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Using MaxQuant for proteomics ‐‐‐‐A step‐by‐step guide
Rongxiao Sa , Department of Chemistry
Pra@k Jagtap , Minnesota Supercompu@ng Ins@tute Nov24, 2009
Outline
• Step by step procedure on using MaxQuant for protein iden@fica@on (saliva proteins, 6 raw files)
Raw data processing Mascot searching protein grouping and file genera@ng
• Brief procedure on using MaxQuant for SILAC data
Why choose MaxQuant
• Free and user friendly, great user support from discussion group
• Faster than other algorithms
• Great for large datasets(>5G)
• More protein iden@fica@on, or more MS/MS iden@fied
• Cross reference to various databases ( GO, Swissprot, Ensembl, KEGG)
• Great for SILAC quan@fica@on
System requirements for running MaxQuant
• Windows XP or vista 32 bit opera@ng system
• Xcalibur installed on the same computer
• Mascot Daemon installed or access
• At least dual core processor ( or 2 Duo)
• At least 2G RAM
Correct data acquisi@on mode on LTQ‐orbitrap
• MS scan should be acquired in profile mode, MS/MS centroid mode
• Use lock mass calibra@on
Log in
• Either on sdvlapp32 or on pc1, pc2, pc3 in Walter library 585 with your user account
• Double click on the MaxQuant folder shortcut on the desktop
Tutorial file loca@on
My computer\C:\Document and secngs\all users\shared tutorial
For your own purposes, put your files in U drive
Protein and pep@de grouping, quan@fica@on
Generate MSM files for mascot searching
Generate decoy databases
Modules of MaxQuant
Select Parameters
Select Singlets for non‐labeled data Select Doublets for SILAC data with 2 experiments Select triplets for SILAC data with 3 experiments
Processed files are located in the same folder as raw files are and are in the folder named combined
Quant.exe Done
Quant.exe generated files
C:\documents and secngs\all users\shared documents\proteomics workshop\raw files \combined
Allspectra.CID.iso.par Allspectra.CID. iso_0.msm Allspectra.CID.sil0.par Allspectra.CID.sil0_0.msm Allspectra.CID.sil1.par Allspectra.CID.sil1_0.msm
Mascot parameter files Corresponding MSM files
Select search parameters Parameter file loca@on: C:\documents and secngs\all users\shared documents\ proteomics tutorial\processed files
Cau@on: Parameter files can not be on net drive ( U drive), it has to be saved on C: or E drive on Sdvlapp32, pc1,pc2 and pc3
Protein grouping with [email protected]
Upload raw files
Raw files loca@on: C:\Documents and Secngs\all users\shared documents\ Proteomics workshop\Raw files
Upload Dat files
Dat files loca@on: C:\Documents and Secngs\all users\shared documents\ Proteomics workshop\dat‐files
Upload Protein Database
Protein Database loca@on: C:\Documents and Secngs\all users\shared documents\ Proteomics workshop\databases
Upload Experimental design file
Protein Database loca@on: C:\Documents and Secngs\all users\shared documents\ Proteomics workshop\Processed files\combined