15N-Leu
15N-Ala
(+1X) IBCP/AP
(+1X) IBCP/AP
Supplementary Figure 1: Overlay of the 1H-15N HSQC spectra of 15N-Leu (+1X) IBCP/AP
(black) and 15N-Ala (+1X) IBCP/AP (blue). Although the spectrum of 15N-Leu (+1X)
IBCP/AP shows the expected number of resonances, some of these resonances, which are single
peaks, overlap with those in the 15N-Ala (+1X) IBCP/AP spectrum. Three of the strong peaks
that are obviously overlapping are boxed.
Supplementary Table 1:
Residues
mass
67-77
92-103
1398.6
1374.8
650.4
749.4
1033.6
1245.7
1679.9
269.2
479.3
836.5
949.6
1063.6
1452.8
1566.8
269.2
479.3
836.5
949.6
1063.6
1177.6
1664.9
751.4
1054.6
269.2
479.3
580.4
694.4
751.4
1964.0
1550.8
104-117
106-117
137-150
142-150
158-176
15
N incorporation in regions of IB.
sequence
MKQQLTEDGDSF
EVIRQVKGDLAF
KGDLAF
VKGDLAF
RQVKGDLAF
VIRQVKGDLAF
LNFQNNLQQTPLHL
HL
PLHL
QQTPLHL
LQQTPLHL
NLQQTPLHL
FQNNLQQTPLHL
NFQNNLQQTPLHL
HL
PLHL
QQTPLHL
LQQTPLHL
NLQQTPLHL
NNLQQTPLHL
PELRDFRGNTPLHL
GNTPLHL
FRGNTPLHL
HL
PLHL
TPLHL
NTPLHL
GNTPLHL
ASVGVLTQSCTTPHLHSIL
LTQSCTTPHLHSIL
15
15
No. Leu
N-Leu
(1X 14N-aa)
overincorporated
N-Leu
(10X 14N-aa)
overincorporated
1
1
1
1
1
1
4
1
2
2
3
3
3
3
1
2
2
3
3
3
3
2
2
1
2
2
2
2
3
3
2
3
2
2
2
3
4
1
2
2
3
3
3
3
1
2
2
3
3
3
4
2
3
1
2
2
2
2
4
3
1
2
1
1
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
1
1
1
1
1
4
1
2
2
3
3
3
3
1
2
2
3
3
3
3
2
2
1
2
2
2
2
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
177-189
202-220
203-220
1465.8
580.4
717.4
774.4
1165.6
1266.6
2304.2
269.2
479.3
793.5
1107.6
1763.9
2191.1
269.2
479.3
793.5
1107.6
1648.8
KATNYNGHTPLHL
TPLHL
HTPLHL
GHTPLHL
NYNGHTPLHL
TNYNGHTPLHL
LVSLGADVNAQEPCNGRTPLHL
HL
PLHL
GRTPLHL
PCNGRTPLHL
DVNAQEPCNGRTPLHL
VSLGADVNAQEPCNGRTPLHL
HL
PLHL
GRTPLHL
PCNGRTPLHL
VNAQEPCNGRTPLHL
3
2
2
2
2
2
2
4
1
2
2
2
2
3
1
2
2
2
2
3
2
2
2
2
2
5
1
2
2
2
3
4
1
2
2
2
3
1
0
0
0
0
0
1
0
0
0
0
1
1
0
0
0
0
1
2
2
2
2
2
2
4
1
2
2
2
2
3
1
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
DEX analysis for measurement of 15N incorporation
Commands are indented and written in bold type.
For DEX installation instructions, user guide, and tutorial see
http://biology.sdsc.edu/ccms/dex/.
1. Export mass spectra into an ASCII text format.
2. Write a text file (using vi or other Unix compatible text editor) containing the
sequences (on separate lines) of the peptides or fragments to be analyzed. For MS
spectral analysis, there should be one file containing all peptide sequences. For MS/MS
spectral analysis, there should be one file for each precursor containing the sequences of
the y-ions (with no potential overlap from other ion types) for that precursor.
3. Generate isotopic profiles for each peptide or fragment using the sequence files
generated in step 2:
./isotopic-fast-profiles folder/sequence_file_name folder/profile_file_name 0 1
where "folder" is the name of the folder in the DEX directory where the file is located,
"sequence_file_name" is the name of the file containing the peptide or fragment
sequences, "profile_file_name" is the name for the file that will be created containing the
profile information, 0 is the % deuterium in the sample, and 1 is the mode (natural
isotopic abundance).
4. Analyze the isotopic abundance using DEX using the profiles generated in step 3:
./DEX-master-script txt folder/ folder/profile_file_name
to analyze all spectra (text files) in the designated folder using the specified profiles or
./DEX-resample-script folder/profile_file_name folder/spectrum_file_name.txt
folder/spectrum_file_name.dat
to analyze a single spectrum (spectrum_file_name.txt) using the specified profiles. DEX
resampled spectra are saved as "spectrum_file_name.dat" where "spectrum_file_name" is
the name of the spectral file.
5. Write a text file (using vi or other unix compatible text editor), hereafter file list, that
contains the information for all of the spectra that should be compared to each other. For
example, MSkey.list might contain the information for 14N-Leu IB, 15N-Leu IB
grown with 1X unlabeled amino acids, and 15N-Leu IB grown with 10X unlabeled
amino acids. The file list should contain the header "#Filename Description
Line_num" (without quotations, where the three terms are separated by tabs) and
each line that follows contains the spectrum_file_name.dat, a brief description of the
spectrum (e.g. 14N-Leu), and a line number (can be 1 for all spectra), separated by tabs,
for a single spectrum. For the example above, the file would look like this:
#Filename
C3_MS.dat
D4_MS.dat
E3_MS.dat
Description
14N-Leu
15N-Leu+1X
15N-Leu+10X
Line_num
1
1
1
6. Output the weights for the 15N incorporation in each peptide or fragment in the
resampled spectra generated in step 4.
For MS spectral analysis:
./HD-analysis-script --program cent --graph DECON --grace no --prefix folder/
--filelist folder/file_list_name.list --mass peptide_mass
immediately followed by:
cp HD_analysis_output folder/output_file_name.cnt
where "file_list_name" is the name of the file list generated in step 5, "peptide_mass" is
the mass of the peptide to be analyzed, and "output_file_name" is the name of the output
file (text format) that contains the weights for the 15N incorporation in each peptide.
Since this step will be repeated for each peptide, be sure that each "output_file_name" is
unique (e.g. the peptide mass).
For MS/MS spectral analysis:
./HD-analysis-script --program cent --graph DECON --grace no --prefix folder/
--filelist folder/file_list_name.list --numb fragment_number
immediately followed by:
cp HD_analysis_output folder/output_file_name.cnt
where "file_list_name" is the name of the file list generated in step 5, "fragment_number"
is the line number of the fragment sequence in the sequence file generated in step 2, and
"output_file_name" is the name of the output file (text format) that contains the weights
for the 15N incorporation in each fragment. Since this step will be repeated for each
fragment, be sure that each "output_file_name" is unique (e.g. precursor mass and
fragment number).
7. Repeat step 6 for each peptide or fragment to be analyzed.
8. Determine the 15N incorporation in each peptide or fragment. Open the output file
(output_file_name.cnt) for each peptide or fragment. Each spectrum listed in the file list
will have an entry in the output file (e.g. 14N-Leu, 15N-Leu+1X, 15N-Leu+10X). The
peak column in the output file corresponds to the number of 15N incorporated. The
weights column indicates the weights for each peak (i.e. 0, 1, …, n 15N) in the resampled
spectrum. In this study, only peptides or fragments that showed no 15N incorporation in
the (control) 14N-protein sample were analyzed. 14N-protein samples were considered to
have no 15N incorporation if the weights of peak 1 and any other peaks considered in the
15
N-Leu samples for that peptide or fragment were less than 10. In the 15N-Leu samples,
peak weights that were greater than 10 for consecutive peaks indicated an incorporation
of 15N. For example, if the weights for peaks 0, 1, 2, and 3 were 18, 48, 34, and 0,
respectively, then three 15N were incorporated into the protein in the covered region.
9. If desired, create graphs of the spectrum and DEX isotopic analysis.
To view (for MS spectra):
./View-many-HD-spectra-script --filelist folder/file_list_name.list --prefix
folder/ --mass peptide_mass
To view (for MS/MS spectra):
./View-many-HD-spectra-script --filelist folder/file_list_name.list --prefix
folder/ --numb fragment_number
To write an output file containing the graph, append to above:
--hardcopy
which writes a temporary file called graph_multi.ps. Immediately follow with:
cp graph_multi.ps folder/output_file_name.ps
to save an output file containing the graph. Repeat for each peptide or fragment, but be
sure that each "output_file_name" is unique (see step 6). The output files are postscript
files that can be printed or converted to other graphical formats as desired.
5