An exclusion list based label-free proteome quantification approach using an LTQ Orbitrap.
BACKGROUND
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.
Institute for Microbiology, Ernst Moritz Arndt University Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany.
Jan
J
Muntel
Michael
M
Hecker
Dörte
D
Becher
D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D009569:Nitric Oxide / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D020543:Proteome; D009569:Nitric Oxide