Systematic comparison of ultraviolet photodissociation and electron transfer dissociation for peptide anion characterization.
Ultraviolet photodissociation at 193 nm (UVPD) and negative electron transfer dissociation (NETD) were compared to establish their utility for characterizing acidic proteomes with respect to sequence coverage distributions (a measure of product ion signals across the peptide backbone), sequence coverage percentages, backbone cleavage preferences, and fragmentation differences relative to precursor charge state. UVPD yielded significantly more diagnostic information compared with NETD for lower charge states (n ≤ 2), but both methods were comparable for higher charged species. While UVPD often generated a more heterogeneous array of sequence-specific products (b-, y-, c-, z-, Y-, d-, and w-type ions in addition to a- and x- type ions), NETD usually created simpler sets of a/x-type ions. LC-MS/UVPD and LC-MS/NETD analysis of protein digests utilizing high pH mobile phases coupled with automated database searching via modified versions of the MassMatrix algorithm was undertaken. UVPD generally outperformed NETD in stand-alone searches due to its ability to efficiently sequence both lower and higher charge states with rapid activation times. However, when combined with traditional positive mode CID, both methods yielded complementary information with significantly increased sequence coverage percentages and unique peptide identifications over that of just CID alone.
Department of Chemistry and Biochemistry, The University of Texas at Austin, 78712, USA.
Jared B
JB
Shaw
James A
JA
Madsen
Hua
H
Xu
Jennifer S
JS
Brodbelt
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D030562:Databases, Protein; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010782:Photolysis; D011506:Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014466:Ultraviolet Rays
D000838:Anions; D010455:Peptides; D011506:Proteins