Provided by: toppic_1.3.0+dfsg1-4build1_amd64 
NAME
toppic - Top-down mass spectrometry based Proteoform Identification and Characterization
SYNOPSIS
toppic [options] database-file-name spectrum-file-names
DESCRIPTION
TopPIC (Top-down mass spectrometry based Proteoform Identification and Characterization)
identifies and characterizes proteoforms at the proteome level by searching top-down
tandem mass spectra against a protein sequence database. TopPIC is a successor to
MS-Align+. It efficiently identifies proteoforms with unexpected alterations, such as
mutations and post-translational modifications (PTMs), accurately estimates the
statistical significance of identifications, and characterizes reported proteoforms with
unknown mass shifts. It uses several techniques, such as indexes, spectral alignment,
generation function methods, and the modification identification score (MIScore), to
increase the speed, sensitivity, and accuracy.
1. Input
• A protein database file in the FASTA format
• A mass spectrum data file in the msalign format
• A text file containing LC/MS feature information (optional)
• A text file of fixed PTMs (optional)
• A text file of PTMs for the characterization of unexpected mass shifts (optional)
2. Output
TopPIC outputs two comma separated value (csv) files, an xml file, and a collection of
html files for identified proteoforms. For example, when the input data file is
spectra_ms2.msalign, the output includes:
• spectra_ms2_toppic_prsm.csv: a csv file containing identified proteoform
spectrum-matches (PrSMs) with an E-value or spectrum level FDR cutoff.
• spectra_ms2_toppic_proteoform.csv: a csv file containing identified proteoforms with
an E-value or proteoform level FDR cutoff.
• spectra_ms2_toppic_proteoform.xml: an xml file containing identified proteoforms with
the E-value or proteoform level FDR cutoff.
• spectra_html/toppic_prsm_cutoff: a folder containing java script files of identified
PrSMs using the E-value or spectrum level FDR cutoff.
• spectra_html/toppic_proteoform_cutoff: a folder containing java script files of
identified PrSMs using the E-value or proteoform level FDR 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 spectrum_html/topview/index.html. Google Chrome is recommended (Firefox and IE are
not recommended).
When the input contains two or more data files, TopPIC 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_toppic_prsm.csv: a csv file containing PrSMs identified from all the
input files with an E-value or spectrum level FDR cutoff.
• combined_ms2_toppic_proteoform.csv: a csv file containing proteoforms identified from
all the input files with an E-value or proteoform level FDR cutoff.
• combined_ms2_toppic_proteoform.xml: an xml file containing proteoforms identified
from all the input files with the E-value or proteoform level FDR cutoff.
• combined_html/toppic_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/toppic_proteoform_cutoff: a folder containing java script files of
PrSMs identified from all the input files using the E-value or proteoform level FDR
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> Set the fragmentation method(s) of MS/MS
spectra. When "FILE" is selected, the fragmentation methods of spectra are given in the
input spectrum 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 containing the information of
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 part-per-million (ppm). Default value: 15. When the lookup table
approach (-l) is used for E-value estimation, valid error tolerance values are 5, 10, and
15 ppm.
-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 value for unexpected mass shifts (in
Dalton). Default value: 500.
-m [ --min-shift ] <a number> Se the minimum value for unexpected mass shifts (in Dalton).
Default value: -500.
-s [ --num-shift ] <0|1|2> Set the maximum number of unexpected mass shifts in a PrSM.
Default value: 1.
-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.
-l [ --lookup-table ] Use a lookup table method for computing p-values and E-values. It is
faster than the default generating function approach, but it may reduce the number of
identifications.
-r [ --num-combined-spectra ] <a positive integer> Set the number of combined spectra. The
parameter is set to 2 (or 3) for combining spectral pairs (or triplets) generated by the
alternating fragmentation mode. Default value: 1.
-i [ --mod-file-name ] <a common modification file> Specify a text file containing a list
of common PTMs for proteoform characterization. The PTMs are used to identify and localize
PTMs in reported PrSMs with unknown mass shifts. See an example file.
-H [ --miscore-threshold ] <a number between 0 and 1> Set the score threshold (MIScore)
for filtering results of PTM characterization. Default value: 0.45.
-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 about 16 GB memory is needed for 4 threads.
-x [ --no-topfd-feature ] Specify that there are no TopFD feature files for proteoform
identification.
-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 TopPIC.
EXAMPLES
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta with a feature file spectra.feature. The user does not need to
specify the feature file name. TopPIC will automatically obtain the names of feature
files from the spectrum file name spectra_ms2.msalign.
toppic 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."
toppic -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.
toppic proteins.fasta *_ms2.msalign
• Search a deconvoluted MS/MS spectrum file spectra_ms2.msalign against a protein database
file proteins.fasta without feature files.
toppic -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.
toppic -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 2 mass
shifts are allowed and the maximum allowed mass shift value is 10,000 Dalton.
toppic -s 2 -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.
toppic -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.
toppic -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 with alternating CID, HCD,
and ETD spectra against a protein database file proteins.fasta with feature files.
Combine alternating CID, HCD, and ETD spectra to increase proteoform coverage.
toppic -r 3 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. After proteoforms with unexpected mass shifts
are identified, TopPIC matches the mass shifts to four common PTMs: acetylation,
phosphorylation, oxidation and methylation, and uses an MIScore cutoff 0.3 to filter
reported PTM sites. The modification file common_mods.txt can be found here.
toppic -i common_mods.txt -H 0.3 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.
toppic -u 6 proteins.fasta spectra_ms2.msalign
SEE ALSO
• topfd (1)
• topmg (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