posted on 2020-03-17, 21:29authored byYuri E.
M. van der Burgt, Kasper M. Siliakus, Christa M. Cobbaert, L. Renee Ruhaak
Elevated serum prostate-specific
antigen (PSA) levels in body fluids
may indicate prostate cancer (PCa), but it is noted that the clinical
performance is rather poor. Specificity and sensitivity values of
20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in
overdiagnosis and unnecessary interventions. Previous exploratory
studies have indicated that the glycosylation of PSA potentially leads
to improved PCa diagnosis based on qualitative analyses. However,
the applied methods are not suited for a quantitative evaluation or
implementation in a medical laboratory. Therefore, in this proof-of-principle
study, we have evaluated the use of hydrophilic interaction liquid
chromatography (HILIC) in combination with targeted quantitative mass
spectrometry for the sialic acid linkage-specific analysis of PSA
glyco-proteoforms based on either trypsin or ArgC peptides. The efficiency
of PSA proteolysis was optimized as well as the glycopeptide separation
conditions (buffer type, strength, and pH). The HILIC-based analysis
of PSA glyco-proteoforms presented here has the potential for the
clinical validation of patient cohorts. The method shows the feasibility
of the use of a HILIC stationary phase for the separation of isomeric
glycopeptides to detect specific glyco-proteoforms. This is the first
step toward the development and evaluation of PSA glyco-proteoforms
for use in a clinical chemistry setting aiming for improved PCa diagnosis
or screening.