Provided by: toppic_1.3.0+dfsg1-4build1_amd64 
NAME
topmg - Top-down mass spectrometry based proteoform identification using Mass Graphs
SYNOPSIS
topmg [options] database-file-name spectrum-file-names
DESCRIPTION
TopMG (Top-down mass spectrometry based proteoform identification using Mass Graphs) is a
software tool for identifying ultra-modified proteoforms by searching top-down tandem mass
spectra against a protein sequence database. It is capable of identifying proteoforms with
multiple variable PTMs and unexpected alterations, such as histone proteoforms and
phosphorylated ones. It uses mass graphs, which efficiently represent candidate
proteoforms with multiple variable PTMs, to increase the speed and sensitivity in
proteoform identification. In addition, approximate spectrum-based filtering methods are
employed for protein sequence filtering, and a Markov chain Monte Carlo method (TopMCMC)
is used for estimating the statistical significance of identifications.
1. Input
• A protein database file in the FASTA format
• A mass spectrum data file in the msalign format
• A text file of variable PTMs
• A text file of fixed PTMs (optional)
• A text file containing LC/MS feature information (optional)
2. Output
TopMG outputs two csv files, an xml file, and a collection of html files for identified
proteoforms. For example, when the input mass spectrum data file is spectra_ms2.msalign,
the output includes:
• spectra_ms2_topmg_prsm.csv: a csv file containing identified PrSMs with an E-value or
spectrum level FDR cutoff.
• spectra_ms2_topmg_proteoform.csv: a csv file containing identified proteoforms with
an E-value or proteoform level FDR cutoff.
• spectra_ms2_topmg_proteoform.xml: an xml file containing identified proteoforms with
the E-value or proteoform level FDR cutoff.
• spectra_html/topmg_prsm_cutoff: a folder containing java script files of identified
PrSMs using the E-value or spectrum level FDR cutoff.
• spectra_html/topmg_proteoform_cutoff: a folder containing java script files of
identified PrSMs using the E-value or proteoform level cutoff.
• spectra_html/topview: a folder containing html files for the visualization of
identified PrSMs.
To browse identified proteins, proteoforms, and PrSMs, use a chrome browser to open the
file spectra_html/topview/index.html. Google Chrome is recommended (Firefox, IE are not
recommended).
When the input contains two or more spectrum files, TopMG outputs two csv files, an xml
file, and a collection of html files for each input file. When a file name is specified
for combined identifications, it combines spectra and proteoforms identified from all the
input files, removes redundant proteoform identifications, and reports two csv files, an
xml file, and a collection of html files for the combined results. For example, when the
input is spectra1_ms2.msalign and spectra2_ms2.msalign and the combined output file name
is "combined," the output files are:
combined_ms2_topmg_prsm.csv: a csv file containing PrSMs identified from all the input
files with an E-value or spectrum level FDR cutoff. combined_ms2_topmg_proteoform.csv:
a csv file containing proteoforms identified from all the input files with an E-value
or proteoform level FDR cutoff. combined_ms2_topmg_proteoform.xml: an xml file
containing proteoforms identified from all the input files with the E-value or
proteoform level FDR cutoff. combined_html/topmg_prsm_cutoff: a folder containing java
script files of PrSMs identified from all the input files using the E-value or spectrum
level FDR cutoff. combined_html/topmg_proteoform_cutoff: a folder containing java
script files of PrSMs identified from all the input files using the E-value or
proteoform level cutoff. combined_html/topview: a folder containing html files for the
visualization of identified PrSMs.
OPTIONS
-h [ --help ] Print the help message.
-a [ --activation ] <CID|HCD|ETD|UVPD|FILE> Fragmentation method of tandem mass spectra.
When FILE is used, fragmentation methods of spectra are given in the input spectral data
file. Default value: FILE.
-f [ --fixed-mod ] <C57|C58|a fixed modification file> Set fixed modifications. Three
available options: C57, C58, or the name of a text file specifying fixed modifications
(see an example file). When C57 is selected, carbamidomethylation on cysteine is the only
fixed modification. When C58 is selected, carboxymethylation on cysteine is the only fixed
modification.
-n [ --n-terminal-form ] <a list of allowed N-terminal forms> Set N-terminal forms of
proteins. Four N-terminal forms can be selected: NONE, NME, NME_ACETYLATION, and
M_ACETYLATION. NONE stands for no modifications, NME for N-terminal methionine excision,
NME_ACETYLATION for N-terminal acetylation after the initiator methionine is removed, and
M_ACETYLATION for N-terminal methionine acetylation. When multiple forms are allowed, they
are separated by commas. Default value: NONE,M_ACETYLATION,NME,NME_ACETYLATION.
-d [ --decoy ] Use a shuffled decoy protein database to estimate spectrum and proteoform
level FDRs. When -d is chosen, a shuffled decoy database is automatically generated and
appended to the target database before database search, and FDR rates are estimated using
the target-decoy approach.
-e [ --mass-error-tolerance ] <a positive integer> Set the error tolerance for precursor
and fragment masses in ppm. Default value: 15.
-p [ --proteoform-error-tolerance ] <a positive number> Set the error tolerance for
identifying PrSM clusters (in Dalton). Default value: 1.2 Dalton.
-M [ --max-shift ] <a number> Set the maximum absolute value for unexpected mass shifts
(in Dalton). Default value: 500.
-t [ --spectrum-cutoff-type ] <EVALUE|FDR> Set the spectrum level cutoff type for
filtering PrSMs. Default value: EVALUE.
-v [ --spectrum-cutoff-value ] <a positive number> Set the spectrum level cutoff value for
filtering PrSMs. Default value: 0.01.
-T [ --proteoform-cutoff-type ] <EVALUE|FDR> Set the proteoform level cutoff type for
filtering proteoforms and PrSMs. Default value: EVALUE.
-V [ --proteoform-cutoff-value ] <a positive number> Set the proteoform level cutoff value
for filtering proteoforms and PrSMs. Default value: 0.01.
-i [ --mod-file-name ] <a modification file> Specify a text file of variable PTMs. See an
example file.
-u [ --thread-number ] <a positive number> Set the number of threads used in the
computation. Default value: 1. The maximum number of threads is determined by the CPU and
memory of the computer used for computation. About 4 GB memory is required for each
thread. If the computer has 16 GB memory and a CPU with 8 cores, the maximum number of
threads is 4 because 16 GB memory is required for 4 threads.
-x [ --no-topfd-feature ] Specify that there are no TopFD feature files for proteoform
identification.
-D [ --use-asf-diagonal ] Use the ASF-DIAGONAL method for protein sequence filtering. The
default filtering method is ASF-RESTRICT. When -D is selected, both ASF-RESTRICT and
ASF-DIAGONAL will be used. The combined approach may identify more PrSMs, but it is much
slower than using ASF-RESTRICT only. See this paper for more details.
-P [ --var-ptm ] <a positive number> Set the maximum number of variable PTM sites in a
proteoform. Default value: 5.
-s [ --num-shift <0|1|2> Set the maximum number of unexpected mass shifts in a proteoform.
Default value: 0.
-c [ --combined-file-name ] <a filename> Specify an output file name for combined
identifications when the input consists of multiple spectrum files.
-k [ --keep ] Keep intermediate files generated by TopMG.
ADVANCED OPTIONS
-j [ --proteo-graph-dis ] <a positive number> Set the length of the largest gap in
constructing proteoform graphs. Default value: 40. See this paper for more details.
-G [ --var-ptm-in-gap ] <a positive number> Set the maximum number of variable PTM sites
in a gap in a proteoform graph. Default value: 5. See this paper for more details.
EXAMPLES
To use the following examples, a variable modification file variable_mods.txt in the
current foler is needed. (See an example.)
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with feature files. The user does not need to specify the feature
file name. TopMG will automatically obtain the names of feature files from the spectrum
file name spectra_ms2.msalign.
topmg -i variable_mods.txt proteins.fasta spectra_ms2.msalign
• Search two deconvoluted MS/MS spectrum files spectra1_ms2.msalign and
spectra2_ms2.msalign against a protein database file proteins.fasta with feature files.
In addition, all identifications are combined and reported using a file name "combined."
topmg -i variable_mods.txt -c combined proteins.fasta spectra1_ms2.msalign
spectra2_ms2.msalign
• Search all deconvoluted MS/MS spectrum files in the current folder against a protein
database file proteins.fasta with feature files.
topmg -i variable_mods.txt proteins.fasta *_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta without feature files.
topmg -i variable_mods.txt -x proteins.fasta spectra_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with feature files and a fixed modification: carbamidomethylation on
cysteine.
topmg -i variable_mods.txt -f C57 proteins.fasta spectra_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with feature files. In an identified proteoform, at most 1
unexpected mass shift and 4 variable PTMs are allowed and the maximum value for
unexpected mass shifts is 10,000 Dalton.
topmg -i variable_mods.txt -P 4 -s 1 -M 10000 proteins.fasta spectra_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with feature files. The error tolerance for precursor and fragment
masses is 5 ppm.
topmg -i variable_mods.txt -e 5 proteins.fasta spectra_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with feature files. Use the target decoy approach to compute
spectrum level and proteoform level FDRs, filter identified proteoform spectrum-matches
by a 5% spectrum level FDR, and filter identified proteoforms by a 5% proteoform level
FDR.
topmg -i variable_mods.txt -d -t FDR -v 0.05 -T FDR -V 0.05 proteins.fasta
spectra_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with feature files. Use 6 CPU threads to speed up the computation.
About 24 GB memory is required for 6 threads. If the computer lacks enough memory, TopMG
may crash.
topmg -i variable_mods.txt -u 6 proteins.fasta spectra_ms2.msalign
SEE ALSO
• topfd (1)
• toppic (1)
• topdiff (1)
MAN PAGE PRODUCTION
This man page was written by Filippo Rusconi <lopippo@debian.org>. Material was taken from
http://proteomics.informatics.iupui.edu/software/toppic/manual.html.
AUTHOR
Filippo Rusconi <lopippo@debian.org> and upstream authors (Dr. Xiaowen Liu's Lab at
Indiana University-Purdue University Indianapolis and others)
COPYRIGHT
Filippo Rusconi and Indiana University-Purdue University Indianapolis