To run TopPIC suite, a computer with at least 4 GB memory and a 64-bit Linux or Windows operating system is required. TopFD, TopPIC, TopMG, and TopDiff provide a command line interface for both Linux and Windows users as well as a graphical user interface (GUI) for Windows users. Please see our tutorial for more details on the GUI.

TopFD (Top-down mass spectral Feature Detection) is a software tool for top-down spectral deconvolution, which groups top-down mass spectral peaks into isotopomer envelopes and converts isotopomer envelopes to monoisotopic neutral masses. In addition, it extracts proteoform features from MS1 spectra.

The input of TopFD is mass spectrometry data files in the mzXML or mzML format. Raw mass spectral data generated from various mass spectrometers can be converted to mzML or mzXML files using msconvert.

TopFD outputs two LC/MS feature text files with a file extension "feature", one LC/MS feature file with a file extension "xml", and a deconvoluted mass spectral data file for MS/MS spectra in the msalign format with a file extension "msalign", which is similar to the MGF file format. In addition, TopFD creates two folders containing javascript files of spectral data for spectral visualization and other files generated in spectral deconvolution. For example, when the input file name is spectra.mzML, the output includes:

• spectra_ms1.feature: a feature file containing LC/MS features.
• spectra_ms2.feature: a feature file containing MS/MS scan IDs and their corresponding LC/MS feature IDs.
• spectra_feature.xml: a feature file containing LC/MS features in the xml format.
• spectra_ms2.msalign: a list of deconvoluted MS/MS spectra.
• spectra_file: a folder containing deconvoluted MS1 spectra and annotations of LC/MS data.
• spectra_html: a folder containing java script files of MS1 and MS/MS data for spectral visualization.

To run TopFD, open a terminal window and run the following command.

topfd [options] spectrum-file-names

Deconvolute a centroid data file spectra.mzML and output four files: spectra_ms2.msalign, spectra_ms1.feature, spectra_ms2.feature, spectra_feature.xml.

topfd spectra.mzML

Deconvolute two centroid data files spectra1.mzML and spectra2.mzML and output three files for each input data file.

topfd spectra1.mzML spectra2.mzML

Deconvolute all centroid data files in the current folder.

topfd *.mzML

Deconvolute a centroid data file spectra.mzML that does not contain MS1 spectra.

topfd -o spectra.mzML

Deconvolute a centroid data file spectra.mzML with a precursor isolation window size 2 m/z.

topfd -w 2 spectra.mzML

Deconvolute a centroid data file spectra.mzML with a signal/noise ratio 2 for MS1 spectra.

topfd -r 2 spectra.mzML

Deconvolute a centroid data file spectra.mzML with the following settings: the maximum charge state: 50, the maximum mass: 30,000 Dalton, and the signal/noise ratio for MS/MS spectra: 2.

topfd -c 50 -m 30000 -t 2 spectra.mzML

• 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)

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 javascript 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 javascript 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 javascript 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.

To run TopPIC, open a terminal window and run the following command.

toppic [options] database-file-name spectrum-file-names

-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.

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

• 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)

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 javascript files of identified PrSMs using the E-value or spectrum level FDR cutoff.
• spectra_html/topmg_proteoform_cutoff: a folder containing javascript 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 javascript 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.

To run TopMG, open a terminal window and run the following command.

topmg [options] database-file-name spectrum-file-names

-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.

-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.

To use the following examples, a variable modification file variable_mods.txt in the current foler is needed. (See anexample.)

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

• A protein database file in the FASTA format
• Several mass spectrum data files in the msalign format
• Proteoform identification files of the mass spectrum data files in the xml format, e.g., spectra_ms2_toppic_proteoform.xml

TopDiff outputs a csv file containing proteoform identifications and their abundances in the input mass spectrum data. The default output file name is sample_diff.csv.

To run TopDiff, open a terminal window and run the following command.

topdiff [options] database-file-name spectrum-file-names

-h [ --help ]

Print the help message.

-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.

-e [ --error-tolerance ] <a positive number>

Set the error tolerance for mapping identified proteoforms across multiple samples (in Dalton). Default value: 1.2 Dalton.

-t [ --tool-name ] <toppic|topmg>

Specify the name of the database search tool: toppic or topmg. Default: toppic.

-o [ --output ] <a file name>

Specify the output file name. Default: sample_diff.csv.

Compare proteoform abundances using TopPIC identifications of two spectrum files spectra1_ms2.msalign and spectra2_ms2.msalign. The protein sequence database file name is proteins.fasta.

topdiff proteins.fasta spectra1_ms2.msalign spectra2_ms2.msalign

Compare proteoform abundances using TopPIC identifications of two spectrum files spectra1_ms2.msalign and spectra2_ms2.msalign. The protein sequence database file name is proteins.fasta and a fixed modification, carbamidomethylation on cysteine, is used in database search.

topdiff -f C57 proteins.fasta spectra1_ms2.msalign spectra2_ms2.msalign

Compare proteoform abundances using TopMG identifications of two spectrum files spectra1_ms2.msalign and spectra2_ms2.msalign. The protein sequence database file name is proteins.fasta and a fixed modification, carbamidomethylation on cysteine, is used in database search.

topdiff -f C57 -t topmg proteins.fasta spectra1_ms2.msalign spectra2_ms2.msalign