Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling

Abstract

Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including alpha-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.

Figure 1

(A) A representative nano-LC/MS/MS base peak chromatogram, showing the detection of the peptide ions across the 40 min gradient separation. (B) MS/MS sequencing data of a peptide from uromodulin, identified in the eluted fraction of a bladder-tumor-bearing patient urine sample.

Figure 2

Reproducibility of nano-LC/MS/MS of Con A-captured fractions from a human urine sample. The retention times of a +2 charged precursor mass of YFIDFVAR from the protein kinnogen-1 from three independent runs were measured to be 22.84, 22.73, and 22.83 min.

Figure 3

Subcellular location of proteins identified in naturally micturated urine samples using a Con A lectin column and MS/MS analysis. A total of 186 urinary proteins were identified.