Kushnir, Mark M.

Abstract Background Alpha fetoprotein (AFP) is a circulating tumor marker utilized clinically to detect liver cirrhosis, liver cancer and testicular seminomas. AFP molecule contains one single N-glycosylation site, with highly variable glycan structures, resulting in diverse AFP glycoforms in circulating blood. The core fucosylation of AFP (AFP-L3) is a clinical biomarker to monitor and predict risks of liver cancer progression and recurrence. Clinical testing for AFP-L3 utilizes lectin-binding and gel-shift electrophoresis. We developed a novel method to measure AFP glycoforms, based on immune-enrichment and liquid chromatography with high resolution mass spectrometry (LC-HRMS). Methods Immune enrichment of AFP was achieved by anti-AFP antibody, immobilized to magnetic beads. After binding and enrichment, the magnetic beads were washed, AFP was eluted, and the samples were analyzed by LC-HRMS (Exploris 480 MS and Vanquish UHPLC, Thermo Fisher Scientific), using full scan, positive polarity acquisition mode. Chromatographic separation was performed on PLRP column (Agilent); flow rate 200 µL/min. The HRMS data were processed and deconvoluted with intact mass analysis workflow in Biopharma Finder 5.1 (Thermo Fisher Scientific) to derive the intact AFP masses. The method’s sensitivity and imprecision were assessed by replicate analysis of serial diluted patient serum samples. AFP and its associated glycoforms were matched and identified with an in-house developed bioinformatics workflow. The deconvoluted intact masses and the sum intensity of detected charged species for each AFP glycoform were used to quantify relative abundance of AFP glycoforms. Results The method specificity for AFP was confirmed by bottom-up analysis after immune-enrichment with observed peptide sequence coverage over 35%. The recovery of the immune enrichment was shown to be independent of the AFP glycoform compositions. The identified AFP glycoforms and their relative abundance were consistent among replicates between AFP concentration 100 ng/mL and 5000 ng/mL. Analysis of authentic serum specimens from patients with different chronic liver diseases and liver cancer (n=102), revealed multiple complex biantennary branched AFP glycan modifications, with different numbers of terminal sialic acid and core fucose. Four AFP glycoforms were identified in over 60% patient samples with elevated AFP concentrations. Although occurring consistently, relative abundance of these AFP glycoforms varied significantly among patients. Assessment of the AFP fucosylation and sialylation features suggested that the two are orthogonal processes. Clinical correlations suggested that fewer terminal sialic acids were associated with impaired liver functions, while the fucosylation was linked to inflammatory responses and presence of liver malignancy. Strong linear correlation (Spearman’s rs=0.952, N=35) was observed between AFP-L3% quantitated by the LC-HRMS method, and AFP-L3% determined by the lectin-binding based isotachophoresis assay (Wako Diagnostics). Discrepancies between the two methods were observed in specimens containing < 400 ng/mL of AFP, in which the LC-HRMS method detected only a single predominant glycoform. Conclusions We developed and evaluated the LC-HRMS method for profiling circulating AFP glycoforms in patients with chronic liver diseases and liver cancer. The detected AFP glycoforms and their relative abundance were used to establish glycan features, which can be further linked to cancer development, impaired liver function, and inflammation.

AbstractBACKGROUNDFor many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope–labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays.CONTENTThe Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials—in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry—is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.