Mass accuracy is a key parameter in proteomic experiments, improving 
specificity, and success rates of peptide identification. Advances in 
instrumentation now make it possible to routinely obtain high resolution data in 
proteomic experiments. To compensate for drifts in instrument calibration, a 
compound of known mass is often employed. This 'lock mass' provides an internal 
mass standard in every spectrum. Here we take advantage of the complexity of 
typical peptide mixtures in proteomics to eliminate the requirement for a 
physical lock mass. We find that mass scale drift is primarily a function of the 
m/z and the elution time dimensions. Using a subset of high confidence peptide 
identifications from a first pass database search, which effectively substitute 
for the lock mass, we set up a global mathematical minimization problem. We 
perform a simultaneous fit in two dimensions using a function whose 
parameterization is automatically adjusted to the complexity of the analyzed 
peptide mixture. Mass deviation of the high confidence peptides from their 
calculated values is then minimized globally as a function of both m/z value and 
elution time. The resulting recalibration function performs equal or better than 
adding a lock mass from laboratory air to LTQ-Orbitrap spectra. This 'software 
lock mass' drastically improves mass accuracy compared with mass measurement 
without lock mass (up to 10-fold), with none of the experimental cost of a 
physical lock mass, and it integrated into the freely available MaxQuant 
analysis pipeline ( www.maxquant.org ).