FAQ

• Q001: How can I do relative label-free quantification with MaxQuant?

  After you have loaded the raw files set the 'Multiplicity' parameter on the 'Modifications & labels' page to 1. Check the 'Label-free quantification' box on the 'Identification and quantification' page. Now you have to specify an experimental design. For that purpose generate a template file which you can edit with Excel by pressing the 'Exp. design' button on the 'Raw files' page. A tab separated text file will be generated. Open in in Excel. Specify which raw files belong to which 'Experiment' by assigning names in the 'Experiment' column. An 'Experiment' basically corresponds to a sample that is being quantified against the other samples. If any pre-fractionation has been done specify the fractions in the 'Fraction' column. These should be integer numbers. Now save and close the file. On the page 'Identification and quantification' upload the file that you just edited in the 'Experimental design' box. Finally you need to decide if you want to use the 'Match between runs' feature. This is recommended in case of no pre-fractionation or fairly reproducible fractionation. The latter is reproducible if the peptides/proteins that are being fractionated typically differ by at most one fraction index. The result will be in the proteinGroups.txt file in the columns called 'LFQ Intensity ...'.

• Q002: For my SILAC labeling I used arginine and lysine having the same mass difference between the heavy and light peptide. Can I analyze my data with MaxQuant?

  This kind of data can be analyzed with MaxQuant by de-selecting the 'Filter labeled amino acids' option in the 'Identification and quantification' page. If this is checked the peptide candidates that are suggested by the search engine are filtered by the numbers of arginines and lysines, which is known before the database search from the mass difference in the SILAC pair. It is highly recommended to always use differently labeled arginines and lysines due to the increased information content.

• Q003: How should I acquire MS and MS/MS spectra?

  MS data have to be recorded in profile mode. Profile data are required for the MaxQuant 3D peak detection to find peaks, isotope patterns and SILAC pairs. MS/MS spectra can be acquired in profile or centroid mode.

• Q004: Is there a limitation for the number of raw files to be processed through MaxQuant?

  No. MaxQuant was tested and worked for up to 1000 raw files.

• Q005: How does the experimental design file help?

  The experimental design file helps in assigning the raw files to the experiment which they belong and to the details of gel slice or fraction number wherever applicable. It allows the files to be analyzed together but still retain the individual ratio values for each sample.

• Q007: Which peptides to choose for protein quantification?

  Protein quantification can be performed in three ways using (a) only unique peptides (b) unique peptides and razor peptides (c) all peptides. Using all peptides is usually not recommended.

• Q008: How does the minimum ratio count threshold affect the overall quantification?

  The minimum ratio count threshold specifies the number of ratio counts (e.g. SILAC pairs) below which the quantification will not be performed for a protein. Increasing this threshold would reduce the number of proteins quantified. However the higher the ratio counts, the more robust is the ratio estimation.