key: cord-0793471-hzhdf5ke authors: Stevens, Katherine G.; Pukala, Tara L. title: Conjugating immunoassays to mass spectrometry: solutions to contemporary challenges in clinical diagnostics date: 2020-10-07 journal: Trends Analyt Chem DOI: 10.1016/j.trac.2020.116064 sha: fefa77eeae44f1103d8c2c42e4f2170bc52acbbc doc_id: 793471 cord_uid: hzhdf5ke Developments in immunoassays and mass spectrometry have independently influenced diagnostic technology. However, both techniques possess unique strengths and limitations, which define their ability to meet evolving requirements for faster, more affordable and more accurate clinical tests. In response, hybrid techniques, which combine the accessibility and ease-of-use of immunoassays with the sensitivity, high throughput and multiplexing capabilities of mass spectrometry are continually being explored. Developments in antibody conjugation methodology have expanded the role of these biomolecules to applications outside of conventional colorimetric assays and histology. Furthermore, the range of different mass spectrometry ionisation and analysis technologies has enabled its successful adaptation as a detection method for numerous clinically relevant immunological assays. Several recent examples of combined mass spectrometry-immunoassay techniques demonstrate the potential of these methods as improved diagnostic tests for several important human diseases. The present challenges are to continue technological advancements in mass spectrometry instrumentation and develop improved bioconjugation methods, which can overcome their existing limitations and demonstrate the clinical significance of these hybrid approaches. The term biomarker can denote any biological molecule or combination of factors that 3 indicate a particular biological state, which are often used to differentiate normal or abnormal 4 processes or conditions [1] . Whereas new biomarkers are identified using untargeted, semi-5 quantitative (comparative) analytical approaches, to develop a viable clinical test there needs 6 to be a reproducible method for their absolute quantitation. For implementation in clinical 7 laboratories, analytical tests designed for diagnostic applications need to meet performance 8 requirements with respect to their accuracy and predictive capabilities [2] . Diagnostic 9 accuracy is determined based on a test's abilities to positively identify individuals who have a 10 condition and eliminate those who do not, or its sensitivity and specificity, respectively. A 11 test's predictive value is evaluated by calculating the proportions of correct diagnoses out of 12 the total positive and total negative test results, or positive and negative predictive values, 13 respectively [2] . In the context of biomarkers, these factors are dependent on the dynamic 14 range, accuracy, and reproducibility of whichever analytical method is used to detect changes 15 in their abundance. 16 Most common clinical laboratory tests make use of spectrophotometric and/or 17 immunologic detection methods [3] . Of these, immunoassays, which take advantage of the 18 highly selective interactions between specific immunoglobulins and their target antigens, are 19 some of the most clinically relevant techniques [4, 5] . Furthermore, the chemical composition 20 of these large proteins enables a variety of strategies for modifying their structure with limited 21 perturbation of their antigen-binding activity (discussed in detail in Section 2.2). This feature 22 allows for immunoassays to be coupled to a range of different detection methods, including 23 radiometric, fluorescent, colorimetric, chemiluminescent, non-labelled (light scattering) and 24 electrochemical detection [4, 6] . Detection can be further enhanced using enzymatic, 25 polymerase chain reaction (PCR), liposome and nanomaterial-based signal amplification 26 strategies [7] [8] [9] . 27 In many instances, absolute levels of biomarkers in biological fluids or tissue biopsies 28 are insufficient for determining a reliable diagnosis, particularly for diseases characterised by 29 complex changes in tissue morphology and localised changes in protein expression [10] . More relevant information can therefore be obtained by comparing the spatial distribution of 31 biomarkers in normal and diseased specimens. Immunohistochemistry (IHC) involves the 32 J o u r n a l P r e -p r o o f labelling of specific antigens in tissue sections with antibodies, which are then visualised 33 using some combination of staining and imaging techniques(s). Owing to its simplicity, 34 affordability and versatility, this technique is commonly employed in diagnostic pathology 35 [10, 11] . 36 Developments in immunoassay and IHC technologies, such as automated enzyme-37 linked immunosorbent assays (ELISAs), microfluidics, lab-on-a-chip technologies, and 38 computer-assisted image analysis, have resulted in significant reductions in analysis time and 39 complexity, sample volumes and specialised equipment or expertise required [5, 10] . 40 However, many of these methods still utilise some form of spectrophotometric detection and 41 are therefore limited in the number of analytes that can be detected in a single experiment due 42 to overlaps in the emission ranges of different fluorophores and narrow dynamic range [12] . The development of immunoassays that utilise detection methods not constrained by the 44 inherent limitations of spectrophotometric measurements has therefore become an important 45 goal for modern diagnostic medicine. Since its inception in the early 20 th century, mass spectrometry (MS) has developed 49 into an important tool for biomedical researchers and clinicians [13] [14] [15] . Soft ionisation 50 methods, such as electrospray ionisation (ESI), matrix-assisted laser desorption/ionisation 51 (MALDI) and chemical ionisation, enable ionisation of molecules with minimal 52 fragmentation. These methods are therefore very useful for the MS analysis of intact 53 biomolecules, with MALDI and ESI commonly utilised in both research and clinical settings 54 [3, 13, 14, 16] . Compared to spectrophotometric methods for detecting biomolecules, MS 55 differentiates analytes based on the mass-to-charge ratio (m/z) of intact molecules and/or the 56 characteristic products of their gas-phase fragmentation, and therefore provides high 57 specificity and sensitivity and enables the detection of different isoforms [13, 17] . The ability 58 of MS to detect many different analytes simultaneously, or multiplex, is useful for analysing 59 complex biological mixtures as entire proteomes, lipidomes, or metabolomes can be 60 investigated for a single sample [13, 18] . This technology has obvious applications in 61 diagnostic medicine; hence, renewed enthusiasm for advancement in MS methodology is now 62 aimed at developing clinically viable platforms [17, 19] . In this study, the authors concluded that multiplexed analysis reduced false negative results 90 and has the potential to detect novel viruses [28] . Comparatively, sample preparation for MALDI-MS is a lot simpler as this ionisation 120 method is more tolerant of biological sample components, such as buffers [3] . However, the 121 absence of pre-analysis enrichment steps also makes it more difficult to detect low-abundance 122 ions, as MALDI spectra are often dominated by signals from more concentrated, albeit less 123 clinically significant, biomolecules, a phenomenon sometimes described using the analogy of 124 a needle in a haystack [31] . For imaging experiments, MALDI also requires additional sample preparation steps to 126 that of immunohistochemistry, such as enzymatic digestion or chemical release of proteins and glycans [13] . Some common tissue conservation techniques, such as paraformaldehyde 128 fixation followed by long-term storage, can result in incompatibility with MSI analysis [33] . For clinical applications, a major caveat of soft ionisation techniques, including ESI 139 and MALDI, is that some analytes will ionise more efficiently than others, meaning that Absolute quantification can be achieved in a more straightforward manner using 161 inductively coupled plasma (ICP)-MS, which involves atomisation of molecules using 162 extremely high temperatures (7,000-10,000 K) to detect hetero-elements (any element other 163 than C, N, O and H) [12, 45, 46] . This ionisation technique is highly sensitive, has a wide 164 dynamic range, and produces signals that are directly proportional to the sample concentration The broad range of reactive chemical groups on antibodies makes them amenable to 255 various conjugation methods (Figure 1 and Table 1 ) [66] . 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Targeted matrix-assisted laser desorption/ionisation mass spectroscopy MS) has been used to localise carboxypeptidase D protein in rat brain tissue sections. MALDI 676 spectra of fluorescein isothiocyanate (FITC)-labelled (a) and mass-tagged (b) secondary 677 antibodies corresponding to MALDI-MS (c), photographic (d) carboxypeptisase D (CPD) antibody binding. Reprinted with permission from Ref Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.