Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale.
Intact protein characterization using mass spectrometry thus far has been achieved at the cost of throughput. Presented here is the application of 193 nm ultraviolet photodissociation (UVPD) for top down identification and characterization of proteins in complex mixtures in an online fashion. Liquid chromatographic separation at the intact protein level coupled with fast UVPD and high-resolution detection resulted in confident identification of 46 unique sequences compared to 44 using HCD from prepared Escherichia coli ribosomes. Importantly, nearly all proteins identified in both the UVPD and optimized HCD analyses demonstrated a substantial increase in confidence in identification (as defined by an average decrease in E value of ∼40 orders of magnitude) due to the higher number of matched fragment ions. Also shown is the potential for high-throughput characterization of intact proteins via liquid chromatography (LC)-UVPD-MS of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identified and found in 292 distinct proteoforms, 168 of which contained some type of post-translational modification.
Department of Chemistry, University of Texas at Austin , 1 University Station A5300, Austin, Texas 78712, United States.
Joe R
JR
Cannon
Michael B
MB
Cammarata
Scott A
SA
Robotham
Victoria C
VC
Cotham
Jared B
JB
Shaw
Ryan T
RT
Fellers
Bryan P
BP
Early
Paul M
PM
Thomas
Neil L
NL
Kelleher
Jennifer S
JS
Brodbelt
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D006736:Horses; D008969:Molecular Sequence Data; D056951:Photoelectron Spectroscopy / Q000379:methods*; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics*; D013997:Time Factors
D029701:Saccharomyces cerevisiae Proteins