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Abstract Introduction
Prostate cancer (PCa) is one of the most prevalent cancers in men. Serum PSA levels are used for (early) diagnosis of PCa, but the performance of this medical test is poor, with false positive rates of 56-73% [1]. Therefore, there is a need for better tests with improved clinical performance for early detection of PCa. PSA is a glycoprotein and altered glycosylation patterns, specifically α2,3-linked sialic acids on urinary PSA, were shown to be associated with PCa [2]. Therefore, inclusion of α2,3-linked sialylated glycoforms of PSA might improve clinical performance. Here, we develop an LC-MRM-MS method for quantitation of PSA and its glycoforms using HILIC to enable sialic acid linkage isomer separation.
Methods
An Agilent 1290 infinity LC system coupled to an Agilent 6495 QQQ-MS was used to develop a targeted LC-MRM-MS method for peptides and glycopeptides from PSA. Commercially available PSA isolated from human seminal fluid was reduced, alkylated and proteolytically digested by either trypsin or argC. MRM-transitions were developed and optimized for two peptides and the isomeric glycopeptides with glycan composition HexNAc4Hex5Fuc1Sia2. Three different HILIC columns were evaluated for their separation of sialic acid isomers and the separation conditions (buffer type, strength and pH) were optimized. Digestion efficiency of PSA was evaluated to achieve test robustness and improve analytical sensitivity.
Results
Two peptides were selected for protein quantitation, namely FLRPGDDSHDLMLLR and LSEPAELTDAVK for trypsin and FLRPGDDSHDLMLLR and KWIKDTIVANP for argC. For these peptides, three transitions were selected; for glycopeptides two oxonium ions (m/z 274, sialic acid – H2O, and m/z 366, HexNAcHex) were selected as fragment ions. Best separation of glycopeptide linkage isomers was achieved using a Waters BEH amide column, with optimal solvent conditions of solvent A: 10 mM AF pH 4.4 in water, and solvent B: 90% acetonitrile in 10 mM AF pH 4.4. In an LC gradient of 8 min, glycopeptides with two α2,3-linked sialic acids eluted at 6.0 min, while glycopeptides with two α2,6-linked sialic acids eluted at 6.2 min. Stable digestion of PSA, a prerequisite for true quantitative results, was reached within 30 minutes for trypsin, and within 3 hours for argC.
Conclusions
The here developed HILIC-based LC-MS method for the separation of PSA glycopeptide linkage isomers, specifically α2,3- and α2,6-linked sialic acids, demonstrates the feasibility of quantifying individual glycopeptide isomers and thus specific glycoforms of PSA. While further method development is required, the focus on specific glycoforms using a targeted MS approach as outlined here, provides the granularity necessary for the development of molecular tests for detection and monitoring of prostate cancer in this era of precision medicine.
[1] Lilja et al. Nat Rev Cancer, 2008. 8: p. 268-78.
[2] Yoneyama et al. Biochem Biophys Res Commun, 2014. 448: p. 390-6. |
= Discovery stage.
= Translation stage.
= Clinically available.
