title: Development and characterization of novel 8-plex DiLeu isobaric labels for quantitative proteomics and peptidomics.
abstract: BACKGROUND
  Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu.
  
  
  METHODS
  The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label's reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS(2) ) on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined.
  
  
  RESULTS
  An 8-plex set of DiLeu reagents with 1 Da spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 µg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values.
  
  
  CONCLUSIONS
  Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals.
authors.affiliation: School of Pharmacy, University of Wisconsin, 777 Highland Ave, Madison, WI, 53705, USA.
authors.forename: Dustin C
authors.initials: DC
authors.lastname: Frost
authors.affiliation: Department of Chemistry, University of Wisconsin, 1101 University Ave, Madison, WI, 53706, USA.
authors.forename: Tyler
authors.initials: T
authors.lastname: Greer
authors.affiliation: School of Pharmacy, University of Wisconsin, 777 Highland Ave, Madison, WI, 53705, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Xiang
authors.affiliation: School of Pharmacy, University of Wisconsin, 777 Highland Ave, Madison, WI, 53705, USA.
authors.forename: Zhidan
authors.initials: Z
authors.lastname: Liang
authors.affiliation: School of Pharmacy, University of Wisconsin, 777 Highland Ave, Madison, WI, 53705, USA.|Department of Chemistry, University of Wisconsin, 1101 University Ave, Madison, WI, 53706, USA.
authors.forename: Lingjun
authors.initials: L
authors.lastname: Li
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D003903:Deuterium / Q000737:chemistry; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods
chemical_list: D010455:Peptides; D003903:Deuterium; D007930:Leucine