IMAC Fractionation in Combination with LC–MS Reveals H2B and NIF‑1 Peptides As Potential Bladder Cancer Biomarkers

Improvement
in bladder cancer (BC) management requires more effective
diagnosis and prognosis of disease recurrence and progression. Urinary
biomarkers attract special interest because of the noninvasive means
of urine collection. Proteomic analysis of urine entails the adoption
of a fractionation methodology to reduce sample complexity. In this
study, we applied immobilized metal affinity chromatography in combination
with high-resolution LC–MS/MS for the discovery of native urinary
peptides potentially associated with BC aggressiveness. This approach
was employed toward urine samples from patients with invasive BC,
noninvasive BC, and benign urogenital diseases. A total of 1845 peptides
were identified, corresponding to a total of 638 precursor proteins.
Specific enrichment for proteins involved in nucleosome assembly and
for zinc-finger transcription factors was observed. The differential
expression of two candidate biomarkers, histone H2B and NIF-1 (zinc
finger 335) in BC, was verified in independent sets of urine samples
by ELISA and by immunohistochemical analysis of BC tissue. The results
collectively support changes in the expression of both of these proteins
with tumor progression, suggesting their potential role as markers
for discriminating BC stages. In addition, the data indicate a possible
involvement of NIF-1 in BC progression, likely as a suppressor and
through interactions with Sox9 and HoxA1.