Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale.
Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale.
Durbin
Kenneth R
KR
Department of Chemistry, The Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Tran
John C
JC
Zamdborg
Leonid
L
Sweet
Steve M M
SM
Catherman
Adam D
AD
Lee
Ji Eun
JE
Li
Mingxi
M
Kellie
John F
JF
Kelleher
Neil L
NL
D000465:Algorithms; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000032:analysis; D006367:HeLa Cells; D006657:Histones / Q000032:analysis / Q000235:genetics; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000235:genetics; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D012984:Software; D050777:Stathmin / Q000032:analysis / Q000235:genetics; D014404:Tubulin / Q000032:analysis / Q000235:genetics
D005656:Fungal Proteins; D006657:Histones; D020033:Protein Isoforms; D050777:Stathmin; D014404:Tubulin; D007633:Keratins