An exclusion list based label-free proteome quantification approach using an LTQ Orbitrap.

Rationale: Label-based mass spectrometry is a powerful tool for large-scale protein identification and quantification. However, it requires the chemical or metabolic incorporation of the labeled compound(s) which can be difficult to attain, e.g. for non-cultivable organisms or scarce sample, such as biopsies. Therefore, we set out to develop and validate an efficient label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) workflow based on optimized instrument settings and incremental exclusion lists.

Methods: To increase the number of quantified peptides an incremental exclusion list was incorporated along with optimized instrument settings for the used LTQ Orbitrap. As a proof of concept, label-free quantification data from this optimized approach were compared to the results of control measurements without exclusion lists and of an in vivo metabolic labeling GeLC/MS/MS experiment. The data were drawn from Staphylococcus aureus whole cell lysates of non-stressed and nitric oxide (NO)-stressed cells.

Results: Compared to MS analysis without exclusion lists the new approach resulted in an increased number of identified peptides, enabling label-free quantification of more than 990 S. aureus proteins. With respect to the number of quantified proteins and differences in protein levels between the control and NO-treated samples the results of the new method were consistent with those of the GeLC/MS/MS experiment.

Conclusions: The application of exclusion lists and optimized instrument settings in LC/MS/MS analysis significantly enhances the sensitivity and resolution of label-free protein identification and quantification. Therefore, the new workflow is a powerful alternative to label-based quantification methods.

(Copyright © 2012 John Wiley & Sons, Ltd.)