Evaluation of the carrier proteome limit on a ZenoTOF instrument

ABSTRACT: This analysis sought to evaluate the carrier proteome limits on a SCIEX 7600 ZenoTOF mass spectrometer. A 9 channel TMTPro digest standard previously described in Orsburn, et al., Nature Communcations 2022 was used. . Briefly, a K562 human peptide digest standard (Promega) and E.coli peptide standard (Waters) were resuspended in 100mM TEAB (SimpliFi) and aliquots of 10 micrograms of each were labeled with the TMTPro reagents, 126, 127n, 128n, 129n, 130n, 131n, 132n, 133n and 134 and the reactions quenched with hydroxylamine according to vendor instructions. Labeled peptides were combined to maintain and equal concentration of K562 digest in each lane by combining 2 micrograms of each. E.coli peptides were diluted to 10to5to1 in TEAB and combined with the respective lanes to result in a repeating E.coli concentration of 20% and 10% of the first channel. The combined peptides were diluted to 100ng/uL total concentration and analyzed on a SCIEX 7600 ZenoTOF instrument coupled to a Waters H Class microflow UHPLC. A 60 minute gradient was used at a flow rate of 5 uL/min using a Phenomenex column. A top 10 data dependent method was used for all analysis. The resulting output files were converted to MGF using the ProteoWizard software and the MGF files were processed in Proteome Discoverer 2.4SP1 using a 15ppm MS1 and 0.03 Da MS/MS tolerance window. Static carbamidomethylation of cysteine and TMTPro tags on lysine and peptide N-termini were used as well as dynamic modifications of methionine oxidation to search against a combination of the SwissProt Human and E.coli FASTA. The cRAP database was used for contaminant identification.

INSTRUMENT(S): ZenoTOF 7600

ORGANISM(S): Escherichia Coli (ncbitaxon:562) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Benjamin C Orsburn  

PROVIDER: MSV000094044 | MassIVE | Thu Feb 08 11:19:00 GMT 2024

SECONDARY ACCESSION(S): PXD049286

REPOSITORIES: MassIVE