Ultrafast ultraviolet photodissociation at 193 nm and its applicability to proteomic workflows.
Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated. Peptide sequence aromaticity and the precursor charge state were found to influence photodissociation efficiency more so than the number of amide chromophores, and the ion trap q-value and number of laser pulses significantly affected the number and abundances of diagnostic product ions (e.g., sequence and immonium ions). Also, photoionization background subtraction was shown to dramatically improve SEQUEST results, especially when peptide signals were low. A liquid chromatography-mass spectrometry (LC-MS)/UVPD strategy was implemented and yielded comparable or better results relative to LC-MS/collision induced dissociation (CID) for analysis of proteolyzed bovine serum albumin and lysed human HT-1080 cytosolic fibrosarcoma cells.
Madsen
James A
JA
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, USA.
Boutz
Daniel R
DR
Brodbelt
Jennifer S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D010777:Photochemistry / Q000379:methods; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis*; D014466:Ultraviolet Rays
D010455:Peptides; D012710:Serum Albumin, Bovine