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Quantitative Proteomics

Within cells, proteins are the real effectors of all activities. The expression of certain proteins changes upon cell stimulation or stress, or when cells differentiate or turn into a disease state. Measuring the protein expression levels as well as characterizing their post-translational modifications gives us information on their cellular state. The goal of quantitative proteomics is to evaluate the relative expression of proteins between two cellular samples being compared. To achieve this, the Aebersold labs employs two general strategies: i) labelfree shotgun proteomics and ii) targeted proteomics using SRM (or SWATH-MS). In these workflows, a biological sample undergoes proteolytic digestion with an enzyme and the resulting peptides are analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Multiple samples can be analyzed sequentially and quantification is achieved by comparing the individual runs with each other.

Label-free shotgun
Quantitative proteomics using labelfree shotgun proteomics (Gstaiger and Aebersold 2009)

Labelfree shotgun proteomics is often used for discovery proteomics where a sample of potentially unknown composition is analyzed by LC-MS/MS. In each cycle, the mass spectrometer performs a MS1 scan and usually selects the most intense ions in this scan for detailed analysis and identification. By using sophisticated algorithms, the individual tandem mass spectra are assigned to the original peptides and linked to isotopic intensity patterns in the MS1 spectra. After correcting for retention time distortion, these isotopic intensity patterns can be mapped across multiple runs and thus comparison between multiple samples is possible.

SRM  
Quantitative proteomics using targeted proteomics, for example SRM (Gstaiger and Aebersold 2009).

Targeted proteomics on the other hand is used for directed quantification of a few select analytes. In contrast to shotgun proteomics, targeted proteomics is deterministic and is able to track the fragments of an analyte with high sensitivity and specificity across the whole chromatographic elution of the LC. It is thus often used if reproducible and accurate quantification is required across a large number of samples (such as a biomarker study).

 
 
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27.02.2017
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