scholarly journals Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Hong Shu ◽  
Lei Zhang ◽  
Yiwei Chen ◽  
Yijie Guo ◽  
Limin Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Cirrhosis ◽  
Acute Phase Response ◽  
Lectin Binding ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free ◽  
Comprehensive Understanding ◽  
Lectin Blot ◽  
Free Quantification

Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS316TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS316TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on 18O/16O C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.

scholarly journals Multiplexed Proteomic Approach for Identification of Serum Biomarkers in Hepatocellular Carcinoma Patients with Normal AFP

2020 ◽  
Vol 9 (2) ◽  
pp. 323
Author(s):  
Young-Sun Lee ◽  
Eunjung Ko ◽  
Eileen L. Yoon ◽  
Young Kul Jung ◽  
Ji Hoon Kim ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Cirrhosis ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Hepatoma Cell ◽  
Multiple Reaction Monitoring ◽  
Serum Levels ◽  
Human Hepatoma Cell ◽  
Hepatoma Cell Lines ◽  
Proteomic Approach

Alpha fetoprotein (AFP) has been used as a serologic indicator of hepatocellular carcinoma (HCC). We aimed to identify an HCC-specific serum biomarker for diagnosis using a multiplexed proteomic technique in HCC patients with normal AFP levels. A total of 152 patients were included from Guro Hospital, Korea University. Among 267 identified proteins, 28 and 86 proteins showed at least a two-fold elevation or reduction in expression, respectively. Multiple reaction monitoring (MRM) analysis of 41 proteins revealed 10 proteins were differentially expressed in patients with liver cirrhosis and HCC patients with normal AFP. A combination of tripartite motif22 (Trim22), seprase, and bone morphogenetic protein1 had an area under receiver operating characteristic of 0.957 for HCC diagnosis. Real-time PCR and western blot analysis of the paired tumor/non-tumor liver tissue in HCC revealed a reduced expression of Trim22 in the tumor tissue. Also, serum levels of Trim22 were significantly reduced in HCC patients with normal AFP compared to those with liver cirrhosis (p = 0.032). Inhibition of Trim22 increased cellular proliferation in human hepatoma cell lines, whereas overexpression of Trim22 decreased cellular proliferation in hepatoma cell lines. In conclusion, the combination of three serum markers improved the chance of diagnosing HCC. MRM-based quantification of the serum protein in patients with normal AFP provides the potential for early diagnosis of HCC.

scholarly journals Clinical Assay for AFP-L3 by Using Multiple Reaction Monitoring–Mass Spectrometry for Diagnosing Hepatocellular Carcinoma

2018 ◽  
Vol 64 (8) ◽  
pp. 1230-1238 ◽  
Author(s):  
Hyunsoo Kim ◽  
Areum Sohn ◽  
Injoon Yeo ◽  
Su Jong Yu ◽  
Jung-Hwan Yoon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatocellular Carcinoma ◽  
Multiple Reaction Monitoring ◽  
False Negative ◽  
Internal Standard ◽  
Analytical Sensitivity ◽  
Reaction Monitoring ◽  
Clinical Implementation ◽  
Clinical Value ◽  
Serum Samples

Abstract BACKGROUND Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA). However, LiBA does not always reflect AFP-L3 concentrations because of its low analytical sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring–mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum. METHODS The assay entailed the addition of a stable isotope-labeled internal standard protein analog, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using L. culinaris agglutinin lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation. RESULTS The lower limit of quantification of the MRM-MS assay (0.051 ng/mL) for AFP-L3 was less than that of LiBA (0.300 ng/mL). Thus, AFP-L3, which was not observed by LiBA in HCC samples (n = 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more analytical sensitive platform. The method was validated, meeting all the criteria in integrated multinational guidelines. CONCLUSIONS Because of the lower incidence of false-negative findings, the MRM-MS assay is more suitable than LiBA for early detection of HCC.

scholarly journals Label-Free Absolute Quantitation of Oligosaccharides Using Multiple Reaction Monitoring

2014 ◽  
Vol 86 (5) ◽  
pp. 2640-2647 ◽  
Author(s):  
Qiuting Hong ◽  
L. Renee Ruhaak ◽  
Sarah M. Totten ◽  
Jennifer T. Smilowitz ◽  
J. Bruce German ◽  
...  
Keyword(s):  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free ◽  
Absolute Quantitation

scholarly journals Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria

2020 ◽  
Vol 21 (14) ◽  
pp. 4924 ◽  
Author(s):  
Anna A. Toymentseva ◽  
Anastasia O. Koryagina ◽  
Alexander V. Laikov ◽  
Margarita R. Sharipova
Keyword(s):  
Serine Proteases ◽  
Multiple Reaction Monitoring ◽  
Expression System ◽  
Complex Mixture ◽  
Rapid Identification ◽  
Glutamyl Endopeptidase ◽  
Reaction Monitoring ◽  
Label Free ◽  
Fragment Ions ◽  
Specific Proteins

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

scholarly journals Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments

Biology ◽  
2014 ◽  
Vol 3 (2) ◽  
pp. 383-402 ◽  
Author(s):  
Lisa Chung ◽  
Christopher Colangelo ◽  
Hongyu Zhao
Keyword(s):  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free

scholarly journals Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring

2011 ◽  
Vol 10 (9) ◽  
pp. 4005-4017 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Lynn A. Beer ◽  
Kurt T. Barnhart ◽  
David W. Speicher
Keyword(s):  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free ◽  
Serological Biomarkers

Quantitative analysis of genetically modified soya using multiple reaction monitoring mass spectrometry with endogenous peptides as internal standards

2018 ◽  
Vol 25 (1) ◽  
pp. 50-57 ◽  
Author(s):  
Shobha Devi ◽  
Yi-Cheng Lin ◽  
Yen-Peng Ho
Keyword(s):  
Mass Spectrometry ◽  
Genetically Modified ◽  
Multiple Reaction Monitoring ◽  
Linear Regression Analysis ◽  
Exchange Chromatography ◽  
Protein Quantification ◽  
Reaction Monitoring ◽  
Label Free ◽  
Internal Standards ◽  
Phosphate Synthase

A simple label-free method was developed for the quantification of the herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase using multiple reaction monitoring liquid chromatography–mass spectrometry. Sample pretreatment procedures including ion exchange chromatography and CaCl2 precipitation were used to purify the 5-enolpyruvylshikimate-3-phosphate synthase protein. Quantification of various percentages of genetically modified soya (0.5–100%) was performed by selecting suitable endogenous soybean peptides as internal standards. Results indicated that Gly P (QGDVFVVPR) and Lec P (LQLNK) are useful internal standards for the quantification of low and high percentages of genetically modified soya, respectively. Linear regression analysis of both calibration curves yielded good linearity with R2 of 0.99. This approach is a convenient and accurate quantification method for genetically modified soya at a level as low as 0.5% (less than the current EU threshold for labeling genetically modified soya).

scholarly journals Analysis of Serum Paraoxonase 1 Using Mass Spectrometry and Lectin Immunoassay in Patients With Alpha-Fetoprotein Negative Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Xinyi Cao ◽  
Zhao Cao ◽  
Yuyin Shao ◽  
Chao Liu ◽  
Guoquan Yan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatocellular Carcinoma ◽  
Inflammatory Diseases ◽  
Alpha Fetoprotein ◽  
Paraoxonase 1 ◽  
Label Free ◽  
The Difference ◽  
Validation Set ◽  
Serum Paraoxonase ◽  
Free Quantification

The diagnosis of AFP (alpha-fetoprotein)-negative HCC (hepatocellular carcinoma) mostly relies on imaging and pathological examinations, and it lacks valuable and practical markers. Protein N-glycosylation is a crucial post-translation modifying process related to many biological functions in an organism. Alteration of N-glycosylation correlates with inflammatory diseases and infectious diseases including hepatocellular carcinoma. Here, serum N-linked intact glycopeptides with molecular weight (MW) of 40–55 kDa were analyzed in a discovery set (n = 40) including AFP-negative HCC and liver cirrhosis (LC) patients using label-free quantification methodology. Quantitative lens culinaris agglutin (LCA) ELISA was further used to confirm the difference of glycosylation on serum PON1 in liver diseases (n = 56). Then, the alteration of site-specific intact N-glycopeptides of PON1 was comprehensively assessed by using Immunoprecipitation (IP) and mass spectrometry based 16O/18O C-terminal labeling quantification method to distinguish AFP-negative HCC from LC patients in a validation set (n = 64). Totally 195 glycopeptides were identified using a dedicated search engine pGlyco. Among them, glycopeptides from APOH, HPT/HPTR, and PON1 were significantly changed in AFP-negative HCC as compared to LC. In addition, the reactivity of PON1 with LCA in HCC patients with negative AFP was significantly elevated than that in cirrhosis patients. The two glycopeptides HAN253WTLTPLK (H5N4S2) and (H5N4S1) corresponding to PON1 were significantly increased in AFP-negative HCC patients, as compared with LC patients. Variations in PON1 glycosylation may be associated with AFP-negative HCC and might be helpful to serve as potential glycomic-based biomarkers to distinguish AFP-negative HCC from cirrhosis.

Sign in / Sign up

Export Citation Format

Share Document