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Supplemental Information
Absolute quantification of prion protein (90-231) using stable isotope-labeled
chymotryptic peptide standards in a LC-MRM AQUA workflow
Robert M Sturm1, Gloria Kreitinger1, Clarissa Booth2, Lloyd Smith1, Joel Pedersen2,3,*,
Lingjun Li1,4,*
1Department
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2Molecular
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3Department
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4School
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of Chemistry, University of Wisconsin-Madison
and Environmental Toxicology Center, University of Wisconsin-Madison
of Soil Science, University of Wisconsin-Madison
of Pharmacy, University of Wisconsin-Madison
Supplemental Table 1:
Potential MRM target peptides from chymotrypsin, Glu-C and thermolysin in silico
digestions using Protein Prospector MS-Digest algorithm. All peptides lack chemically
reactive peptides Met and Cys and are present in the proteinase K-resistant core of
PrPTSE.
Supplemental Figure 1:
Oxidation of bovine rPrP peptide VVEQMCITQYQR and synthetic peptide
VVEQMCITTQYQK. Bovine rPrP was digested with trypsin and analyzed by nanoLCMS/MS on a Bruker amaZon ETD ion trap. Both non-oxidized (a) and oxidized (b) forms
of VVEQMCITQYQR were detected in analysis. (c) Non-oxidized and oxidized forms of
VVEQMCTTQYQK. Red Cys residue indicates that cysteine has carbamidomethylation
modification. Green Met residue in MS/MS spectra indicates methionine sulfoxide
detected.
Supplemental Figure 2:
Extracted ion chromatograms (EIC) of YRPVDQY and RPVDQY produced from bovine
and hamster rPrP chymotryptic digestions. Red trace shows z = +1 EIC. Blue trace
shows z = +2 EIC. Green trace shows z = +3 EIC. Digestion was analyzed on a Bruker
amaZon ETD ion trap.
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Supplemental Figure 3:
MALDI autoflex III TOF/TOF mass spectra of bovine rPrP chymotryptic digestions
collected during digestion time optimization experiment. After 1 h the predominant ion is
YRPVDQY (m/z 940.4+1). As digestion continues, YRPVDQY remains dominant ion, but
RPVDQY (m/z 777.4+1) is observed at lower intensity.
Supplemental Figure 4:
Digestion of SIS peptide YRPVDQY with chymotrypsin. After 9.5 h digestion, MRM
transitions b6 and b5 transitions were monitored for YRPVDQY and RPVDQY,
respectively. RPVDQY b5 transition is 0.09% as intense as the YRPVDQY b6 transition.
These data suggest that the N-terminal Tyr of YRPVDQY may hydrolyze during
digestion to produce RPVDQY.
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Supplemental Table 1:
Enzyme
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hamster target peptide (m/z +1, +2, +3)
bovine target peptide (m/z +1, +2, +3)
Chymotrypsin YRPVDQY (940.45, 470.73, 314.16)
RPVDQY (777.39, 389.20, 259.80)
QKESQAY (853.40, 427.21, 285.14)
GQGGGTHNQW (1041.45, 521.23, 347.82)
YRPVDQY (940.45, 470.73, 314.16)
RPVDQY (777.38, 389.20, 259.80)
QRESQAY (881.41, 441.21, 294.48)
GQGGTHGQW (927.41, 464.21, 309.81)
Glu-C
QYNNQNNFVHD (1392.59, 696.80, 464.87)
QYNNQNNFVHD (1392.59, 696.80, 464.87)
Thermolysin
WNKPSKPKTN (1199.65, 600.33, 400.56)
VTTTTKGEN (950.48, 475.74, 317.50)
GQGGGTHNQ (855.37, 428.19, 285.80)
WNKPSKPKTN (1199.65, 600.33, 400.56)
VTTTTKGEN (950.48, 475.74, 317.50)
GQGGTHGQ (741.33, 371.17, 247.78)
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Supplemental Figure 1:
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Supplemental Figure 2:
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Supplemental Figure 3:
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Supplemental Figure 4:
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