MASS SPECTROMETRIC IDENTIFICATION OF THE HCVcoreAg ISOTYPE IN IMMOBILIZED COMPLEXES

A.L. Kaysheva1,2*, Yu.D. Ivanov1, P.A. Frantsuzov1, N.V. Krohin1, T.I. Pavlova1, V.F. Uchaikin1, V.A. Konev1, O.B. Kovalev1, V.S. Ziborov1, A.I. Archakov1

1IBMC RAMS, Pogodinskaya St. 10, Moscow, 119121 Russia;

2PostgenTech Ltd, Moscow, Russia

*Corresponding author: Anna L. Kaysheva, Pogodinskaya 10, Moscow, 119121, Russia Tel.: (+7) (499) 2463761; fax: (+7) (499) 2450857; E-mail address: kaysheva@gmail.com.

 

Abstract

A method for determination of the isotype of hepatitis C virus core antigen in the serum of patients from central Russia is proposed. The method is based on a combination of specific enrichment and concentration of the HCVcoreAg on the surface of the biosensor chip for an atomic force microscope (AFM chip) with subsequent identification of this antigen by mass spectrometry (MALDI-MS). MALDI-MS analysis allowed to achieve reliable identification of HCVcoreAg in the complexes formed on the AFM chip surface as well as to determine the isotype of this antigen and, finally, to reveal the most frequently identified peptides.

 

According to the WHO, the disease caused by hepatitis C virus (HCV) refers to the social diseases. Today, about 10% of the world population is HCV-infected, including 6 million in Russia (1). The incubation period for HCV ranges from 2 weeks to 6 months, and in most cases is asymptomatic (2), so it is important to diagnose HCV in groups of individuals within the zone of risk of infection. However, early diagnosis of HCV infection is still difficult. This infection often remains undetected until serious liver damage. Timely diagnosis of HCV is complicated not only by the lack of highly sensitive test systems for the early detection of HCV infection, but also by high variability of clinical protein markers – HCVcoreAg and glycoproteins E1 and E2 (5).

The population of the hepatitis C virus is extremely heterogeneous. To date, 6 genotypes (classification by Simmonds (4)) and more than 90 subtypes of HCV (5) are known. Dominance and distribution of HCV genotypes are geographically uneven. For instance, in Europe 1b genotype dominates over 2a, 2b, 2c, 3a genotypes. In the Russian Federation, genotype 1b is identified most often. Virus genotype is clinically important in determining potential response against chemotherapy (6).

Acute hepatitis C becomes chronic in 85% of cases. Since this disease is often asymptomatic (2), it is particularly important to diagnose HCV during the acute phase in groups of individuals, within the zone of risk of infection, and to determine its genotype to select effective chemotherapy.

HCV resistance is conditioned by its ability to replicate with high levels of mutations due to inaccurate reading of HCV RNA polymerase. As a result, there are several immunologically different variants or quasi-species that allow the virus to avoid the immune response (7).

The development of highly sensitive methods for direct MS identification of HCV protein antigens as well as methods of determining their isotypes, are important for medical applications, including the effective chemotherapy and the study of virus drug resistance.

This study aims the development of a method to identify conservative regions of HCVcoreAg. By the example of the population of central Russia, the efficiency of a approach for the HCVcoreAg isotype determination ‑ involving MS identification of HCV marker protein specifically captured onto the AFM chip surface from serum samples – has been demonstrated.

Application of MALDI-MS analysis, without prior affinity enrichment, to identify target proteins in biological material is limited due to the low sensitivity of 10-9 M and high dynamic range of proteins’ concentrations in human serum (14). To obviate this limitation, the detection and identification of proteins of HCV virus particles in human serum was accomplished by using the AFM-MALDI-MS method (3). MS analysis of proteins was performed on the AFM chup surface after the incubation of this chip in serum samples ‑ either positive or negative (by PCR) for the presence of HCV RNA. AFM measurements and biospecific fishing of HCVcoreAg from serum samples onto the surface of AFM chip with immobilized monoclonal antibodies was carried out as described earlier by the group of Professor Yu. D. Ivanov (3). AFM analysis of serum samples was described in detail in our previous study (3). Now, we shall concentrate on the results of MS-identification of protein objects, specifically captured and concentrated on the AFM chip surface from serum samples.

Figure 1 displays a typical MALDI-MS spectrum obtained from the AFM chip surface incubated in HCV-positive serum No. 33. The analysis of this spectrum allowed to identify HCVcoreAg by 12 peptides.

As a result of MALDI-MS analysis of 15 serum samples positive for the presence of HCV, it was identified in 14 serum samples. The revealed isoforms of the HCVcoreAg corresponded to genotype 1b and isolate HC-J2 ‑ Q8V7V3, Q66VB3, O71242, and genotype 2c, isolate HC-J7 – Q68692 (Table 1).

As seen from Table 1, Q8V7V3 isotype of HCVcoreAg was detected in most serum samples (12 of 15 samples). The identified HCVcoreAg isotypes (Q8V7V3, Q66VB3, O71242) mostly correspond to genotype 1b and isolate HC-J2 of HCV, and the homology of the revealed amino acid sequences with Q8V7V3 isotype of the HCVcoreAg made up 89-96%.

It is known from the literature data that the amino acid sequence of the HCVcoreAg is extremely variable. In April 2015, the UniProt KB protein database contained 6,423 amino acid sequences corresponding to HCVcoreAg. It is known that the most conservative regions of HCVcoreAg are: the hydrophobic domain containing 24-39 amino acid residues (aa) as well as clusters 2-23, 39-74 aa 101-121 aa (13).

As a result of the AFM-MALDI-MS analysis of serum samples, it was found that 6 peptides, constituting the HCVcoreAg, occurred most frequently during the identification. These peptides are listed in Table 2.

As seen from the MALDI-MS analysis, the most frequently occurring peptides belong to the conservative regions of the core protein, and are found in all identified isotypes of the core protein.

As a result of analysis of 15 serum samples containing HCV particles, the most frequently occurring isotype of HCVcoreAg was determined. Uneven frequency of occurrence of core antigen peptides identified in various HCV-containing serum samples was registered. It was found that the most often identified peptides correspond to conservative regions of the amino acid sequence of the HCVcoreAg. This information is of interest for the development of effective highly sensitive serological test systems for the direct detection of core antigen, and, hence, for selection of the most efficacious course of chemotherapy depending on the region of patient residence.

 

References

1. The World Health Organization, 2014 http://www.who.int/mediacentre/factsheets/fs164/en/
2. FDA approves first combination pill to treat hepatitis C http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm418365.htm
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4. Simmonds P, Holmes EC, Cha TA, Chan SW, McOmish F, Irvine B. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS–5 region. J. Gen. Virol. 1993;7:2391–2399.
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10. Ivanov YD, Pleshakova TO, Krohin NV, Kaysheva AL, Usanov SA, Archakov AI. Registration of the protein with compact disk. Biosens Bioelectron. 2013;43:384–390.
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13. Yan B.-S., Tam M.H., Syu W.-Jr. Self–association of the C–terminal domain of the hepatitis C virus core protein // FEBS. – 2004. – 258. – P.100-106.
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IS fig1

Figure 1. MALDI-MS spectrum of hydrolyzed objects from the surface of AFM chip with immobilized anti-HCVcore, incubated in serum No. 33 containing HCVcoreAg. Asterisks indicate peptides corresponding to HCVcoreAg. Measurements were carried out on the Autoflex III TOF mass spectrometer (Bruker).

 

Table 1. The frequency of occurrence of HCV core antigen isotypes in HCV-positive serum samples.

IS tab1

 

Table 2. Frequency of occurrence of core antigen peptides in the AFM-MALDI-MS analysis of serum samples.

IS tab2

 

 

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