Dimethyl isotope labeling assisted de novo peptide sequencing.
Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.
Hennrich
Marco L
ML
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Mohammed
Shabaz
S
Altelaar
A F Maarten
AF
Heck
Albert J R
AJ
D000595:Amino Acid Sequence; D000818:Animals; D060387:Ascorbate Peroxidases; D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D007553:Isotope Labeling / Q000379:methods*; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism; D027421:Persea / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002247:Carbon Isotopes; D010446:Peptide Fragments; D010940:Plant Proteins; D012710:Serum Albumin, Bovine; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D008666:Metalloendopeptidases