Multiple Reaction Monitoring of Multiple Low-Abundance Transcription Factors in Whole Lung Cancer Cell Lysates

Lung
cancer-related transcription factors (TFs) were identified
by integrating previously reported genomic, transcriptomic, and proteomic
data and were quantified by multiple reaction monitoring (MRM) in
various cell lines. All experiments were performed without affinity
depletion or subfractionation of cell lysates. Since the target proteins
were expected to be present in low abundance, we experimentally optimized
MRM transition parameters with chemically synthesized peptides. Quantitation
was based on stable isotope-labeled standard peptides (SIS peptides).
Out of 288 MRM measurements (36 peptides representing 28 TFs ×
8 cell lines), 241 were successfully obtained within a quantitation
limit of 15 amol, 221 measurements (91.7%) showed coefficients of
variation (CVs) of ≤20%, and 149 (61.8%) showed CVs of ≤10%,
quantifying as low as 19.4 amol/μg protein for STAT2 with a
CV of 6.3% in an A549 cell. Comparisons between MRM measurements and
levels of the corresponding mRNAs revealed linear, nonlinear, or no
relationship between protein and mRNA levels, indicating the need
for an MRM assay. An integrative analysis of MRM and gene expression
profiles from doxorubicin-resistant H69AR and sensitive H69 cells
further showed that 14 differentially expressed TFs, such as STAT1
and SMAD4, regulated genes associated with drug resistance and cell
differentiation-related processes. Thus, the analytical performance
of MRM for the quantitation of low abundance TFs suggests its usefulness
for biological application.