193-nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization

• Published in: Proteomics (Weinheim) Vol. 11; no. 7; pp. 1329 - 1334
• Main Authors: Madsen, James A., Kaoud, Tamer S., Dalby, Kevin N., Brodbelt, Jennifer S.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01-04-2011; WILEY‐VCH Verlag; Wiley-VCH; Wiley Subscription Services, Inc
• Subjects: Acids - analysis; Acids - chemistry; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animal proteomics; Animals; Anions; Anions - chemistry; Anions - metabolism; Basic HPLC; Biological and medical sciences; Electrons; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Ions; Miscellaneous; Mitogen-Activated Protein Kinase Kinases - analysis; Mitogen-Activated Protein Kinase Kinases - chemistry; Molecular Sequence Data; MS/MS; Peptides - analysis; Peptides - chemistry; Phosphopeptides; Phosphoproteins - analysis; Phosphoproteins - chemistry; Phosphorylation; Photochemical Processes; Photodissociation; Proteins; Proteome - analysis; Proteome - chemistry; Proteomics - instrumentation; Proteomics - methods; Spectrometry, Mass, Electrospray Ionization - methods; Static Electricity; Tandem Mass Spectrometry; Ultraviolet Rays; Anions; Animal proteomics; Peptides; Basic HPLC; Proteomics; HPLC chromatography; Proteome; Phosphopeptides; Photodissociation; MS/MS
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.201000565

193‐nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a‐/x‐type, followed by d and w side‐chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1− through 3−) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge‐reduced radical products generated by UV photoexcitation were also subjected to collision‐induced dissociation (termed, activated‐electron photodetachment dissociation (a‐EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH∼11.5), piperidine‐modified mobile phase, LC‐MS/UVPD was implemented and resulted in the successful analysis of mitogen‐activated pathway kinases (MAPKs) using ultrafast activation times (5 ns).