title: Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of the TGF-β/Smad3 Signaling Pathway.
abstract: Isobaric labeling has become a widespread tool for quantitative proteomic studies. Here, we report the development and evaluation of several dimethylated amino acids as novel isobaric tags for quantitative proteomics. Four-plex dimethylated alanine (DiAla), valine (DiVal), and leucine (DiLeu) have been synthesized, sharing common features of peptide tagging and reporter ion production. DiAla and DiLeu are shown to achieve complete labeling. These two tags' impacts on peptide fragmentation and quantitation are further evaluated using HEK293 cell lysate. DiAla labeling generates more abundant backbone fragmentation whereas DiLeu labeling produces more intense reporter ions. Nonetheless, both tags enable accurate quantitative analysis of HEK293 cell proteomes. DiAla and DiLeu tags are then applied to study the TGF-β/Smad3 pathway with four differentially treated mouse vascular smooth muscle (MOVAS) cells. Our MS data reveal proteome-wide changes of AdSmad3 as compared to the GFP control, consistent with previous findings of causing smooth muscle cell (SMC) dedifferentiation.1 Additionally, the other two novel mutations on the hub protein Smad3, Y226A, and D408H, show compromised TGF-β/Smad3-dependent gene transcription and reversed phenotypic switch. These results are further corroborated with Western blotting and demonstrate that the novel DiAla and DiLeu isobaric tagging reagents provide useful tools for multiplex quantitative proteomics.
authors.affiliation: School of Pharmacy, University of Wisconsin , Madison, Wisconsin 53705, United States.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yu
authors.affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin , Madison, Wisconsin 53705, United States.
authors.forename: Xudong
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Chemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.
authors.forename: Tyler
authors.initials: T
authors.lastname: Greer
authors.affiliation: Department of Chemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Lietz
authors.affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin , Madison, Wisconsin 53705, United States.
authors.forename: K Craig
authors.initials: KC
authors.lastname: Kent
authors.affiliation: School of Pharmacy, University of Wisconsin , Madison, Wisconsin 53705, United States.|Department of Chemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.
authors.forename: Lingjun
authors.initials: L
authors.lastname: Li
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives; D000596:Amino Acids / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives; D008745:Methylation; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives
chemical_list: D000596:Amino Acids; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D007930:Leucine; D014633:Valine; D000409:Alanine
keywords: dimethylated amino acid; isobaric tag; phenotype; quantitative proteomics; smooth muscle cell