Abstract 1835: In-depth quantitative analysis of protein glycoforms in human prostate cancer plasma
Authors: Totten Sarah M. , Kulloli Majlinda , Tanimoto Cheylene , Brooks James D. , Pitteri Sharon J. 
Published: August 2015

In this study, a novel analytical workflow was used to interrogate differences in the expressed levels of proteins and glycoproteins detectable in plasma in men with benign prostate hyperplasia versus those diagnosed with low- to high-grade prostate cancer. Currently, the use of early detection techniques such as measuring the level of prostate-specific antigen (PSA) in the blood lead to over-diagnosis and over-treatment in men with slow-growing or none-threatening prostate tumors, making evident the need for a biomarker that is indicative of cancer aggressiveness. Using the in-depth chromatographic separations and LC-MS/MS methods described below, circulating levels of moderate- to low-abundant proteins and their glycoforms were measured, providing the opportunity to observe alterations in the patterns of glycosylation - a posttranslational modification known to be aberrant with tumorigenesis - in those who developed more aggressive prostate cancers. We hypothesize that alterations in protein glycosylation with the development of prostate cancer are measurable in the blood, and, once identified, quantified, and verified, can be used as early molecular indicators of prostate cancer grade, aggressiveness, and overall risk.

Twenty blinded plasma samples collected from men with benign, low-, or high-grade prostate cancer biopsies, along with a pooled plasma sample from healthy men for reference, were first depleted of the fourteen most abundant proteins, as these have been extensively studied. The depleted prostate samples and the reference pooled plasma were respectively isotopically labeled with 13C and 12C acrylamide for quantitation, and combined. Multi-lectin affinity chromatography was used to target various glycoforms, specifically those containing core-fucosylated (Aleuria aurantia lectin or AAL), or branched complex-type glycans (Phaseolus vulgaris Leucoagglutinin and Phaseolus vulgaris Erythroagglutinin lectin, or PHA-L/E). For further sample decomplexing, each lectin fraction was subjected to reversed-phase fractionation prior to tryptic digestion. Peptides were analyzed by nanoflow liquid chromatography on a C18 column coupled to a LTQ-Orbitrap Elite mass spectrometer. Alterations in the glycoprotein profiles were then correlated to disease status as determined by prostate biopsies.

Using this approach, an average of 270 proteins were identified in the protein fraction bound to AAL, and 285 proteins were identified in the fraction bound to PHA-L/E, as well as the fraction of proteins unbound to either lectin. The preliminary data demonstrate significant differences between case and control samples in the types and levels of the targeted glycoforms present in each lectin fraction in comparison to the reference plasma, suggesting there are measurable alterations in the glycosylation of several plasma proteins between men with benign, indolent and aggressive prostate tumors.

Citation Format: Sarah M. Totten, Majlinda Kulloli, Cheylene Tanimoto, James D. Brooks, Sharon J. Pitteri. In-depth quantitative analysis of protein glycoforms in human prostate cancer plasma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2015-1835