Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example

Abstract

LC-MS and immunoassay can detect protein biomarkers. Immunoassays are more commonly used but can potentially be outperformed by LC-MS. These techniques have limitations including the necessity to generate separate calibration curves for each biomarker. We present a rapid mass spectrometry-based assay utilising a universal calibration curve. For the first time we analyse clinical samples using the HeavyPeptide IGNIS kit which establishes a 6-point calibration curve and determines the biomarker concentration in a single LC-MS acquisition. IGNIS was tested using apolipoprotein F (APO-F), a potential biomarker for non-alcoholic fatty liver disease (NAFLD). Human serum and IGNIS prime peptides were digested and the IGNIS assay was used to quantify APO-F in clinical samples. Digestion of IGNIS prime peptides was optimised using trypsin and SMART Digest™. IGNIS was 9 times faster than the conventional LC-MS method for determining the concentration of APO-F in serum. APO-F decreased across NAFLD stages. Inter/intra-day variation and stability post sample preparation for one of the peptides was ≤13% coefficient of variation (CV). SMART Digest™ enabled complete digestion in 30 minutes compared to 24 hours using in-solution trypsin digestion. We have optimised the IGNIS kit to quantify APO-F as a NAFLD biomarker in serum using a single LC-MS acquisition.

Transcriptional and Epigenetic Alterations in the Progression of Non-Alcoholic Fatty Liver Disease and Biomarkers Helping to Diagnose Non-Alcoholic Steatohepatitis

“…Similarly, Liu et al found that hepatic ApoF mRNA levels were decreased by high fat, cholesterol-enriched diets, and ApoF was subject to negative regulation by agonist-activated LXR or PPARα nuclear receptors binding to a regulatory element ~1900 bases 5′ to the ApoF promoter [ 44 ]. The concentration of protein ApoF in serum has also been quantified to decrease across NAFLD stages in a previous study, which was consistent with our study [ 45 ]. In addition, recent studies have revealed that BICC1 might be involved in the immune response in the tumor microenvironment by affecting immune cells, especially macrophages, and the overexpression of BICC1 was closely related to the poor prognosis in tumors such as gastric cancer and oral cancer, as well as multiple functional pathways such as focal adhesion and ECM-receptor interaction, which were also enriched in our study [ 46 , 47 ].…”

Exploring the Potential of Iminosugars as Antivirals for Crimean-Congo Haemorrhagic Fever Virus, Using the Surrogate Hazara Virus: Liquid-Chromatography-Based Mapping of Viral N-Glycosylation and In Vitro Antiviral Assays

“…In addition to searching with trypsin as the protease, searches were also performed with the enzyme parameter set to ‘None’. Parallel reaction monitoring (PRM) was also carried out using the Skyline software version 18 (MacCoss Lab Software, Seattle, USA) and MS settings as previously described [ 32 ]. The HAZV peptides that were targeted were R.LADFFIDT N SSQ C YDEILVK.K (88, 107) (N97 deamidated, C101 alkylated; 1190.0567++ or 793.7069+++) and K.NDGPGDHITF CN GSVVTK.I (334, 351) (N346 deamidated, C345 alkylated; 959.9336++ or 640.2915+++).…”

Modified ELISA for Ultrasensitive Diagnosis

“…The potential applications of this method depend on the specificity and strength of the antigen–antibody reaction being evaluated. Due to the limitation of ELISAs requiring the use of a purified antigen and an extraordinarily functional antibody, some researchers strongly propose that other methods be used, such as liquid chromatography–mass spectrometry (LC-MS) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ] and various lab-on-a-chip methods [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. A recent trend in LC-MS development for diagnosis is to focus on the detection of drugs and biomarkers, including peptides and steroids, owing to difficulties in preparing suitable antibodies against these small molecules, whereas lab-on-a-chip–based platforms are thought to offer easier sample preparation, chemical manipulation and reaction, and high throughput using various novel detection techniques with, sometimes, label-free detection.…”