MassIVE MSV000086417

Complete Public PXD022390

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

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Description

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. [doi:10.25345/C5X20K] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: AML ; TMT ; clinical proteomics ; phosphoproteomics

Contact

Principal Investigators:
(in alphabetical order) 		Paul Piehowski, Pacific Northwest National Laboratory, United States
Submitting User: alchemistmatt

Publications

Sanford JA, Wang Y, Hansen JR, Gritsenko MA, Weitz KK, Sagendorf TJ, Tognon CE, Petyuk VA, Qian WJ, Liu T, Druker BJ, Rodland KD, Piehowski PD.
Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality.
J Am Soc Mass Spectrom. 2022 Jan 5;33(1):17-30. Epub 2021 Nov 23.

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