Development of liquid chromatography mass spectrometric fingerprinting based method for HIV-1 integrase detection

Abstract

Human Immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS) is a relatively modern disease that has caused extensive morbidity, mortality and suffering worldwide. HIV/AIDS is associated with different enzymes that are responsible for viral replication and one of such enzyme is the HIV-1 integrase. Integrase is responsible for the incorporation of viral DNA into the host cell, the enzyme is emerging as a novel target for intervention by chemotherapeutics, thus making the scientific investigation of HIV-1 integrase an important field of research. Currently, Enzyme-Linked Immunosorbet Assay (ELISA) based method are used for diagnosis of HIV infections across the world. However, other methods which are capable of detecting HIV-1 proteins with high sensitivity and specificity are imperative. Mass Spectrometry (MS) is known to offer unprecedented sensitivity and specificity during protein identification and, as such, its use for medical use is of greater interest. Therefore, this study sought to develop an MS method which can be applied for detection and characterization of HIV-1 integrase. The main objective of the current study was to identify and detect HIV-1 integrase using Multiple Reaction Monitoring (MRM). Detection of HIV-1 integrase using MRM will provide a method which may serve as a basis for diagnosis HIV/AIDS infection. Using Nested PCR and Sanger sequencing, pET15b and integrase construct were successfully confirmed. To this end, recombinant HIV-1 integrase was expressed in E. coli BL21 (DE3) cells. The recombinant HIV-1 integrase was successfully purified using nickel-affinity chromatography. Using electrospray ionization (ESI) based MS method, it was observed that integrase produces peptides masses that matches with theoretical masses, however some of the peptide masses differs due to multiple charge state. In addition, using precursor ion 600.84 for MRM, it was noted that the precursor ion produce three product ions acquired during ionization in the ESI chamber. Furthermore, the peak intensity of the product ions were noted to increase as the collision energy increases. These findings make the precursor ion 600.84 and its product ions a fingerprint that can be used as a tool integrase detection. As a proof of concept, the current method can be further developed by means of incorporating other MS ions originating from HIV-1 proteins, thereby increasing the likelihood of a future diagnosis.