Authors:
Susan C Trow1, Keling Dong3, Marjorie S Minkoff3, Christie L Hunter3, M Christine
Zink1, and David R Graham1, 2
1. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, 733 N.
Broadway, Baltimore, MD, 21205
2. Department of Medicine, Division of Cardiology, Johns Hopkins School of Medicine,
607 Mason F. Lrd Bld., Ctr Twr, 5200 Eastern Avenue, Baltimore, MD, 21224
3. AB Sciex, 353 Hatch Drive, Foster City, CA 94404
Title:
Unambiguous Detection and Quantitation of Full-length Thioredoxin (TRX) and
Truncated Thioredoxin (TRX80) in Complex Samples
Introduction
Thioredoxin (TRX) is a 12 kDa ubiquitously expressed redox cycling enzyme key to
maintaining a reducing environment in all cell types. Oxidative stress can lead to the
upregulation and secretion of thioredoxin, which occurs in many disease states. A 10
kDa cleavage product of secreted thioredoxin, TRX80, has also been identified and may
have unique inflammatory properties. To clearly define a role for thioredoxin in health
and disease, the ability to monitor both forms in a highly quantitative way within a
sample is necessary. These forms are difficult to differentiate and quantitate by
traditional approaches. We have developed a mass spectrometry approach for complex
protein samples in which digestion with cyanogen bromide results in unique identifying
peptides for both TRX and TRX80 that can be quantitated. This approach can overcome
limitations of antibody-based detection methods imposed by unique properties of TRX
and TRX80.
Method (limit 120 words)
Samples of undepleted plasma and recombinant TRX/TRX80 were digested with
cyanogen bromide. Limits of detection (LOD) for the two unique peptides spiked into
CNBr digested whole plasma were obtained by MALDI after performing rp-HPLC on a
Tempo LC (AB Sciex™) or fractionation by SCX. Combining these separation methods in a
MudPIT approach was also examined. MRMPilot™ Software (AB Sciex™) has been used
to develop MRM transitions to transfer this approach to a more sensitive assay on a
QqLIT mass spectrometer (QTRAP 5500® system, AB Sciex™), using heavy labeled
standard peptides for absolute quantitation.
Preliminary Data (limit 300 words)
CNBr digestion yields two peptides common to both TRX and TRX80, but also an
additional unique identifying peptide for each, which can not be obtained by tryptic
digestion (3484 and 914 [M+H, respectively]. Both peptide forms have been identified
and verified by MALDI TOF/TOF analysis (4800 MALDI TOF/TOFTM instrument, AB
SciexTM). Diagnostic peaks were detected by MALDI for both TRX and TRX80 when the
CNBr digested recombinant proteins were spiked into CNBr digested plasma, with LODs
of 10 ng in 100ug by rpHPLC and 1 ng in 100ug by SCX. Improved fractionation
strategies are currently under investigation to improve the detection limits, to enable
detection of endogenous TRX/TRX80 using MALDI TOF/TOF analysis. In parallel, an LC
MRM strategy is also being explored to determine the most sensitive, high throughput
strategy for monitoring TRX/TRX80. MRM transitions to both peptide forms are under
development using MRMPilot™ Software. After validating these transitions, the LODs
for TRX/TRX80 in plasma by MRM will be determined using heavy labeled standard
peptides for absolute quantitation.
Novel Aspect
Utilizing CNBr to produce identifying peptides for TRX and TRX80, development of a MS
based assay to quantitate these peptides.