Proteomic Profiling pp 249-258 | Cite as
Identification of Protein N-Termini Using TMPP or Dimethyl Labeling and Mass Spectrometry
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Abstract
Determination of a protein’s N-terminal sequence can be important for the characterization of protein processing. To increase the confidence of protein N-terminal identification, chemical derivatization of the N-terminal amine group by (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) or dimethyl labeling followed by mass spectrometric analysis is commonly performed. Using this approach, proteins can be separated by SDS-PAGE, and the protein N-terminus of interest is labeled by TMPP or dimethyl in-gel before tryptic digestion and LC-MS analysis. The N-terminus of a protein can thus be easily identified because the N-terminal tryptic peptides are preferentially labeled. Peptides with N-terminal derivatization produce a better fragmentation pattern during tandem mass spectrometric analysis, which significantly facilitates sequencing of these peptides.
Key words
TMPP labeling Dimethyl labeling N-terminal sequence analysis Mass spectrometryNotes
Acknowledgement
This work was supported by NIH Shared Instrumentation Grant S10RR027990 and NINDS grant P30 NS050276 to T.A.N. We thank the ABRF Protein Sequencing Research Group (2013) for helpful protocols, reagents and advice.
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