A Rapid LC-MS/MS-PRM Assay for Serologic Quantification of Sialylated O-HPX Glycoforms in Patients with Liver Fibrosis

Abstract

Development of high throughput robust methods is a prerequisite for a successful clinical use of LC-MS/MS assays. In earlier studies, we reported that nLC-MS/MS measurement of the O-glycoforms of HPX is an indicator of liver fibrosis. In this study, we show that a microflow LC-MS/MS method using a single column setup for capture of the analytes, desalting, fast gradient elution, and on-line mass spectrometry measurements, is robust, substantially faster, and even more sensitive than our nLC setup. We demonstrate applicability of the workflow on the quantification of the O-HPX glycoforms in unfractionated serum samples of control and liver disease patients. The assay requires microliter volumes of serum samples, and the platform is amenable to one hundred sample injections per day, providing a valuable tool for biomarker validation and screening studies.

Glycopeptides with Sialyl Lewis Antigen in Serum Haptoglobin as Candidate Biomarkers for Nonalcoholic Steatohepatitis Hepatocellular Carcinoma Using a Higher-Energy Collision-Induced Dissociation Parallel Reaction Monitoring-Mass Spectrometry Method

“… 43 , 44 In addition, the PRM-MS can provide a wide dynamic range for quantitative analysis for target precursors. 41 , 45 These aspects of PRM allow accurate quantification of a larger number of target precursors in an anticipated elution time interval.…”

“…The significant advantage of PRM-MS is that it is a targeted quantitative analysis as compared to DDA-MS, which scans over the entire mass range. The increased sensitivity of PRM is due to its targeted nature where the mass spectrometer can spend longer times collecting the target peptide ions, which is termed dwell time, compared to DDA-MS. Also, PRM allows all fragment ions of a predefined precursor to be measured in parallel, where a full MS2 product ion spectrum of a specified precursor ion can be acquired, and all detectable product ions can be simultaneously monitored at high accuracy and resolving power. , In addition, the PRM-MS can provide a wide dynamic range for quantitative analysis for target precursors. , These aspects of PRM allow accurate quantification of a larger number of target precursors in an anticipated elution time interval.…”

A multiplexed microflow LC–MS/MS-PRM assay for serologic quantification of IgG N- and HPX O- glycoforms in liver fibrosis

“…RSLCnano chromatography system and a Q-Exactive HF Mass Spectrometer (Thermo) with a nanospray source housing a multinozzle M3 emitter spray tip (Newomics, Berkeley, CA, USA) 21 . The samples were loaded onto a PepMap C18 Cartridge (1 mm x 5 mm) and desalted using a sample loading pump at 10 μl/min flow rate 0-1 min with 0.1% formic acid in water, following which the peptides were eluted at a 5 μl/min flowrate.…”

“…Table 1 shows the peptide glycoforms analyzed in this study, MS data collection parameters, and transitions used for the quantitation. We analyzed simultaneously selected IgG N-glycoforms of the peptide EEQYNSTYR (G0, G0N, G0FN, G0F, G1, G1N, G1FN, G1F), and mono-and di-sialyated HPX O-glycopeptide TPLPPTSAHGNVAEGETKPDPVTER HexNAc(1) Hex(1)Neu5Ac(1), HexNAc(1)Hex(1)Neu5Ac(2) 11,20,21 . The respective glycan structures are shown in Table 1.…”

“…Here we build on our earlier studies 20,21 to introduce a multiplexed microflow LC-MS/MS-PRM (mLC-MS/ MS) assay for simultaneous quantification of the N-glycoforms of IgG and sialylated O-glycoforms of HPX. Our Sample processing.…”