【4】蛋白质组学鉴定软件之MSGFPlus 1.简介 2.安装运行 3.结果
MSGF+也是近年来应用得比较多的蛋白鉴定软件。java写的，2008年初次发表JPR，2014年升级发表NC，免费开源，持续更新维护，良心软件。而且，有研究者对不同蛋白质组学鉴定软件进行比较分析，MSGF+的表现也是非常不错的（一下子找不到文献出处~~）。
Github源码：https://github.com/MSGFPlus/msgfplus
支持的输入格式包括：mzML, mzXML, Mascot Generic File (mgf), MS2 files, Micromass Peak List files (pkl), Concatenated DTA files (_dta.txt)
主要支持HUPO PSI 的标准输入mzML格式，以及输出mzIdentML格式（简写mzid ），易转化为TSV格式。
关于mzIdentML格式，参考http://www.psidev.info/mzidentml
2.安装运行
软件下载：https://github.com/MSGFPlus/msgfplus/releases
关于使用，MS-GF+有非常详细的文档：MS-GF+ Documentation
参数配置文件：
https://github.com/MSGFPlus/msgfplus/tree/master/docs/ParameterFiles
关于运行，提供了很多示例以及参数的解释：
https://msgfplus.github.io/msgfplus/MSGFPlus.html
运行示例1：
java -Xmx4000M -jar MSGFPlus.jar
-s test.mzML
-d uniprot_swissprot_human_20190313_20417.fasta
-t 20ppm -ti -1,2 -ntt 0 -tda 1 -e 0 -m 3 -inst 3 -minCharge 1 -maxCharge 6 -addFeatures 1
-mod Mods.txt
-o test.mzid
修饰文件Mods.txt内容如下：
# This file is used to specify modifications
# # for comments
#
# Max Number of Modifications per peptide
# If this value is large, the search takes long.
NumMods=2
# To input a modification, use the following command:
# Mass or CompositionStr, Residues, ModType, Position, Name (all the five fields are required).
# CompositionStr (C[Num]H[Num]N[Num]O[Num]S[Num]P[Num]Br[Num]Cl[Num]Fe[Num])
# - C (Carbon), H (Hydrogen), N (Nitrogen), O (Oxygen), S (Sulfer), P (Phosphorus), Br (Bromine), Cl (Chlorine), Fe (Iron), and Se (Selenium) are allowed.
# - Negative numbers are allowed.
# - E.g. C2H2O1 (valid), H2C1O1 (invalid)
# Mass can be used instead of CompositionStr. It is important to specify accurate masses (integer masses are insufficient).
# - E.g. 15.994915
# Residues: affected amino acids (must be upper letters)
# - Must be uppor letters or *
# - Use * if this modification is applicable to any residue.
# - * should not be "anywhere" modification (e.g. "15.994915, *, opt, any, Oxidation" is not allowed.)
# - E.g. NQ, *
# ModType: "fix" for fixed modifications, "opt" for variable modifications (case insensitive)
# Position: position in the peptide where the modification can be attached.
# - One of the following five values should be used:
# - any (anywhere), N-term (peptide N-term), C-term (peptide C-term), Prot-N-term (protein N-term), Prot-C-term (protein C-term)
# - Case insensitive
# - "-" can be omitted
# - E.g. any, Any, Prot-n-Term, ProtNTerm => all valid
# Name: name of the modification (Unimod PSI-MS name)
# - For proper mzIdentML output, this name should be the same as the Unimod PSI-MS name
# - E.g. Phospho, Acetyl
# - Visit http://www.unimod.org to get PSI-MS names.
C2H3N1O1,C,fix,any,Carbamidomethyl # Fixed Carbamidomethyl C
#144.102063,*,fix,N-term,iTRAQ4plex # iTRAQ 4 plex
#144.102063,K,fix,any,iTRAQ4plex # iTRAQ 4 plex
# Variable Modifications (default: none)
O1,M,opt,any,Oxidation # Oxidation M
#15.994915,M,opt,any,Oxidation # Oxidation M (mass is used instead of CompositionStr)
H-1N-1O1,NQ,opt,any,Deamidated # Negative numbers are allowed.
#C2H3NO,*,opt,N-term,Carbamidomethyl # Variable Carbamidomethyl N-term
#H-2O-1,E,opt,N-term,Glu->pyro-Glu # Pyro-glu from E
#H-3N-1,Q,opt,N-term,Gln->pyro-Glu # Pyro-glu from Q
#C2H2O,*,opt,Prot-N-term,Acetyl # Acetylation Protein N-term
#C2H2O1,K,opt,any,Acetyl # Acetylation K
#CH2,K,opt,any,Methyl # Methylation K
#HO3P,STY,opt,any,Phospho # Phosphorylation STY
运行示例2：
java -Xmx4g -Xms1g -jar MSGFPlus.jar
-conf MSGFPlus_Parameters.txt
-d test.fasta
-s test.mzML
-o test.mzid
参数配置文件MSGFPlus_Parameters.txt内容如下：
#Parent mass tolerance
# Examples: 2.5Da or 30ppm
# Use comma to set asymmetric values, for example "0.5Da,2.5Da" will set 0.5Da to the left (expMass<theoMass) and 2.5Da to the right (expMass>theoMass)
PrecursorMassTolerance=20ppm
#Max Number of Modifications per peptide
# If this value is large, the search will be slow
NumMods=5
#Modifications (see below for examples)
StaticMod=C2H3N1O1, C, fix, any, Carbamidomethyl # Fixed Carbamidomethyl C
DynamicMod=O1, M, opt, any, Oxidation # Oxidized methionine
DynamicMod=H-1N-1O1, NQ, opt, any, Deamidated # Deamidation of Glutamine (+0.984016)
#Custom amino acids
CustomAA=C3H5NO, U, custom, U, Selenocysteine # Custom amino acids can only have C, H, N, O, and S
#CustomAA=H0, X, custom, X, RemoveAA # Remove AA
#Fragmentation Method
# 0 means as written in the spectrum or CID if no info (Default)
# 1 means CID
# 2 means ETD
# 3 means HCD
# 4 means Merge spectra from the same precursor (e.g. CID/ETD pairs, CID/HCD/ETD triplets)
FragmentationMethodID=3
#Instrument ID
# 0 means Low-res LCQ/LTQ (Default for CID and ETD); use InstrumentID=0 if analyzing a dataset with low-res CID and high-res HCD spectra
# 1 means High-res LTQ (Default for HCD; also appropriate for high res CID); use InstrumentID=1 for Orbitrap, Lumos, and QEHFX instruments
# 2 means TOF
# 3 means Q-Exactive
InstrumentID=1
#Enzyme ID
# 0 means No enzyme used
# 1 means Trypsin (Default); use this along with NTT=0 for a no-enzyme search of a tryptically digested sample
# 2: Chymotrypsin, 3: Lys-C, 4: Lys-N, 5: Glu-C, 6: Arg-C, 7: Asp-N, 8: alphaLP, 9: No Enzyme (for peptidomics)
EnzymeID=1
#Isotope error range
# Takes into account of the error introduced by choosing non-monoisotopic peak for fragmentation.
# Useful for accurate precursor ion masses
# Ignored if the parent mass tolerance is > 0.5Da or 500ppm
# The combination of -t and -ti determins the precursor mass tolerance.
# e.g. "-t 20ppm -ti -1,2" tests abs(exp-calc-n*1.00335Da)<20ppm for n=-1, 0, 1, 2.
IsotopeErrorRange=0,3
#Number of tolerable termini
# The number of peptide termini that must have been cleaved by the enzyme (default 1)
# For trypsin, 2 means fully tryptic only, 1 means partially tryptic, and 0 means no-enzyme search
NTT=2
#Target/Decoy search mode
# 0 means don't search decoy database (default)
# 1 means search decoy database to compute FDR (source FASTA file must be forward-only proteins)
TDA=1
#Number of Threads (by default, uses all available cores)
NumThreads=8
#Minimum peptide length to consider
MinPepLength=6
#Maximum peptide length to consider
MaxPepLength=50
#Minimum precursor charge to consider (if not specified in the spectrum)
MinCharge=1
#Maximum precursor charge to consider (if not specified in the spectrum)
MaxCharge=6
#Number of matches per spectrum to be reported
#If this value is greater than 1 then the FDR values computed by MS-GF+ will be skewed by high-scoring 2nd and 3rd hits
NumMatchesPerSpec=1
#Amino Acid Modification Examples
# Specific static modifications using one or more StaticMod= entries
# Specific dynamic modifications using one or more DynamicMod= entries
# Modification format is:
# Mass or CompositionStr, Residues, ModType, Position, Name (all the five fields are required).
# Examples:
# C2H3N1O1, C, fix, any, Carbamidomethyl # Fixed Carbamidomethyl C (alkylation)
# O1, M, opt, any, Oxidation # Oxidation M
# 15.994915, M, opt, any, Oxidation # Oxidation M (mass is used instead of CompositionStr)
# H-1N-1O1, NQ, opt, any, Deamidated # Negative numbers are allowed.
# CH2, K, opt, any, Methyl # Methylation K
# C2H2O1, K, opt, any, Acetyl # Acetylation K
# HO3P, STY,opt, any, Phospho # Phosphorylation STY
# C2H3NO, *, opt, N-term, Carbamidomethyl # Variable Carbamidomethyl N-term
# H-2O-1, E, opt, N-term, Glu->pyro-Glu # Pyro-glu from E
# H-3N-1, Q, opt, N-term, Gln->pyro-Glu # Pyro-glu from Q
# C2H2O, *, opt, Prot-N-term, Acetyl # Acetylation Protein N-term
#Custom amino acids examples
# Only supports empirical formulas of elements C H N O S.
# If other elements are needed, or a specific mass is needed, they can be added as fixed modifications on the custom AA
# Maximum atom counts: 255 C, 255 H, 63 N, 63 O, 15 S
# Format spec is:
# EmpiricalFormula, ResidueSymbol, custom, OriginalAA, Name (all the five fields are required, though OriginalAA is not actually used for anything)
# Examples:
# C5H7N1O2S0,J,custom,P,Hydroxylation # Hydroxyproline
# C3H6N2O0S1,X,custom,C,Amidation # C-terminal amidation of Cys
# C5H5N1O1S0,Z,custom,E,Glu->pyro-Glu # N-terminal pyroGlu residue, from either Glu OR Gln
3.结果
原始输出格式MzIdentML，示例文件test.mzid。
有2种方法将mzid文件转化为tsv，使结果更加易读。详见https://msgfplus.github.io/msgfplus/MzidToTsv.html：
- 一是MSGFPlus.jar内置的MzIDToTsv工具，实现容易，但对于大文件慢。
Usage: java -Xmx3500M -cp MSGFPlus.jar edu.ucsd.msjava.ui.MzIDToTsv
-i MzIDFile (MS-GF+ output file (*.mzid))
[-o TSVFile] (TSV output file (*.tsv) (Default: MzIDFileName.tsv))
[-showQValue 0/1] (0: do not show Q-values, 1: show Q-values (Default))
[-showDecoy 0/1] (0: do not show decoy PSMs (Default), 1: show decoy PSMs)
[-unroll 0/1] (0: merge shared peptides (Default), 1: unroll shared peptides)
- 二是单独使用MzidToTsvConverter.exe工具，转化快，处理大文件，限于Windows（Linux需要mono）
MzidToTsvConverter.exe -mzid:SearchResults.mzid -unroll -showDecoy
转化为tsv后的示例文件：test_Unrolled.tsv
表头内容包含：
1 #SpecFile
2 SpecID
3 ScanNum
4 FragMethod
5 Precursor
6 IsotopeError
7 PrecursorError(ppm)
8 Charge
9 Peptide
10 Protein
11 DeNovoScore
12 MSGFScore
13 SpecEValue
14 EValue
15 QValue
16 PepQValue
ref：
https://msgfplus.github.io/msgfplus/index.html
http://www.psidev.info/mzidentml
https://omics.pnl.gov/software/ms-gf
https://github.com/MSGFPlus/msgfplus
https://github.com/MSGFPlus/msgfplus/tree/master/docs/ParameterFiles
https://msgfplus.github.io/msgfplus/MzidToTsv.html
https://github.com/MSGFPlus/msgfplus/releases
蛋白质组学鉴定定量系列软件总结：
【1】蛋白鉴定软件之X!Tandem
【2】蛋白鉴定软件之Comet
【3】蛋白鉴定软件之Mascot
【4】蛋白质组学鉴定软件之MSGFPlus
【5】蛋白质组学鉴定定量软件之PD
【6】蛋白质组学鉴定定量软件之MaxQuant