_Pep_File2, Tpp_Pro_File1, Tpp_Pro_File2, Enzyme, Miss_or_not, DTA_Dir, K_H_N, K_H_C, K_L_N, K_L_C, R_H_N, R_H_C, R_L_N, R_L_C, BY_ions_number, Msms_error, Delta, Mod_1, Mod_2, Mod_3, Mod_4, Mod_5, Mod_6).

5. 2. 1 Parameter specification: 5 Tpp_Pep_File, Tpp_Pro_File, the path of the peptide and protein files from the Tpp. Enzyme, here you can fill in ‘trypsin’, or ‘Lys N_and_Arg C’. Miss_or_not, you can decide whether the miss cleavaged peptides to be quantified, ‘1’ means yes while ‘0’ means no. DTA_Dir, the dta file path. K_H_N, K_H_C, K_L_N, K_L_C, R_H_N, R_H_C, R_L_N, R_L_C, terminal modifications, see Figure 3 for details. BY_ions_number, the minimum number of the b,y ions of the peptides used to quantification, you can fill in the number. Msms_error, the tolerance which used to looking for b,y ions, and the unit is Da, etc. you can set 0.05 for QE, and 1 for LTQ. Delta, it is the difference m/z of the b,y ions, it is a number. Mod_?, For peptide identification, maximum of 6 modifications can be included. You can find it in the peptide file from TPP.

6. Figure 3 Fixed modification of N termini and C termini in ITMSQ. K_?_? means peptides with C-terminal lysine, K_?_? indicates peptides with C-terminal argnine. ?_H_? or ?_L_? means the light or heavy counterparts. ?_?_C and ?_?_N indicates tags for C-terminal and N-terminal, respectively. 6

7. 2.2 Example I: Qantitation of IVTAL labeled Samples Figure 4 : Here, we copy the result to the ITMSQ folder, so we just need to input the file name. Then assign the enzyme, and 0 means we don’t allow the miss cleavage peptides to be quantified. E:\zhanglei\NAY\1203\R is the location of dta files. The following 8 numbers are the modification on each termini of the peptide, as show in the figure, 6Da mass tags are introduced on the N- or C-temini during IVTAL labeling. The next “6“ means the minimum number of the b, y ions of the peptides used for quantitation. And “1”means the tolerance which we used to looking for b,y ions. The following “6” means the difference m/z of the b,y ions is 6 Da. The rest of the number is the modification of the peptide. 7 Command: Matlab(‘K_interact.pep.txt’, ’R_interact.pep.txt’, ’K_interact.Prot.txt’, R_interact.pep.txt’, ’0’, ‘E:\zhanglei\NAY\1203\R’, 0, 0, 0, 0, 6, 0, 0, 6, 6, 6, 1, 6, ‘[147.04]’, ‘[134.12]’, ’[160.03]’, ’[162.12]’, ’’, ’’)

8. 8 2.2 Example II: Quantitation of QITL labeled Samples Command: matlab(‘pep1.txt’,’pep2.txt’,’pro1.txt’, ’pro2.txt’,‘trypsin’,’1’,‘D:\matlab\matlab_scrip\matlab _scrip\nieaiying\O16O18’,32.05640,0,28.03130,4.0085, 32.05640,0,28.03130,4.0085,6,1,4 ‘[33.06]’, ‘[170.12]’,’n[29.04]’,’[170.12]c[21.01]’,’[160.03]’,’[147.04]’) Figure 5: The figure shows the isobaric terminal labeling of QITL method. Miss cleavaged peptides can generate quantitation information in QITL, thus why “1” is set in the command. During QITL labeling, 32.05640 Da or 28.03130 Da mass tags are introduced on the peptide N- temini by dimethyl labeling, and 4.0085 Da mass tag is introduced on the C-terminal of the peptide by 18 O. The next number “6“ means the minimum number of the b, y ions of the peptides used for quantitation. And “1”indicates the tolerance for b,y ions. “4” means the difference m/z of the b,y ions is 4 Da. The following ‘[33.06]’, ‘[170.12]’, ’n[29.04]’, ’[170.12]c[21.01]’,’[160.03]’,’[147.04]’) are the modification of the peptide.

9. Protein Name Peptide number which used to quantification, the right is the redundancy The ratio, from left to right is the original ratio,the normalized and the protein P values. 3. Result The result is generated in the folder of D:\ITMSQ_1.0\Protein_result \ as result.txt. It can be opened with Excel as show in figure 6. 9

10. Old OUT files With a perl scrip, we can find the out files with principle that there are target peptide ranking top three, and then pick the corresponding DTA files The chimera DTA files New DTA files With a Matlab scrip, we can remove the opposite b and y ions from the DAT files, and then generate a new DTA files Research the new DTA files to get new out files New OUT files Pep.xml files With the Out2XML from Tpp, we can get pep.xml files now Peptide and protein list With the Tpp software, we will get the peptide and protein list The workflow of spectra splitting 10 4. Appendix

11. Frequency diagram QQ-plot Kolmogorov-Smirnov 95% 2.5% Gaussian distribution The determination of up and down regulated boundaries [0.7184, 1.2868] [0.7306, 1.2746] [0.7145, 1.2907] 11