MaxQuant User s Guide Version

Transcription

1 MaxQuant User s Guide Version Jűrgen Cox and Matthias Mann Nature Biotechnology 26, (2008) Sara ten Have

2 References For all updates and all new versions of MaxQuant visit For commonly asked questions and community help go to the google discussion site, which is linked from MaxQuant.org or can be found here. The MaxQuant paper. Note that it has a large supplement containing in-depth descriptions of algorithms. Cox, J. and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, The first major application of MaxQuant in proteome-wide SILAC-based quantification. de Godoy LM, Olsen JV, Cox J, Nielsen ML, Hubner NC, Fröhlich F, Walther TC, Mann M. (2008) Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455, Andromeda search engine. Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10, A paper describing algorithmic developments for mass accuracy improvements. Cox, J. and Mann, M. (2009) Computational principles of determining and improving mass precision and accuracy for proteome measurements in an Orbitrap. J Am Soc Mass Spectrom 20, A protocol applicable to 1.0.X.Y versions. Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, Mann M.. (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4, First application of the label free quantification algorithm. Luber CA, Cox J, Lauterbach H, Fancke B, Selbach M, Tschopp J, Akira S, Wiegand M, Hochrein H, O'Keeffe M, Mann M. (2010) Quantitative proteomics reveals subset-specific viral recognition in dendritic cells. Immunity 32, Large-scale phosphoproteomics application including calculation of site occupancies. Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, Gnad F, Cox J, Jensen TS, Nigg EA, Brunak S, Mann M. (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3, ra3. All ion fragmentation. Geiger T, Cox J, Mann M. (2010) Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation.mol Cell Proteomics 9, Software lock mass. Joint recalibration of time and mass dependent mass errors. Cox, J., Michalski, A. and Mann, M. (2011) Software lock mass by two dimensional minimization of peptide mass errors. J Am Soc Mass Spectrom 22,

3 MaxQuant s default Settings and databases that are downloaded with it, are usually sufficient for most types of analysis, and cover most model species. If however you have some unusual modification or species which you need to analyse, go to the Andromeda configuration section first (page 10). RAWFILES 1. Open MaxQuant 2. Click the Load Files button and locate your.raw files (these are the Mass Spectrometer s raw data, and the.raw format is specific to Thermo Instruments) 3. Select all your files. (you can tell MaxQuant what files to combine and separate so this is very useful for intra-sample, inter-experiment comparison, and this is one of the most clever aspects of MaxQuant). Tips: Have MaxQuant installed on a local drive- not a network drive. Have your files (.raw files and.fasta files) located on the same drive- as maintaining the communication over a network during processing takes longer and may drop out.

4 5. 4. Make sure all the files you wanted to analyse are loaded. 5. Click the Exp. Design button. This will generate a combined folder in the same location as your.raw files

5 EXPERIMENTAL DESIGN FILE 6. Open your Experimental Design template file. This is best done in Excel as this will maintain the formatting required to be read properly by MaxQuant. 7. Fill in the Slice /Fraction data (non-identical numbers) and the Experiment column (the samples you would like to have combined /separated information for) 8. Save as a.txt file. You can save it as the same name or modify it for your reference. Unmodified Experimental Design Template Tips: In the Experiment column if you name things identically they will be combined, this is case sensitive. The power of MaxQuant is to do comparative analysis, so use the experimental design file to your advantage here. This means that the data for all proteins identified in an experiment will be in the same file- but the quantitative data separated according to your specifications. If you name samples identically in the experiment column their information will be combined, and you loose the ability to individually compare samples- but if they are all fractions of the same thing- this is ideal.

6 v 12. MODIFICATIONS AND LABELS 9. These settings describe the chemistry done to the proteins. Any chemistry done which may have an effect on mass must be included in these settings. Modifications that might be possible are considered Variable Modifications (the database is then searched with and without these modifications). Modifications which must occur are Fixed Modifications (the database is searched only with this modification- see MS/MS Sequences Section). 10. Choose the enzyme you used to digest your protein with- in most cases this is Trypsin. 11. Specify labels if you used a labelling strategy. If you did not then select Multiplicity 1. This will do a label free search. 12. The First Search 20ppm and Main Search 6ppm should be left as they are- this is for the purpose of MaxQuant identifying the maximum number of peptides for mass and retention time calibration, and then to refine the results at the Re-Quantification step. This is particularly effective is you use a first search database- which is mentioned later on. 13. Missed cleavages account for the enzyme not being 100% effective- this is common and the default setting is the accepted tolerance for this. 14. The Type setting is machine dependant. If you are using an Exactive, you will need to select All Ion Fragmentation. If you are using any of the other Thermo instruments (XL, Velos etc.) Standard is the setting you need. 15. If a double digest with 2 enzymes was used you can specify this here tick Separate Enzyme for First Search and select the enzyme from the drop down box. This can be specified for modifications also. Tips: If you modify the Andromeda config files for modifications and labels, they will be seen in this field. You do need to make sure you have saved all in Andromeda, and reloaded MaxQuant, to see the new modifications.

7 MS/MS & SEQUENCES 16. The machine specific settings are found in this field. The defaults found in the top panel are fine for the majority of searches. 17. Specify the fixed modifications here- Carbamidomethyl is a fixed modification if you have reduced and alkylated your sample using Iodoacetamide. 18. Load the.fasta files for the database you wish to search. This can be either one that is supplied with MaxQuant, or if you require a specific one you will need to parse this through Andromeda Config, so MaxQuant knows how to read the file. 19. Load the first search.fasta file. Human and mouse first search.fasta files are provided with MaxQuant. These contain commonly seen proteins which are expected in samples, and are used in the first search as calibration points for the more exact Main Search and re-calibration steps later on. Their use is recommended. Tips: Adds in CON_PROTEIN ID labelled proteins which are known,laboratory originating, contaminants- VERY USEFUL! Isoleucine=Leucine Generates a reversed or scrambled database to match your data to. This enables False discovery rates to be calculated and applied. It also interchanges Lysine with Arginine to avoid false -reverse hits which might originate from palindromic peptides. The error generated if you have not parsed your.fasta file in Andromeda config.

8 IDENTIFICATION AND QUANTIFICATION 20. The values in the top panel describe the stringency of the searches performed, such as False Discovery Rate (FDR), number of peptides required for an identification, and Posterior Error Probability (PEP) score cut off. The default settings are a good standard set up. You can increase the FDR to be less stringent for specific reasons but this is best left to experts. 21. Deselect the Filter Labelled amino acids box if doing label free. 22. Second peptides looks for mixed spectra and is very useful for further peptide identification, it is therefore advisable to leave this setting. 23. Load the Experimental Design Template which was modified in steps Select which variable modifications you would like to be quantified along with the unmodified versions of the peptides. 25. The settings in the Protein Quantification panel are appropriate for most analyses and can be left as the defaults. 26. The Misc. panel defaults are also appropriate for most analyses. The ibaq Quantification (Intensity Based Absolute Quantification) is used for label free quantitation (calculating intensities from peak intensities, including isotopic peaks, with some additional calculations) and can match retention times between samples. 27. Re-Quantify should always be used also, as this allows for a second peak finding to occur after protein identification has been done. Tips: The Threads setting at the bottom of the window tells the computer how many cores to apply to the processing, and the more you specify (limiting the maximum to the number of cores you have) the faster the processing will go. MaxQuant will not use the maximum for all processes and you should be able to use other programs on your computer at the same time, without too much detriment to performance.

9 PARTIAL PROCESSING If Processing fails at anytime, in the combined file, you will find a Proc file. Within this there are text documents which are written as each process is completed. Above you can see the first search process had an error. This means I could load all the.raw files and specific modifications, with the experimental design file again, but instead of processing from the beginning you can tell MaxQuant to start at the first search instead.

10 If you click on the performance tab you can keep track of where your analysis is up to, and how long things have been taking. On completion a Done window will open and you have results.

11 Andromeda Configuration allows you to add in new protein databases, as they are updated, new or unusual modifications, and different enzymes or combinations of enzymes. Modifications you make in this program will be visible in MaxQuant after you have saved it, and load up a new session of MaxQuant. This will enable the search engine Andromeda to interrogate your MS data the way you require it to be done ANDROMEDA CONFIGURATION MODIFICATIONS 1. Open Andromeda Config. Make sure you are on the Modifications tab. 2. Click on the large green Plus button (+). This will load a new modification called Unknown. 3. Give Unknown a new Title in the Title bar, and also in the Full Name bar- this is un-restricted. 4. Click the Change button to enter the composition of your modifications (C, H, N, O etc.) from the drop down list, specifying the number of molecules with the count arrow on the right. 5. The overall mass is shown in the panel below the composition- ensure this is correct. Click Ok.

12 ANDROMEDA CONFIGURATION MODIFICATIONS 5. Next specify which amino acid is affected by the modification- this is done in the Specificity tab. 6. Click the big green Plus (+) and an empty specification will appear. 7. Go to the site drop down menu and select the required amino acid. If there are several affected amino acids specify each by clicking the plus button for each one. 8. If there are neutral losses, or diagnostic peaks associated with your modification, fill these in with the green Plus (+) button, specifying the chemical compositions.

13 ANDROMEDA CONFIGURATION MODIFICATIONS 9. Correction factors are often given in the commercial information for itraq reagents, these values can be inserted in the Correction factors panel.

14 ANDROMEDA CONFIGURATION PROTEASES 10. The Default proteases found in Andromeda will more than likely cover most experiments you do. If however you have a novel combination of proteases you can input this in the Proteases panel 11. Click the green plus (+) on the top Left hand corner and this will load a new protease for you. Give it a new title. 12. Specify in the Sense drop down which side of the amino acid the protease cleaves on (C-term, or N-term). 13. Specify the amino acids at which the protease cleaves by clicking on the amino acid letter and clicking the right pointing arrow button. 14. Repeat for the amino acids which may restrict cleavage. 15. To check the set up for this protease is correct click on the Test Enzyme tab. This will do a theoretical digest of a sequence showing you the resulting peptides.

15 ANDROMEDA CONFIGURATION SEQUENCES 16. Andromeda needs to be told how to read your fasta files, as some databases are delimited in different ways (this means the name, gene name, ID number etc. are separated by different characters). If this is done correctly the results files will be laid out correctly and be easier to manage. 17. Click on the Green plus (+) button in the top left hand corner this will load a new database entry. 18. Click on the open file button in the top right hand corner next to the database bar, and load your fasta file. 19. Specify a parsing rule from the select rule panel, then click on the calculator button. The rule you selected should now appear in the Rule bar. 20. Click on the Test Rule tab and check if Andromeda is able to read the information from the database correctly. 21. Writing specific rules is also possible. Click the green plus (+) button in the top left hand corner of the Select Rule panel, this will generate a new rule line, enter the rule in regular expression (Regex) in the left hand bar, and give it a name in the right hand bar. 22. Save all. 23. Re-load MaxQuant to see all of your changes, you will not see them in an existing session.