Identification of Differentially Expressed Proteins in Direct Expressed Prostatic Secretions of Men with Organ-confined Versus Extracapsular Prostate Cancer

Abstract

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n ‫؍‬ 8) or organ-confined (n ‫؍‬ 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n ‫؍‬ 5) versus patients with no evidence of recurrence upon follow-up (n ‫؍‬ 10). Lastly, we performed proof-of-concept SRM-MSbased relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS. Molecular & Cellular

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies

“…As indicated by many recent articles [14][15][16][17][18][19][20][21][22][23], and extensive reviews [3,24], identification of differentially expressed proteins has commonly been achieved through iTRAQbased studies with multidimensional LC-MS/MS. Additional MSbased methods have also been developed using capillary electrophoresis [24] despite the lower mass loading capacity of the capillary columns compared to conventional LC columns [25,26].…”

Multi-omics biomarker pipeline reveals elevated levels of protein-glutamine gamma-glutamyltransferase 4 in seminal plasma of prostate cancer patients

“…TGM4 was previously suggested as a prostate cancer biomarker, but results were inconsistent and revealed either significant over-expression 65 or under-expression [66][67][68][69] of TGM4 in PCa versus benign disease, or inconclusive results with the opposite directions based on different assays 70 . TGM4 was found down-regulated 1.7-fold in urinary extracellular vesicles of PCa and had AUC 0.58 to diagnose PCa on biopsy 66 .…”

Prostasomes as a source of diagnostic biomarkers for prostate cancer

“…The presence of prostasomes in EV fractions isolated from urine was confirmed by detection of prostate-specific proteins, including prostate-specific membrane antigen (PSMA), prostatic acid phosphatase, and prostate transglutaminase (67,(70)(71)(72)(73). One advantage of collecting EVs from urine, as compared with blood, is that such isolates are more enriched in prostasomes relative to other constituents, although tissues within the urogenital system other than the prostate, including the kidney (74) and bladder (75), also contribute EVs to urine.…”