Evaluation of differential peptide loading on TMT-based proteomic and phosphoproteomic data quality

ABSTRACT: Investigating the impacts on MS data quantitation reproducibility when loading differential amounts of peptides, ranging from 20 to 400 ug, across channels of TMT 11 multiplexes. PTRC_Exp9 files represent global proteome and phosphoproteome data generated from PBMCs isolated from AML patients, acquired through 12 fractions and 6 fractions per plex, respectively. In this experiment, TMT channels were loaded with varying peptide quantities ranging from 20 to 400 ug. PTRC_Exp12 files represent proteomic and phosphoproteomic data from the MOLM-14 AML cell line, also acquired across 12 global proteomics fractions and 6 phosphoproteomics fractions per plex. In this experiment, all TMT channels were loaded with equivalent quantity of peptides (400 ug). Samples were reduced with dithiothreitol, alkylated with iodoacetamide, and double-digested with Lys-C and trypsin. Digested peptides were desalted with C18 SPE columns, labeled with TMT11 isobaric tags, and fractionated by high pH reverse phase separation. After aliquots were removed for global proteomics (12 fractions per plex), samples were concatenated and IMAC was used to enrich phosphopeptides (6 fractions per plex). LC-MS/MS measurements were acquired on an Orbitrap Fusion Lumos mass spectrometer.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Paul Piehowski  

PROVIDER: MSV000086417 | MassIVE | Thu Nov 05 18:35:00 GMT 2020

SECONDARY ACCESSION(S): PXD022390

REPOSITORIES: MassIVE