RT Journal Article
SR Electronic
T1 Simplified high-throughput methods for deep proteome analysis on the timsTOF Pro
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 657908
DO 10.1101/657908
A1 Jarrod J Sandow
A1 Giuseppe Infusini
A1 Laura F Dagley
A1 Rune Larsen
A1 Andrew I Webb
YR 2021
UL http://biorxiv.org/content/early/2021/04/07/657908.abstract
AB Recent advances in mass spectrometry technology have seen remarkable increases in proteomic sequencing speed, while improvements to dynamic range have remained limited. An exemplar of this is the new timsTOF Pro instrument, which thanks to its trapped ion mobility, pushes effective fragmentation rates beyond 100Hz and provides accurate CCS values as well as impressive sensitivity. Established data dependent methodologies underutilize these advances by relying on long analytical columns and extended LC gradients to achieve comprehensive proteome coverage from biological samples. Here we describe the implementation of methods for short packed emitter columns that fully utilize instrument speed and CCS values by combining rapid generation of deep peptide libraries with enhanced matching of single shot data dependent sample analysis. Impressively, with only a 17 minute separation gradient (50 samples per day), the combination of high performance chromatography and CCS enhanced library based matching resulted in an average of 5,931 protein identifications within individual samples, and 7,244 proteins cumulatively across replicates from HeLa cell tryptic digests. Additionally, an ultra-high throughput setup utilizing 5 min gradients (180 samples per day) yielded an average of 3,666 protein identifications within individual samples and 4,659 proteins cumulatively across replicates. These workflows are simple to implement on available technology and do not require complex software solutions or custom-made consumables to achieve high throughput and deep proteome analysis from biological samples.Competing Interest StatementJJS, GI and AIW state that they have potential conflicts of interest regarding this work as they are employees of Ion Opticks.