A proteomic strategy based on a multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization. Casein peptide digests obtained by using an equimolar enzymatic mix of trypsin and chymotrypsin were analyzed by LC-tandem MS, and the powerful capabilities of alternating CID/ETD approach (1-4) were evaluated in terms of protein coverage and MASCOT score. Overlapping peptides are produced throughout the protein, reducing the ambiguity in mapping modifications between natural variants and animal species. Only the first-ranked peptides arising from database search were included in the peptide lists. Then, a manual post-database internal validation of MS/MS dataset was performed, therefore, the matched peptides were cross checked by the evaluation of ion scores, number of experimental product ions and their relative abundances in the MS/MS spectrum. The acquisition of high quality-spectra by the combined use of CID and ETD has been obtained, thus allowing a confirmation of spectral information and reducing ambiguity in peptide sequence assignments. Hence, the optimized enzymatic digestion in combination with integrated CID/ETD experiments following nanoLC separations provides an excellent platform to carry out proteomics analysis aimed at the maximization of sequence coverage and the detection of post-translational modifications, as well as species specific residues and single-point amino acid modifications in protein natural variants. Finally, the advantages of using the enzymatic mix trypsin/chymotrypsin in the bottom-up strategy was confirmed by the nanoLC and CID/ETD tandem mass spectrometry identification of goat milk proteins, previously separated by two-dimensional gel electrophoresis. [1] H. Molina, R. Matthiesen, K. Kandasamy, A. Pandey. Anal. Chem. 80 (2008) 4825-4835. [2] J.E.P. Syka, J.J. Coon, M.J. Schroeder, J. Shabanowitz, D.F. Hunt. Proc. Natl. Acad. Sci. USA 101 (2004) 9528-9533. [3]. F. Kjeldsen, A.M.B. Giessing, C.R. Ingrell, O.N. Jensen. Anal. Chem. 79 (2007) 9243-9252. [4] M.S. Kim, A. Pandey. Proteomics 12 (2012) 530-542.
STRATEGIES IN PROTEIN CHARACTERIZATION BY CID/ETD TANDEM MASS SPECTROMETRY
NARDIELLO, DONATELLA;PALERMO, CARMEN;NATALE, ANNA;CENTONZE, DIEGO
2013-01-01
Abstract
A proteomic strategy based on a multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization. Casein peptide digests obtained by using an equimolar enzymatic mix of trypsin and chymotrypsin were analyzed by LC-tandem MS, and the powerful capabilities of alternating CID/ETD approach (1-4) were evaluated in terms of protein coverage and MASCOT score. Overlapping peptides are produced throughout the protein, reducing the ambiguity in mapping modifications between natural variants and animal species. Only the first-ranked peptides arising from database search were included in the peptide lists. Then, a manual post-database internal validation of MS/MS dataset was performed, therefore, the matched peptides were cross checked by the evaluation of ion scores, number of experimental product ions and their relative abundances in the MS/MS spectrum. The acquisition of high quality-spectra by the combined use of CID and ETD has been obtained, thus allowing a confirmation of spectral information and reducing ambiguity in peptide sequence assignments. Hence, the optimized enzymatic digestion in combination with integrated CID/ETD experiments following nanoLC separations provides an excellent platform to carry out proteomics analysis aimed at the maximization of sequence coverage and the detection of post-translational modifications, as well as species specific residues and single-point amino acid modifications in protein natural variants. Finally, the advantages of using the enzymatic mix trypsin/chymotrypsin in the bottom-up strategy was confirmed by the nanoLC and CID/ETD tandem mass spectrometry identification of goat milk proteins, previously separated by two-dimensional gel electrophoresis. [1] H. Molina, R. Matthiesen, K. Kandasamy, A. Pandey. Anal. Chem. 80 (2008) 4825-4835. [2] J.E.P. Syka, J.J. Coon, M.J. Schroeder, J. Shabanowitz, D.F. Hunt. Proc. Natl. Acad. Sci. USA 101 (2004) 9528-9533. [3]. F. Kjeldsen, A.M.B. Giessing, C.R. Ingrell, O.N. Jensen. Anal. Chem. 79 (2007) 9243-9252. [4] M.S. Kim, A. Pandey. Proteomics 12 (2012) 530-542.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
