MaxQuant User’s GuideVersion 1.5.0.0

Jűrgen Cox and Matthais Mann

Nature Biotechnology 26, 1367-1372, 2008

Kelly Hodge & Sara ten Have

2015

http://www.lamondlab.com/

http://greproteomics.lifesci.dundee.ac.uk/

References

• 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, 1367-72.

• 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, 1251-4.

• 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, 1794-805.

• 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, 1477-85.

• 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, 698-705.

• 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, 279-89.

• 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, 2252-61. 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, 1373-80.

For all updates and all new versions of MaxQuant visit http://maxquant.org/

For commonly asked questions and community help go to the google discussion site, which is linked from MaxQuant.org or can be found here.

http://groups.google.com/group/maxquant-list?pli=1

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.

Raw Files1. Open MaxQuant 2. Click the ‘Load’ 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.

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

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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.

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.

MODIFICATIONS AND LABELS9. 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. Missed cleavages account for the enzyme not being 100% effective- this is common and the default setting is the accepted tolerance for this.

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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 clicked ‘save changes’ in Andromeda, and reloaded MaxQuant, to see the new modifications.

GLOBAL PARAMETERS - GENERAL13. The machine specific settings are found in these fields. 14. 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. 15. Specify the fixed modifications here- Carbamidomethyl is a fixed modification if you have reduced and alkylated your sample using Iodoacetamide. 16. ‘Re-Quantify’ should be selected if you have labelled samples as it normalises the data.17. ‘Match between runs’ is useful if you have more than one file. It allows MaxQuant to search for peptides it saw in previous files (as long as they are being processed at the same time).

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GLOBAL PARAMETERS - IDENTIFICATION & PROTEIN QUANTIFICATION19. 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. 20. Deselect the ‘Filter Labelled amino acids’ box if doing label –free. 21. Second peptides looks for mixed spectra and is very useful for further peptide identification, it is therefore advisable to leave this setting. 22. Select which variable modifications you would like to be quantified along with the unmodified versions of the peptides. 23. Untick ‘Discard unmodified counterpart peptides’. 24. The other settings in the Protein Quantification panel are appropriate for most analyses and can be left as the defaults.

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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.

Tips: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

PARTIAL PROCESSINGIf 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. 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 point it failed instead. In this case you would load the files and the mpar file (see Viewer section on how to load mpar file) then select to start from step 5.

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 CONFIGURATIONMODIFICATIONS

1. Open Andromeda Config tab. Make sure you are on the Modifications tab - it should be pink. 2. Click on the ‘Add’ button. This will load a new modification called “New modification”. 3. Give “Unknown a new name in the Name bar, and also in the Description bar- this is un-

restricted.4. Click ‘Modify table’ – if you don’t click this after every change you make maxquant will not

register the change and you will have to re-enter the information again.5. Click the “Composition” button then the ‘Change’ 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.

6. Click ‘Modify table’. 7. The overall mass is shown in the panel below the composition - ensure this is correct. Click Ok.8. Click ‘Save Changes’.

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ANDROMEDA CONFIGURATION MODIFICATIONS

9. Next specify which amino acid is affected by the modification- this is done in the Specifications tab. 10. Click the plus (+) and an empty specification will appear. 11. 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. 12. 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. Correction factors are often given in the commercial information for iTRAQ reagents, these values can be inserted in the Correction factors panel.

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ANDROMEDA CONFIGURATION PROTEASES

14. 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 15. Click the ‘Add’ button and this will load a new protease for you. 16. Give it a new title under the name tab. 17. Specify in the matrix which side of the amino acid the protease cleaves on and the amino acids at which the protease cleaves by clicking on the amino acid letter you can highlight more than one by clicking on them individually or highlight an entire row by clicking the plus button. 18. Repeat for the amino acids which may restrict cleavage. 19. Click on ‘Modify table’ between each tab as specified in the previous page. 20. Click ‘Save changes’

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ANDROMEDA CONFIGURATION - SEQUENCES 21. 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. 22. Click on the ‘Add’ button in the top left hand corner this will load a new database entry. 23. Click on the open file button in the top right hand corner next to the , and load your fasta file. 24. Specify a parsing rule by typing it in- there is no drop down list for this so you will need to know the rule you want to use. 25. Remember to click ‘Modify table’ between each change as specified in previous pages. 26. Click ‘Save changes’.27. Re-load MaxQuant to see all of your changes, you will not see them in an existing session.

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MAXQUANT - VIEWER

1. Load the raw files as if you were setting up MaxQuant as normal.2. Click on the blue button at the top left hand side of the program. 3. Click ‘Load paramters’ 4. Select the ‘mpar’ file and click open. This will load all the search parameters used in that run -

including databases, modifications and enzymes. 5. The ‘Exists’ column should say ‘True’. if it says ‘False’ then you need to click ‘change folder’ and

select the correct pathway to the files. Do this for all files at once by highlighting them all before selecting the path. If you do not change the folder you will not be able to see anything in the viewer.

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MAXQUANT - VIEWER6. The viewer shows 4 panels. On the top left is the map view, the panel at the bottom left is the multiple viewer window where you can see the MS1 spectra, MS2 spectra, chromatograms of selected peptides, 3D peaks and the multi-map view. The Multi-map viewer also shows the B & Y ions identified for the peptide displayed in the MS2 spectra window. On the top right is the table view where all the data is found separated by .txt file. The final panel on the bottom right is the sequence window and shows the identified peptides for a selected protein (red for unique peptides)7. These buttons will effect the display on the map view such as adding different coloured labels for selected features.

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