Supplemental Information
Novel isotopic N,N-dimethyl leucine (iDiLeu) reagents enable absolute
quantification of peptides and proteins using a standard curve approach
Tyler Greer1, Christopher B. Lietz1, Feng Xiang2, and Lingjun Li1,2
1. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706,
United States
2. School of Pharmacy, University of Wisconsin-Madison, Madison, WI, 53705, United
States
Supplemental Figure 1: Higher-energy collisional dissociation (HCD) comparison of
iDiLeu-, TMT-, and 8-plex iTRAQ-labeled peptide ADVLTAFLNK at high normalized
collision energy values of 35 and 25. At an NCE of 35, the iDiLeu-labeled peptide
showed secondary fragmentation and yielded fewer identifiable b- and y-ions while the
opposite was true for the TMT- and iTRAQ-labeled peptide (a). The iDiLeu-labeled
peptide yielded the most fragments at a reduced NCE of 25 (b), but the commercially
labeled peptides were poorly fragmented at this value.
Supplemental Figure 2: Examples of singly or doubly iDiLeu-labeled peptide clusters
with charges of +2 and +3. Peptides labeled once (a, c) experience interference from
neighbors of the same cluster. Peptides labeled twice (b, d) do not have interference,
but the differing isotopic distributions still require correction for accurate quantification.
Supplemental Figure 3: True precursor intensity equations for peptides experiencing
interference. A Mathcad solve block symbolically solved the raw signal equations
discussed in the paper. S is the raw signal obtained experimentally. The x, y, and z
variables are the fractional contributions of a peptide’s total intensity, I, and represent
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the percentages of a peptide’s distribution existing as a monoisotopic peak,
interference with the heavier mass-labeled peptide, or interference with the lighter
mass-labeled peptide, respectively. Equations can be produced for peptides
experiencing no interference simply by setting y and z equal to 0.
Supplemental Figure 4: Rearrangement of reporter ion raw signal equations into actual
intensity equations using Mathcad. Only two reporter ions interfere with one another, d9
and d12, simplifying the rearranged equations greatly.
Supplemental Figure 5: Illustration highlighting the iDiLeu (a) internal standard and (b)
one-run quantitation techniques. Descriptions of both techniques are discussed within
the text of the article.
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Supplemental Figure 1:
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Supplemental Figure 4:
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Supplemental Figure 5:
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