Introduction to Acquired Immunodeficiency Syndrome (AIDS) and the Conventional Technique to Diagnose Disease
Acquired immunodeficiency syndrome the HIV causing disease of a defective immune system that can be determined through the use of MS. In Viral diseases, capsid proteins are specifically identified through MS, which are specifically supposed to be present in the patient’s blood (Banerjee, 2020). The need of disease diagnosis is the critical discovery in the field of laboratory science technology which are on continuously expansion through the use of several advanced technologies. The conventional electron microscopy was used to detect the AIDS causing virus named, HIV (Bianchi et al., 2018). The visualisation of HIV was performed by utilizing the high resolution on the infected tissue of the patients. However, various disadvantages had occurred Infront of researchers in using electron microscopy, such as handling and maintenance of the microscopy was quite difficult. It took a high cost and long time to detect the viral particles in sample. Moreover, the process was complicated and contained various variations because of the nano-size of virus (Tofail et al., 2018). The discovery of mass spectrometry is a highly recommended technology to detect biomarker-based disease in patients. Mass spectrometry (MS) is a widely used technique to detect diseases by screening various substances such as antibiotics and immune-suppressive drugs. The main purpose of using MS is to determine the presence of biomarkers, which are supposed to be associated with certain types of diseases or toxicants. The main advantage of using the mass spectrometry technique is the ultrahigh sensitivity, highly powerful in disease detection, low turnaround time and high specificity. Further discussion of the report will provide the highlight with the following concepts, including proteomics and metabolomics types of (MALDI and ESI) MS, strength and limitations of MS types. Two articles (by Nachtigall et al., 2020 and De Nicolò et al., 2021) have been researched to provide insights into two major concepts of MS on HIV, in which two different types of MS (including ESI and MALDI) are used to show the effectiveness in detecting the virus. The suggested studies of HIV detection also provide insights on proteomics and metabolomics ways of determining the HIV particles in the sample, which is researched for diagnostic purpose through MS.
The basic working of mass spectrometry is dependent on detecting the protein structure (proteome) of the antigens in the cells. However, the other technique is also in use, named metabolomics. Metabolomics mass spectrometry tends to detect the metabolites present in the antigenic cells or pathogens, such as amino acids, sugars and lipids. It is a multipotent analytical system used in clinical laboratories. In general terms, MS can be defined as the medical in-vitro diagnostic test that can provide rapid ultrahigh performance up to the femtomolar range and critical decision-making abilities in healthcare organizations. The new evolving technique of mass spectrometry using ion source design provides more enhanced resolution and miniaturization (Xu & Madden, 2012).
MS can be utilised in blood or saliva sample, which is being carried for the diagnosis of viral nucleoprotein. The sample of the patient revealed the abundance of viral nucleoproteins by using two distinguish among the cancerous and the normal cell lines, viral gene particles or visualizing biomolecule distribution and diagnosing the therapeutic drug monitoring. The evaluation of HIV virus and antiviral drug distribution can be detected through MS. HIV is the human immunodeficiency virus that generally affects the CD4+ T cells, which are the types of immune cells making the person immunocompromised and weaker. The elimination of virus is not quite easy because of its enveloped structure and ability to reverse transcription particles. Moreover, the disease is contagious that can spread through UTIs, using medicated equipment’s and from close contacts. Although complete virus elimination is not possible, several active antiretroviral therapies are being utilised to improve the immunological responses of the patients, which is why the exact information of HIV virus presence is necessary to be known in patients’ blood sample. Hence, the use of MS can aid in examining disposition of viral particles in plasma (Barry et al., 2014). In order to determine the presence of the HIV, patients are being prepared to provide the urine, types of the mass spectrometry, called MALDI and ESI (Nachtigall et al., 2020).
Determination of HIV through Mass Spectrometry Technique
Introducing the two types of MS called Matrix-Assisted Laser Desorption/Ionization (MALDI), and electrospray ionization (ESI) have laid the detection for large proteins structures. Moreover, the liquid chromatography coupled with ESI mass spectrometry also provided the increased dimension to analyse the presence of required component on the mixture of biological sample. The biological samples include (tissue, saliva, sweat, blood, urine, and breath gases). In large-scale proteomics, ESI and MALDI are the ionization techniques that determine the ion source on antigenic peptide detection. The two methods can work to identify more than 72,000 peptides based upon their composition concerning amino acids, charge parameters, hydrophobicity and other yield-related factors. The use of modern separation technology is being applied for various clinical purposes. Before using any detection techniques among MALDI and ESI, a sample containing viral RNA is being collected, as HIV is the RNA virus. The sample is prepared by the RNA extraction technique, in which viral RNA is being extracted by Viral RNA Kit and then is amplified through RT-PCR (Lam et al., 2021).
The advanced technique called MALDI mass spectrometry determines the cell extracts or intact cells of the HIV are being identified that define the strain typing or predicted proteomes. Through MALDI MS, more than 4000 genes can be predicted at a time. The chemical molecules of the HIV capsid are measured in terms of mass to charge ratio (Singhal et al., 2015). MALDI technique uses both the types of proteomics and metabolomics analysis. Through MALDI, quantification of various compounds is made, including nucleic acid, polymers, amino acids, ions, and proteins, making the technique highly reproducible.
- Working principle of MALDI: the technology is based on preparing the HIV contained sample of patient by mixing and coating the solution with an organic compound called the energy absorbent matrix. The matrix is then ionized and absorbed with the laser beam to measure the HIV load. Laser beam generates the protonated ions in patient’s sample. These ions get accelerated and separated based on mass to charge ratio, which is then being detected and analysed through the mass analyser. Different types of mass analysers can detect various molecules in MALDI spectrometry, including quadrupole, ion trap, and time of flight (TOF) types analysers.
https://www.frontiersin.org/articles/10.3389/fmicb.2015.00791/full
Figure 1: The Viral Gene Assay Utilizing MALDI-TOF Mass Spectrometry.
The given figure shows the working activity of MALDI-TOF mass spectrometry used to screen the HIV drug-resistance based variants and provides the ion signalling in the form of peptide mass fingerprint (Singhal et al., 2015).
The microbial identification can be done by comparing the peptide mass fingerprint or PMF (characteristic spectrum of TOF analyser) of an unknown organism’s biomarker constrained in the proteome database. The general mass range (m/z) to determine microbial identification is 2–20 kDa, which provides information on ribosomal and some housekeeping proteins. The knowledge of proteins information is needed as proteins constitute approximately 60–70% of the dry weight of the cells. Matching PMF patterns can aid in identifying particular microorganisms because of containing ribosomal proteins, which is necessary to establish an extensive open database. Hence, the knowledge of the gene sequences and proteins’ activities provided through the database can identify particular genus, species and strain level of the microbes (Rybicka et al., 2021).
- Working principle of ESI-MS: ESI-MS is also an excellent technology providing the coverage of a wide variety of smaller and large biomolecules, lipids, and metabolites (De Nicolò et al., 2021). The type of mass spectrometry can also be categorised as the DESI-MS (because of the Desorption property). ESI-MS provides crucial diagnostic information and promising discriminatory controls that can be provided in a short of time by using the NMR (nuclear magnetic resonance). The working principle of ESI MS depends on the formation of ions created through the dilute solution spraying of sample from metal capillary tip making the fine droplets through the high electric field making it charged. The technique depends on using the high-speed microdroplet charges onto the sample surface. The molecular species are extracted through the splashed microdroplets to form the charged gaseous species, which the MS then detects. Although BESI can be used through both proteomics and metabolomics, metabolomics is a highly recommended technology used in ESI-MS. The technique is generally used for lipid profiling.
https://www.researchgate.net/figure/Principle-of-MALDI-TOF-MS-and-ESI-MS-identification-of-bacteria-For-MALDI-TOF-laser_fig1_232277257
Figure 1: The Viral Gene Assay Utilizing ESI Mass Spectrometry.
The images show the working activity of ESI mass spectrometry used to screen the HIV metabolites present in the patient’s serum and provide the ion signalling upon detection through a mass analyser.
The considering omics studies of proteomics and metabolomics is, called nutrigenomics, possess a wide range of information that provides insight into the mechanism of the food components, exerting effects of food components on the body and interactions of dynamic proteins to understand the metabolic process of the organisms. The advanced application of the technologies in the omics fields is on rapid increase and appreciated, which are used to detect several types of diseases. Proteomics and metabolomics provide the strength based-studies of thousands of proteins and metabolites productions in the cellular system of the organisms describing the comprehensive and quantitative composition of the given cell population. Proteomics tends to investigate the dynamic nature of protein expression and recognise the pathogenic genes altering the metabolic process of the organisms (Ramírez-Camacho et al., 2022). On the other side, the strength of metabolomic studies highlights the use of metabolites, ions, amino acids and sugar groups, which are detected in the pathogens-attacked suspected cell population. Metabolomics is found to be a very crucial strategy to detect the presence of HIV virus in patients’ serum or saliva samples because metabolomics tends to identify the concentration of lipids, amino acids and glucose metabolites. Hence, the paper f=has been found to be on strength-based researches (Noto et al., 2014).
- Proteomics studies against HIV infections: Proteomics studies have been used in various types of microbiological processes for microbial identification. Their epidemiological and biological studies, pathogenicity and the detection of antibiotic resistance activities. The disease identification and determination of the characteristic cells’ property can be analysed through various other treatment techniques, such as gel electrophoresis, liquid chromatography, SDS-PAGE, and mass spectrometry. The development and progression of the microbial proteome are characterised through the use of protein-specific separation technique such as SDS-PAGE. The whole sample is being run on the SDS page instrument that identifies the excellent DNA-RNA hybridization in this technique. SDS-PAGE is the gel-based separation method for analysing microbial classification. Although the SDS page is not much effective because of proving the identification of unknown data or lacks extensive standardized conditions, the inclusion of MS is made in the identification processes (Barnett et al., 2014).
- Metabolomics studies against HIV infections: Metabolomics is an emerging technology and other omics technologies, including genomics, proteomics and transcriptomics. The technique is used to detect and understand the host-pathogen interactions on the molecular level through which interactions with infectious diseases can be investigated. The specific disease screening by metabolomics occurs through mass spectrometry (MS) which imposes great significance in identifying the druggable metabolic molecules. Metabolomics is generally considered the biomarker screening of infectious viral bacterial or parasitic particles. To discover the metabolic processes of the antigenic proteins, metabolomics involves using several smaller cellular molecules as the biomarker, including lipids, nucleotides and amino acids. The use of such smaller molecules can aid in obtaining in-depth knowledge of infection impacts. The metabolomic studies through MS render the high sensitivity at the picogram level, making it ideal for biofluid analysis (Noto et al., 2014).
Two Types of MS
Mass spectrometry tends to open up the door for analysing clinical diseases in very little time, which has the potential to detect certain diseases with an acute level of accuracy and reproducibility. The continuous advancement in MS is making the technique more robust, precise and reproducible with the use of automated robotics to give a high-throughput and cost-effective performance. A single MS can detect multiple types of diseases, which is why it is considered the next-generation pathology technique. Despite the provision of best clinical practices, MS also possess some limitations. The most studied limitation of MS involves the inability to identify the identification of disease at an earlier stage until the level of antigens does not exponentially increase in the blood (Banerjee, 2020).
Although both types of mass spectrometry (including proteomics and metabolomics) are widely used techniques in clinical uses, both have some drawbacks.
- Metabolomics (utilizing ESI) is an extensively used technique using in MS but also poses some risks in detecting the pathogens. For example, the fragmentation of metabolites used in metabolomics is quite unpredictable, and these are highly prone to change. Metabolites have a large variety of physicochemical properties, of which measurements and calculations become quite challenging. Moreover, a single tool is not present to detect all the types of metabolites identification; hence, sometimes process gets tricky (Kowalczyk et al., 2020).
- Drawbacks in using Mass proteomics (using MALDI) spectrometry; the study and stability of the structure of the protein is harder as environmental factors like temperature and pressure can impart the proteins’ structure. Moreover, the validation of proteins and decoding of the protein folding is not quite easy because of its highly condensed structure.
- The advantage of DESI over MALDI is that DESI-MS require minimal or no sample preparation time making the working much faster to produce high-throughput data (Nadler et al., 2017).
- MALDI is used to analyse the simple mixture of peptides on the contrary, ESI can analyse the complex peptide formations in the sample.
- MALDI MS use liquid chromatography by provides the lesser sequence coverage that occurs offline. On the contrary, ESI can sequence more coverage through the use of high throughput liquid chromatography used online.
- The ions spread by the ESI technique are provided in the form of the continuous stream as it is the solution-based technique, whereas the MALDI technique, which is solidly based, provides the ions in pulses (Aziz et al., 2019).
Comparing the two types of mass spectrometry, ESI is found to be more successful and faster technique in comparison to the MALDI mass spectrometry. The ESI mass spectrometry tends to use the metabolomic approach to detect the sugar moieties or amino acids, which are specifically supposed to find in viral RNA. Hence, ESI mass spectrometry is supposed to have no gaps leaving behind and can be used extensively for a variety of purposes (De Nicolò et al., 2021).
Gaps in current research and potential technical solutions in MS: Considering the major drawbacks of metabolomics, it can be concluded that although MS possesses various limitations, the majority of the detection techniques need to use the basic concepts of mass spectrometry by making some evolutions in these techniques. Hence, MS is proved to be very effective in detecting diseased conditions through the modification of new technologies in proteomics and metabolomics. For example, a newly formulated technology called SWATH has been incorporated in proteomics, which possesses high repeatability and sensitivity methods for the selected monitoring reactions (Kang et al., 2021). The SWATH technology generally stands for Sequential Window Acquisition of all Theoretical Mass Spectroscopy, which can be used in a variety of applications to quantify proteins in a versatile manner. Moreover, the advancement in metabolomics, which is regarded the Raman spectroscopy or quantitative metabolic fingerprinting, is used to quantify a large number of metabolites in the sample. Metabolic fingerprinting is the intense investigation that is developed in many clinical laboratories to diagnose several diseases (Lima et al., 2021).
References
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