title: Mass Defect-Based N,N-Dimethyl Leucine Labels for Quantitative Proteomics and Amine Metabolomics of Pancreatic Cancer Cells.
abstract: Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Ling
authors.identifier: 0000-0002-0106-5266
authors.initials: L
authors.lastname: Hao
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Jillian
authors.initials: J
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Lietz
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors.forename: Amanda
authors.initials: A
authors.lastname: Buchberger
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Dustin
authors.initials: D
authors.lastname: Frost
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: W John
authors.initials: WJ
authors.lastname: Kao
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors.forename: Lingjun
authors.identifier: 0000-0003-0056-3869
authors.initials: L
authors.lastname: Li
mesh_terms: D000588:Amines / Q000032:analysis / Q000378:metabolism*; D045744:Cell Line, Tumor; D056148:Chromatography, Reverse-Phase / Q000379:methods; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D055432:Metabolomics / Q000379:methods*; D008745:Methylation; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000588:Amines; D011506:Proteins; D007930:Leucine