Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen

Accepted Manuscript Title: MASS SPECTROMETRIC DETECTION OF THE AMINO ACID SEQUENCE POLYMORPHISM OF THE HEPATITIS C VIRUS ANTIGEN Author: A.L. Kaysheva Yu. D. Ivanov P.A. Frantsuzov N.V. Krohin T.I. Pavlova V.F. Uchaikin V.A. Konev O.B. Kovalev V.S. Ziborov A.I. Archakov PII: DOI: Reference:

S0166-0934(15)00395-X http://dx.doi.org/doi:10.1016/j.jviromet.2015.12.012 VIRMET 12936

To appear in:

Journal of Virological Methods

Received date: Revised date: Accepted date:

28-5-2015 28-12-2015 29-12-2015

Please cite this article as: Kaysheva, A.L., Ivanov, Yu.D., Frantsuzov, P.A., Krohin, N.V., Pavlova, T.I., Uchaikin, V.F., Konev, V.A., Kovalev, O.B., Ziborov, V.S., Archakov, A.I., MASS SPECTROMETRIC DETECTION OF THE AMINO ACID SEQUENCE POLYMORPHISM OF THE HEPATITIS C VIRUS ANTIGEN.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2015.12.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

MASS SPECTROMETRIC DETECTION OF THE AMINO ACID SEQUENCE POLYMORPHISM OF THE HEPATITIS C VIRUS ANTIGEN A.L. Kayshevaa*, Yu.D. Ivanova, P.A. Frantsuzova, N.V. Krohina, T.I. Pavlovaa, V.F. Uchaikina, V.А. Koneva, O.B. Kovaleva, V.S. Ziborova, A.I. Archakova a

Institute of Biomedical Chemistry, Pogodinskaya St. 10, Moscow, 119121

Russia; *Corresponding author: Pogodinskaya 10, Moscow, 119121, Russia Tel.: (+7)(499)2463761; fax: (+7)(499)2450857; E-mail address:[email protected]

Highlights: 

MS/MS of SAPs allowed increasing the number of identified peptides.



AFM-MS/MS can be used for identification of HCVcoreAg in human serum.



AFM-MS/MS could improve the lower of DL of HCVcoreAg when compared to MS/MS.

ABSTRACT A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

Keywords: hepatitis C; HCVcoreAg detection; mass spectrometry; SAP 1

Hepatitis C is a disease of the liver, caused by hepatitis C virus (HCV). According to recent WHO data, to date about 150 million people worldwide are chronically HCV infected. Every year up to 500,000 people die from hepatitis C and associated liver diseases. In 15-30% of cases, chronic HCV infection leads to cirrhosis or liver cancer (WHO, 2014). Early diagnosis can prevent, in 90% of cases, the development of HCV infection in severe forms. Medical programs for the population in developed countries recommend screening for people being at risk of infection (FDA, 2015). Despite the fact that during the early stages of HCV infection antiviral therapy is effective in 50-90% of cases, the level of early diagnosis of HCV infection worldwide is very low. This is explained by the following factors: (1) the incubation period for acute hepatitis C averages 6 to 10 weeks, and is asymptomatic in 80% of cases (FDA, 2015); (2) the concentration detection limit of clinically available serological methods for HCV diagnostics (ELISA, EIA and immunoblotting) does not exceed 10-12M (Archakov et al., 2007), while most HCV protein markers occur in blood at low (less than 10-13M) and ultra low (less than 10-15M) concentrations (Kaisheva et al., 2014); (3) the serological HCV tests, based on registering specific monoclonal antibodies against HCV proteins (which appear in blood within two months after infection), make possible only the indirect detection of these proteins. Modern diagnostics of HCV involves two stages: (a) blood screening with antibodies against HCV proteins by the serologic ELISA and EIA tests (enzyme-linked immunosorbent assay and enzymeimmunoassay, respectively); and (b) in case of a positive serological test, an additional RT-PCR (reverse transcription polymerase chain reaction, including AmpliSens HСV-monitor-FRT, InterLabService, Russia) test is carried out for registration of HCV RNA in serum and for the determination of the virus serotype (U.S. Food and Drug Administration, FDA, 2015). Thus, HCV infection often remains undetected until serious damage to the liver occurs (FDA, 2015). Apparently, a solution to this problem lies in development of highly sensitive, label-free methods for the HCV protein markers’ registration based on direct detection of HCV antigens (HCVcoreAg, surface proteins E1/E2) (Ivanov et al., 2015), appearing in the serum 16 weeks before antibodies against HCV (Kaysheva et al., 2014). In this paper, we propose an approach for the highly sensitive registration ofHCVcoreAg (with sensitivity in serum samples of less than 10-13 M) by using the biospecific AFM-fishing combined with mass spectrometric identification - with due consideration of possible SAPs (single amino acid substitutions) in the HCVcoreAg amino acid sequence.

2

Covalent immobilization of monoclonal antibodies against HCVcoreAg onto amino modified mica surface with subsequent AFM scanning were carried out according to the techniques described in detail by Ivanov (2014). In this study, two types of AFM chips were used: AFM chips with immobilized antibodies and control AFM chips with amino modified surface but without immobilized antibodies. The efficiency of antibodies immobilization onto the AFM chip surface was estimated by AFM. The trypsinolysis of proteins, biospecifically fished out onto anti-HCVcoreAg, was carried out directly on the AFM chip surface. For that, 1 µl of 10-9 M trypsin solution and 1 µl of acetonitrile were added to 5 µl of 50 mM NH4HCO3 buffer solution (рН 7.4). This mixture was transferred onto the AFM chip surface for trypsinolysis. The trypsinolysis was carried out at constant air humidity and controlled temperature using standard technique, analogous to that described in (Ivanov et al., 2013 and 2014). Briefly, the procedure was as follows: 7 µl of trypsinolytic mixture was dispensed onto the AFM chip surface and incubated for 5 h at 42oC and 90% humidity. Then, again, 7 µl of trypsinolytic mixture was dispensed onto the chip surface, and incubated for 13 h. After that, trypsinolytic mixture (sample) was washed off with 20 µl of 80% acetonitrile in 0.7% TFA. The sample was dried out in SpeedVac vacuum evaporizer (Eppendorf, USA). For MS analysis, the sample was dissolved by adding 5 µl of 0.7% TFA. Then, the sample was sonicated in ultrasonic bath for five minutes at R.T. The samples were stored at -80oC (Ivanov et al., 2015). Tandem mass spectrometric analysis (MS/MS) of proteins was carried out using chromatography/mass spectrometry system of ion trap type LC/MSD Trap XCT Ultra (Agilent 6300, USA), equipped with electrospray interface chip-ESI-online. Ionization was carried out at 180°C. Capillary voltage was 1.8 kV. The flow rate of the drying gas in the mass spectrometer was 3.5 L/min. The parameter value of the accumulated ions in the trap ions was set to 50000 in 1 ms. Detection of ions in the ion trap was carried out in ESI-positive ionization mode. The 3

results of tandem mass spectrometric measurements were processed using the Data software (version 3.3; Bruker, Germany). Identification of peptides and proteins was performed using proteomic search engine Mascot (Matrix Science, http://www.matrixscience.com/) with the following search options: NCBI or MSDB data library; trypsin was used as an enzyme; the number of missed hydrolysis sites no more than 1; charge state of the peptide + 1, + 2, + 3; oxidated methionine was indicated as possible modification; parent ion mass determination accuracy 150 ppm, daughter ions mass determination accuracy 300 ppm; the possibility of protein detection was more than 95% (peptide score was no less than 30); the minimal number of unique peptides corresponding to the same protein was two. Measurements were performed to determine the concentration sensitivity of AFM-MS/MS method in the analyte solution. To this end AFM chips with immobilized anti-HCVcore monoclonal antibodies were incubated in HCVcoreAg solutions in the concentration range 10-5 to 10-13 M (Ivanov et al., 2015). For comparison, MS measurements of proteins, fished out onto the surface of the AFM chip from the analyte solution, also containing the target protein in the concentration range 10-5 to 10-13 M, were conducted. These measurements allowed to identify the core antigen, fished out onto the surface of the AFM chip, at concentrations of up to 10-13 M, and in the analyte solution at concentrations of up to 10-10 M (Figure 1). As seen from Figure 1, the AFM-MS/MS method allows to achieve the sensitivity of detection of HCVcoreAg in solution at 10-13 M, with the number of identified peptides of no less than two. At the same time, the MS analysis of solutions of the target protein without biospecific capturing procedure made it possible to achieve the detection sensitivity of only 10-10 M, which is three orders less than the sensitivity obtained upon AFM-MS/MS registration. Tandem mass spectrometric analysis of proteins from the surface of AFM chips after their incubation in serum samples (positive or negative for the presence of HCV RNA by RT-PCR was also performed. The blood plasma samples were analogous to the samples described in 4

previous studies by Ivanov et al. (Ivanov Yu.D. et al. 2010 and 2015). Monoclonal antibodies against HCV core antigen (anti-HCVcore, clone 1Е5, Virogen, USA) were used as molecular probes immobilized in the working area of AFM chip. To estimate the impact of non-specific adsorption of serum components onto the AFM chip surface, control experiments were conducted (Kaisheva et al., 2010). In a first series of control experiments, the AFM chips, containing no anti-HCVcore, were incubated in a HCV-positive serum sample. Mass spectrometric analysis of such chips (that was confirmed by AFM data) missed the presence of any objects of protein nature. A second series of control experiments were conducted with AFM chips containing immobilized anti-HCVcore, after their incubation in HCV negative serum samples – in order to detect cross-reactivity of monoclonal anti-HCVcore to serum components. In these control experiments, no protein molecules were identified (Kaisheva et al., 2010). Thus, the MS analysis of AFM chips after control experiments did not reveal nonspecific sorption of serum components onto exposed surfaces of AFM chips; nor did it reveal any sorption onto immobilized monoclonal antibodies at the cost of cross-reactivity to serum samples, thereby indicating the specificity of AFM analysis. The application of tandem mass spectrometric analysis of proteins captured onto the AFM chip surface after its incubation in serum samples allowed to identify the target protein. A typical mass-spectrum is shown in Figure 2. HCVcoreAg was identified by its two proteotypic peptides: RPQDVKFPTGGQIVGGVYLLPR

(a.a.18-39;

m/z=799.7)

and

RPQDVKFPSGGQIVGGVYLLPR (a.a.18-39; m/z=795.0). It should be noted that the same peptides were identified by the MS/MS analysis of recombinant HCVcoreAg in the analyte solution. In total, 35 serum samples were analyzed. The coincidence of the results obtained by AFMMS/MS and RT-PCR/AFM was observed for 25 serum samples. Of these, 15 serum samples were HCV positive by RT-PCR/AFM and AFM-MS/MS data, and 10 serum samples were HCV 5

negative by the same data. In the remaining 10 HCV positive samples no target protein was MSidentified. Analysis of these samples was performed with due account taken of possible polymorphisms in the amino acid sequence of HCVcoreAg. In cases where the identification of HCVcoreAg did not produce reliable results – with only one peptide being identified - the search for possible SAPs in the polypeptide sequence of the target protein was undertaken. These polymorphisms are primarily caused by the presence of variable regions in the amino acid sequence of HCV antigen. To detect these substitutions, the Q8V7V3_9HEPC sequence was selected as a sequence template. An example illustrating the occurrence of SAP in the Q8V7V3_9HEPC sequence is presented in Figure 3. Figure 3 displays the MS/MS-spectrum of the HCVcoreAg peptide F PQDVKFPGGGQIVGGVYLLPR (m/z =691.1) with a single amino acid substitution in the alignment template Q8V7V3_9HEPC, as was identified in three serum samples. This peptide with

m/z=691.1

differs

from

the

theoretically

predicted

peptide

RPQDVKFPGGGQIVGGVYLLPR with the similar mass (m/z = 799.9) of sequence template by the substitution of only one amino acid located at position 17 (Arg → Thr) of the nonconservative region of HCV core antigen (Bukh et al., 1993). Comparison of the fragmentation spectra for the theoretically predicted peptide of alignment template (m/z = 799.9) with the experimentally obtained fragmentation spectrum for the peptide with m/z = 691.1 has revealed several identical fragments in the three peptides: y3 (m/z = 386.28), y4++ (m/z= 249.24) and y5 (m/z=661.4). The results of AFM-MS/MS analysis with consideration for single amino acid substitutions are presented in Table 1. The AFM-MS/MS method allowed to identify (by RT-PCR) the presence of HCVcoreAg in all the ten HCV-positive sera. The analysis of amino acid sequences of the core antigen in these ten serum samples revealed the following polymorphisms in the nonconservative regions: at position 17 Arg → Thr, for three serum samples; at position 75 Thr → 6

Ala, for three serum samples; at position 78 Gln → Ala, for two serum samples; at position 91 Lys → Cys, for one serum sample. As seen from Table 1, the RT-PCR and AFM data are consistent with AFM-MS/MS data – with due account taken of polymorphisms revealed in different HCVcoreAg sequences. The amino acid sequence of HCVcoreAg is highly variable. In April 2015, the protein database UniProt KB for HCVcoreAg contained 6423 amino acid sequences which differed in: (a) weight: starting at 2057 Da (17 aa) to 23358 Da (214 aa); and (b) in amino acid sequence identity. However, no mass spectra for the HCVcoreAg are annotated in protein databases. Variability of the protein is caused by extreme heterogeneity of the HCV population. By now, six genotypes (classification by Simmonds) (Simmonds et al., 1993) and more than 90 subtypes of HCV (Choo et al., 1989) have been identified. In the literature, a canonical form of HCV core antigen is considered to be a sequence of 121 amino acid residues, located at the N-terminus of the polypeptide (Matsumoto et al., 1996). Previously, we examined 30 HCV-positive (by RT-PCR) serum samples, and found that, according to NCBI protein database, the Q8V7V3_9HEP isoform is identified most often (Archakov et al., 2007; Kaisheva et al., 2010) (Fig. 1). The identity of amino acid sequence of the HCV core antigen between isolates makes up from 85.3 to 100%. The largest part of the antigen – about 132 amino acid residues – is invariant. In the amino acid sequence of the HCV core antigen, one can single out three large conservative hydrophobic regions (2-23 aa, 39-74 aa, 101-121 aa); these regions were found to occur in 52 HCV isolates. The remainder of the amino acid sequence of the antigen is hydrophilic (Fig. 4) (Bukh et al., 1993). The identified polymorphisms belong to the variable part of the amino acid sequence of the HCV core antigen – in agreement with literature data.

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Thus, it was demonstrated that AFM-MS/MS analysis can well be used for detection and identification of HCVcoreAg in human serum. Analysis of AFM-MS/MS measurements can improve the detection accuracy of HCVcoreAg in the serum.

Acknowledgement The work was performed in the framework of the Program for Basic Research of State Academies of Sciences for 2013-2020.

References Archakov, A.I., Ivanov, Y.D., Lisitsa, A.V., Zgoda, V.G. 2007. Fishing AFM Nanotechnology is the way to reverse the Avogadro number in proteomics. Proteomics. 7, 4–9. Bukh, J., Purcell, R.H., Miller, R.H. 1993. At least 12 genotypes of hepatitis C virus Predicted by sequence analysis of the putative E1 Gene of isolates Collected worldwide. Proc Natl Acad Sci USA. 90, 8234–8238. Choo, Q.L., Kuo, G., Weiner, A. J. 1989. Isolation of Clone A cDNA derived from A Bloodborne non-A, non-B viral hepatitis Genome. Science. 244, 359–362. FDA approves first pill combination to Treat hepatitis C http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm418365.htm Ivanov, Y.D., Bukharina, N.S., Pleshakova, T.O., Kaysheva, A.L., Zgoda, V.G., Izotov, A.A., Pavlova, T.I., Ziborov, V.S., Radko, S.P., Moshkovskii, S.A., Archakov, A.I. 2014. Atomic Force Microscopy Mass Spectrometry Fishing and Identification of gp120 aptamers immobilized on. Int J Nanomedicine. 3(9), 4659–4670. Ivanov, Y.D., Kaysheva, A.L., Frantsuzov, P.A., Pleshakova, T.O., Krohin, N.V., Izotov, A.A., Shumov, I.D., Uchaikin, V.F.,

Konev, V.A., Ziborov, V.S., Archakov, A.I. 2015.

Detection of hepatitis C virus core protein in Serum by Atomic Force Microscopy combined with Mass Spectrometry . Int J Nanomedicine. 10, 1597–1608. Ivanov, Y.D., Pleshakova, T.O., Krohin, N.V., Kaysheva, A.L., Usanov, S.A., Archakov, A.I. 2013. Registration of the protein with compact Disk. Biosens Bioelectron. 43, 384–390. Kaisheva, A.L., Ivanov, Y.D., Zgoda, V.G., Frantsuzov, P.A., Pleshakova, T.O., Krokhin, N.V., Ziborov, V.S., Archakov, A.I. 2010. Identification and visualization of hepatitis C viral

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Particles by Atomic Force Microscopy combined with MS/MS analysis. Biomeditsinskaya Khimiya . 56 (1), 26–39. Kaysheva, A.L., Ivanov, Y.D., Zgoda, V.G., Frantsuzov, P.A., Pleshakova, T.O., Krokhin, N.V., Ziborov, V.S., Archakov, A.I. 2010. Identification and visualization of hepatitis C viral Particles by Atomic Force Microscopy combined with MS/MS analysis. Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry. 4 (1), 15–24. Matsumoto, M., Hwang, S.B., Jeng, K.S., Zhu, N., Michael, L. 1996. Homotypic Interaction of Hepatitis and Multimerization C Virus Core Protein. Virology. 218, 43–51. Simmonds, P., Holmes, E.C., Cha, T.A., Chan, S.W., McOmish, F., Irvine, B. 1993. Classification of hepatit is C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J. Gen. Virol. 7, 2391–2399. The World Health Organization, 2014 http://www.who.int/m ediacentre / factsheets / fs164 / en / Zgoda, V.G., Moshkovskii, S.A., Ponomarenko, E.A., Andreewski, T.V., Kopylov, A.T., Tikhonova, O.V., Melnik, S.A., Lisitsa, A.V., Archakov, A.I. 2009. Proteomics of mouse liver microsomes: Different performance of protein Separation workflows for LC-MS/MS. Proteomics. 9 (16), 4102–4105.

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Figure legends Fig. 1. Dependence of the number of identified peptides on the HCVcoreAg concentration in the incubation solution. The measurements were carried out on a LC/MSD Trap XCT Ultra mass spectrometer (Agilent).

10

Fig. 2. The MS/MS spectrum of hydrolyzed objects from the AFM chip surface with immobilized anti-HCVcore after the incubation in serum samples. Peptides of HCVcoreAg: RPQDVKFPTGGQIVGGVYLLPR

(m/z=799.8)

(A),

RPQDVKFPSGGQIVGGVYLLPR

(m/z=795.5) (B). The measurements were carried out on an LC/MSD Trap XCT Ultra mass spectrometer (Agilent). A total of 35 sera were analyzed by AFM MS/MS. 25 of these sera were HCV-positive, and 10 were HCV negative by RT-PCR and AFM data.

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Fig. 3. The tandem MS/MS spectrum of the FPGGGQIVGGVYLLPRMGPRR peptide obtained from the sample after the AFM analysis of the serum: the MS spectrum (A); the MS/MS spectrum (B). Numbers indicate the identified peptide fragments. The measurements were carried out on an LC/MSD Trap XCT Ultra mass spectrometer (Agilent).

12

Fig. 4. The amino acid sequence of the sequence template Q8V7V3_9HEPC for the HCVcoreAg. Bold type marks off the highly conserved regions of the amino acid sequence (Kaisheva et al., 2010).

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Table 1

The results of MS/MS identification of the HCV core antigen (with consideration of the amino acid substitutions) from the AFM chip surfaces, incubated in 10 different serum samples. Number of serum samples

SAP

The canonical peptide

3

17 Arg→Thr F(R)PGGGQIVGGVYLLPRM GPRR

3

75 Thr →Ala T(A)WAQPGYPWPLYGNEGL GWA GWLLSPR

RPQDVKFPSGGQIVGGVYLLPR (m/z=795.5) VQVLDSHYQDVLK (m/z=515.4) RPQDVKFPGGGQIVGGVYLLPR (m/z=799.9) CGSGPWMTPRCLVHYPYR (m/z=708.5) QPIPKDR (m/z=427.3)

2

1

78 Gln→Ala AWAА(Q)PGYPWPLYGNEGL GWA GWLLSPR 91 Lys→Cys AWAQPGYPWPLYGNEGC(L) GWA GWLLSPR

RPQDVKFPGGGQIVGGVYLLPR (m/z=799.9) QPIPKDR (m/z=427.3)

14