Identification of Unexpected Protein Modifications by Mass Spectrometry-Based Proteomics
Shiva Ahmadi and Dominic Winter
In: Wang X., Kuruc M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY
The authors provide a strategy for the discovery of unexpected modifications in complex samples by bottom-up LC-MS/ MS. While expected modifications are readily identified by selecting them as variable modifications, the identification of unknown modifications is less straightforward and requires further data analysis and validation.
They describe in great detail an experimental workflow which includes:
- Parallel searches for the identification of known modifications at unexpected amino acids
- Error tolerant searches for modifications unexpected in the sample but known to the community
- Mass tolerant searches to discover novel modifications.
Additionally, they suggest follow-up verification of identified modifications in the initial dataset and targeted experiments using synthetic peptides.
Procedures were illustrated by analysing a cytosolic fraction of HeLa cells. With LC/MS/MS, the authors identified and quantified a large number of side reactions by monomers and dimers of the alkylation reagents (see figure at right). They also determined that alkylation of peptides with iodine-containing reagents results in a loss of the side chain of methionine. Some 22 notes provide helpful tips on sample preparation, data acquisition, and data processing.