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Description:

SAMPEI 0.0.9

SAMPEI

Summary
Recent studies have revealed diverse amino acid, post-translational and non-canonical modifications of proteins in diverse organisms and tissues. However, their detection and analysis remain hindered by technical limitations. Here, we present a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Termed SAMPEI for Spectral Alignment-based Modified PEptide Identification, this open-source algorithm is designed for the discovery of functional protein and peptide signaling modifications, without prior knowledge of their identities. Using synthetic standards and controlled chemical labeling experiments, we demonstrate its high specificity and sensitivity for the discovery of sub-stoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEI’s robust parameterization and versatility are expected to facilitate the discovery of biological modifications of diverse macromolecules.
Tandem mass spectrometry enables high-throughput peptide identifications in complex biological specimens. In conventional methods, peptide identification relies on database search which is limited by the identification of only small number of post translational modifications (less than 3). Such methods are unable to identify unanticipated PTMs. Here we developed SAMPEI, a computational method leveraging high resolution query spectra within the same or different dataset to assign target spectra with peptide sequence and unanticipated modifications (mass shift). Prior to SAMPEI, we utilized database search (X!tandem) to assign spectra with peptide sequences in each sample. Only spectra with unique modifications and with the highest peptide identification confidence (lowest e value) are selected as queries. SAMPEI then performs a series of orthogonal measures to evaluate the similarity between all unassigned spectra and query spectra within predefined mass difference window (default = +/- 200 Dalton). First, SAMPEI aligns discrete m/z ranges within unassigned spectra to the query spectra. The proportion of matched MS2 ion intensity from query over the total MS2 intensity defined as matched query intensity is used to pre-select candidate spectra. Then, two additional measures to assess the quality of the assignment against query peptide sequence are determined to evaluate the goodness of the match. Specifically, the proportion of the MS2 intensity of target spectrum matched to the theoretical m/z of the query peptide sequence over the total MS2 intensity in the target scan is one of the measures. Finally, the proportion of the largest consecutive b/y ions missing over the length of the peptide sequence defined as largest gap percentage is the last measure. These parameters can be adjusted to maximize sensitivity or specificity, as dictated by experimental needs.

Figure 1. Schematcis of SAMPEI. (A) Example spectra represent the naive and modified peptide where Dm denotes the potential modification at residue F. With database (DB) search, only defined modification is able to be identified (Top). When incorporating DB search with SAMPEI, high quality query spectra was leveraged to identify target spectra with high similarities through three measures, matched query intensity, matched peptide intensity and largest gap percentage
Installation
SAMPEI can be installed using either pip or conda.
Conda

If Anaconda is not installed, first install it by following instructions found here: https://docs.conda.io/en/latest/miniconda.html

Install the package using : conda install -c bioconda sampei

Pip

Pip is generally installed with Python
Install the package using pip install sampei

Usage
Once installed SAMPEI can be run using the sampei command in the terminal.
To ensure that the package has been installed and to view the command line arguments run:
sampei --help
This command will ouput the command line help if the package has been installed correctly.
Running SAMPEI
Once sampei has been installed it can be run using the following command:
sampei <<query_mgf_file_path>> <<target_mgf_file_path>> <<id_file_path>> <<additional_flags>>

Three files are required for SAMPEI to run:
Query/Target mgf files
A pair of mgf files is needed as input files with full path. One is used as query mgf file to perform conventional database search and provide high confidence query spectra. The other mgf file could be the same or different mgf file in which user want to uncover peptide with novel modifications not discovered by conventional search.
Please make sure mgf file formated as one of the following examples.
Example 1:
BEGIN IONS
TITLE=Filename.390.390.3
RTINSECONDS=303.016
PEPMASS=442.230102539063 171650277.125
CHARGE=3+
129.1024323 379843.65625
130.0500488 108622.1875
136.0758667 226632.171875
148.0606232 279413.0625
173.0922699 26675.123046875
END IONS
Example 2:
BEGIN IONS
TITLE=Scan 4, Time=1.181370, MS2, HCD
PEPMASS=414.713379
CHARGE=2
120.438423 276.109863
121.133255 185.422791
122.366203 176.366150
127.615936 226.262482
127.961296 208.581024
127.989967 221.123886
END IONS
ID file
A separate tab-delimited (tsv) file produced by X!tandem or other database search tools needs to be provided. SAMPEI is applicable to different database search tools, as long as id file contains the following columns with designated column names as listed below.

Column name
Description
Example

scan
Scan id of the query scan (integer value)
1234

peptide
Peptide sequence identified by database search (string)
LPRSPPLKVLAE

modifications
Modification assigned to the sequence by database search (need to end with comma, and if multiple, separated by comma)
42.01057@A1,57.02147@C4,

charge
Charge state of the query scan
3

proteins
Protein names or accession number
sp_Q03052_PO3F1_HUMAN

Filename
Query mgf file name with no extension
KRG_MV411_G

expect
If using X!tandem in database search, otherwise optional
1.2e-08

total_MS2_intensity
If provided by X!tandem, otherwise optional
1659586.90743756

Example input files
Demo mgf and id files meet the package requirements can be download here.
Addiotional Flags
There are additional flags which can be set to fine tune the search parameters:

Flag
Default
Description

--error-type
ppm

--fragment-mass-error
20

--largest-gap-percent
0.4

--matched-peptide-intensity
0.5
The ratio of MS2 intensity of target scan matched to the theoretical fragments of peptide sequence over the summation of total MS2 intensity in the target scan

--matched-query-intensity
0.3
The ratio of MS2 intensity of query scan matched to target scan over the summation of total MS2 intensity in the query scan

--max-peaks-per-scan
20

--min-diff-dalton-bin
10
The absolute minimum dalton difference between the query scan and the target scan

--no-filter
False
Do not remove DB identified scans in the target mgf file

--output-directory
output
The directory where output files will be created

--write-intermediate
False
If specified both the unfiltered and filtered results will be written to file

--xtandem-xml

An X!tandem xml file which will be used for filtering results

Output
Output File
The output produced by SAMPEI has columns listed below.

Column name
Description
Example

MGF_query_file
Query mgf file name
190509_DL_OCIAML2_ACL_1.mgf

MGF_target_file
Target mgf file name
190509_DL_OCIAML2_ACL_1.mgf

Diff_dalton
Mass difference between query and target scan
71

Diff_dalton_bin
Integer mass difference between query and target scan
71.0378 71

Query_scan
Scan number of query spectra
49620

Query_scan_mz
Query scan m/z
759.7163086

Query_scan_charge
Query scan charge
3

Target_scan
Scan number of target spectra
33549

Target_scan_mz
Target scan m/z
783.3955688

Target_scan_charge
Target scan charge
3

Matches
Normalized number of matches
0.55

Matched_query
The percentage of MS2 intensity of query scan matched to target scan over the summation of total MS2 intensity in the query scan
0.611027774

Matched_intensity_product
percent matched query intensity times percent matched target intensity
0.237107605

Sum_log_intensity
Sum of log transformed query and target MS2 intensities
11.7649257

Peptide
Peptide sequence of target scan
GPAIGIDLGTTYSCVGVFQHGK

Modifications
Query scan modification
57.02147@C14,

Expect (Optional)
E value by X!tandem
0.00066

Total_MS2_intensity (Optional)
Query scan total MS2 intensity
2398832.919

Proteins
Protein name or id
sp_P54652_HSP72_HUMAN

Matched_peptide_intensity_max
The percentage of MS2 intensity of target scan matched to the theoretical fragments of peptide sequence over the summation of total MS2 intensity in the target scan
0.823

Largest_gap
The largest consecutive b/y ion missing
5

Largest_gap_percent
Largest gap over the length of the peptide sequence
0.227272727

Full_mod
Target scan modification
57.02147@C14,71.0377807617@I6,

Unique_mod
Target scan modification identified uniquely by SAMPEI
71.0377807617@I6,

Citing SAMPEI
Cifani, Li, Luo, Grivainis, Intlekofer, Fenyö, Kentsis. Discovery of protein modifications using high resolution differential mass spectrometry proteomics.