MSTopDown.
---------

The directory contains 10 spectra of histone H4 proteins. The
spectra are given in the pks format (regular peak lists) and in the
mono.txt format (deconvoluted monoisotopic peak masses). MSTopDown
expects to receive its input in the latter format. The file
"TopDownPTMs.txt" must reside in the same directory as the
executable "MSTopDown.exe" (you might need to change the extension
to .exe).

To run MSTopDown you must supply the program with the following
arguments:

-peak_list <path> - the path to a text file with a monoisotpic peak
list. The first value is the monoisotopic precursor mass (equals
mass of amino acids + mass of H2O). Following there should be a list
of monoisotopic masses representing deconvoluted peaks (one mass per
line).

-sequence <path> - a path to a text file with the desired protein
sequence.

-specific <N> - the maximum number N of specific PTMs that should be
considered. These PTMs appear in the file "TopDownPTMs.txt"
(additional PTMs can be added to that file).

-general <N> - the maximal number N of general mass shifts that can
be incorporated into the alignment.

-num_forms <N> - shows results for the N highest scoring forms.

-PPM <XX> - sets the expected mass measurement accuracy to XX ppm
(the default is 10 ppm). Note that this is an approximate accuracy
(some aligned peaks might have a slightly higher mass error).



******************************************************************

Example: Running the following command

MSTopDown.exe -peak_list data/HELA_spec10_mono.txt
 -sequence data/Hela_seq.txt -specific 5 -general 2 -PPM 15 -num_forms 2

Produces the following output:

                #General PTMs (any mass)
#Specific       0       1       2
  PTMs   form
0          1     -      17      21
           2     -       -      21

1          1     -      21      25
           2     -      20      25

2          1     -      25      26
           2     -      24      26

3          1     -      25      26
           2     -      25      26

4          1    25      25      30
           2    25      25      30

5          1    23      26      31
           2    23      26      31


The output is in the form of a table, giving the alignment scores of
the different forms obtained using different numbers of general and
specific PTMs. This stage is used to determine which is the optimal
combination of PTMs to use. In the above case we would tend to
believe that there are 4 specific PTMs (using more PTMs does not
significantly increase the score).

To obtain the alignments we can use the flag "-print" with the exact
number of general/specific PTMs that is desired.

The command

>MSTopDown.exe -peak_list data\HELA_spec10_mono.txt -sequence data\Hela_seq.txt
-specific 4 -general 0 -num_forms 2 -PPM 15 -print

Produces the peak list output given below. For each form we note the
alignment score and the observed mass shifts. Following that is a
list of matched peaks, noting the type of peak (was this a c or z),
and the ppm error. If only specific PTMs are used then the program
gives the ppm error of the monosiotopic precursor mass.


Form 1  (score 25)  approx PPM: 15.0

Mass shifts:

        Delta   Position        Shift type
        -----   --------        ----------
1       84.021  S1-K5           Specific: Acetylation + N-Acetylation
2       42.011  K12             Specific: Acetylation
3       42.011  K16             Specific: Acetylation

Matched peaks:

        Monoiso. Mass   type    ppm     position
        -------------   ----    ---     --------
1       569.297         c       9.310   K5+84.0
2       626.320         c       9.803   G6+84.0
3       683.340         c       6.995   G7+84.0
4       1038.566        c       8.512   G11+84.0
5       1208.677        c       11.844  K12+126.0
6       1265.697        c       10.315  G13+126.0
7       1322.712        c       4.684   G14+126.0
8       1393.757        c       10.106  A15+126.0
9       1563.855        c       4.578   K16+168.0
10      1719.970        c       12.297  R17+168.0
11      1857.026        c       9.553   H18+168.0
12      2013.131        c       10.844  R19+168.0
13      2509.479        c       8.484   R23+168.0
14      2624.516        c       11.755  D24+168.0
15      2851.634        c       7.876   I26+168.0
16      3660.111        c       5.106   I34+168.0
17      3816.218        c       6.546   R35+168.0
18      3972.323        c       7.167   R36+168.0
19      4156.456        c       9.744   A38+168.0
20      4312.549        c       7.557   R39+168.0
21      4809.843        c       3.844   K44+168.0
22      6127.577        c       7.900   R55+168.0
23      7013.106        c       6.495   E63+168.0
24      9874.521        z       -2.621  Y88+168.0
25      10058.643       z       -2.421  L90+168.0

With these specific PTMs, the exact monoisotopic mass should be 11397.383
(ppm error 1.165 compared to mass given in spectrum 11397.370)


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Form 2  (score 25)  approx PPM: 15.0

Mass shifts:

        Delta   Position        Shift type
        -----   --------        ----------
1       42.011  S1-K5           Specific: Acetylation
2       42.011  K8-G11          Specific: Acetylation
3       42.011  K12             Specific: Acetylation
4       42.011  K16             Specific: Acetylation

Matched peaks:

        Monoiso. Mass   type    ppm     position
        -------------   ----    ---     --------
1       527.288         c       11.882  K5+42.0
2       584.309         c       10.106  G6+42.0
3       641.331         c       10.517  G7+42.0
4       1038.566        c       8.512   G11+84.0
5       1208.677        c       11.844  K12+126.0
6       1265.697        c       10.315  G13+126.0
7       1322.712        c       4.684   G14+126.0
8       1393.757        c       10.106  A15+126.0
9       1563.855        c       4.578   K16+168.0
10      1719.970        c       12.297  R17+168.0
11      1857.026        c       9.553   H18+168.0
12      2013.131        c       10.844  R19+168.0
13      2509.479        c       8.484   R23+168.0
14      2624.516        c       11.755  D24+168.0
15      2851.634        c       7.876   I26+168.0
16      3660.111        c       5.106   I34+168.0
17      3816.218        c       6.546   R35+168.0
18      3972.323        c       7.167   R36+168.0
19      4156.456        c       9.744   A38+168.0
20      4312.549        c       7.557   R39+168.0
21      4809.843        c       3.844   K44+168.0
22      6127.577        c       7.900   R55+168.0
23      7013.106        c       6.495   E63+168.0
24      9874.521        z       -2.621  Y88+168.0
25      10058.643       z       -2.421  L90+168.0

With these specific PTMs, the exact monoisotopic mass should be 11397.383
(ppm error 1.165 compared to mass given in spectrum 11397.370)


Often the precursor mass given in the spectrum file is inaccurate,
but can be corrected after producing an initial alignment. To
correct such cases you can run an initial alignment with at least 1
general PTM. If the general shift reported is similar to a specific
PTM, consider modifying the precursor mass accordingly and then
rerun with no general PTMs.
