Innovation and skilled individuals go hand in hand to drive the mass spectrometers market

MS is now growing in familiarity and popularity for a variety of clinical applications, from laboratory tests for analyte quantification to diagnostic applications in infectious diseases, inborn errors of metabolism, and cancer.

Since the turn of the century, mass spectrometry (MS) technologies have continued to improve dramatically, and advanced strategies that were impossible a decade ago are increasingly becoming available.

Different types of MS techniques exist, but it is most frequently performed as gas chromatography MS (GC-MS), liquid chromatography MS (LC-MS), tandem MS (MS/MS, GC-MS/MS, LC-MS/MS), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) for clinical applications.

The basic principles are similar across the various methods. Analytes can be chromatographically separated before ionization or directly ionized and analyzed using their specific mass-to-charge ratios that are recorded in a spectrum. In the targeted approach, particular peaks are used to identify specific analytes, i.e., drugs or steroids. In an untargeted approach, the combined pattern of peaks is analyzed by an algorithm, or compared to a database of known samples, e.g., for microbial identification against a known microorganism.

The power of MS, especially when combined with chromatography, displays enhanced sensitivity and specificity along with its ability to measure several clinical analytes in a multiplexed manner. Additionally, the technique is adaptive because assays can be developed for novel analytes or applications. This is simultaneously one of its disadvantages. One of the greatest challenges that still revolves around MS is the technical knowledge required to develop, validate, and support clinical testing on these instruments, as most applications will be in-house laboratory-developed tests (LDTs). As a result, knowledgeable and skilled individuals are needed, not only to develop robust clinical assays, but also to maintain, troubleshoot, and service these instruments in a timely fashion.

Mass spectrometry-based omics technologies, proteomics, metabolomics and lipidomics have enabled the molecular level systems biology investigation of organisms in unprecedented detail. There has been an increasing interest for gaining a thorough, functional understanding of the biological consequences associated with cellular heterogeneity in a wide variety of research areas such as developmental biology, precision medicine, cancer research, and microbiome science. Recent advances in MS instrumentation and sample handling strategies are quickly making comprehensive omics analyses of single cells feasible, but key breakthroughs are still required to push through the remaining bottlenecks.

Emerging role of MS in pathology
Mass spectrometry-based assays have been increasingly implemented in various disciplines in clinical diagnostic laboratories for their combined advantages in multiplexing capacity and high analytical specificity and sensitivity. It is now routinely used in areas including reference methods development, therapeutic drug monitoring, toxicology, endocrinology, pediatrics, immunology, and microbiology to identify and quantify biomolecules in a variety of biological specimens. As new ionization methods, instrumentation, and techniques are continuously being improved and developed, novel mass spectrometry-based clinical applications will emerge for areas such as proteomics, metabolomics, hematology, and anatomical pathology.

Current clinical applications of MS. One of the first uses of MS in clinical laboratories is in the area of toxicology. Confirmatory urine drug screens for clinical or forensic purposes are commonly measured by gas chromatography-MS (GC-MS), and more recently LC-MS/MS. LC-MS/MS is the most commonly used method for many clinical applications today, ranging from qualitative urine drug screens to quantitative, high-throughput, and esoteric analyses including therapeutic drug monitoring, steroids, inborn errors of metabolism and newborn screening, endocrinology, and immunology.

MALDI-ToF MS has also recently been in development for qualitative and quantitative peptide and protein assays. Examples include monoclonal immunoglobulins and serum-free light chains, hemoglobin A1c, insulin-like growth factor-1, multiplex biomarker panel of C reactive protein, serum amyloid A, fecal calprotectin, and cystatin C.

In microbiology, quantitative measurements of antibiotics and antifungal drugs are available. Recent implementation of MALDI-ToF MS has revolutionized the workflow for identification of micro-organisms. Compared with conventional microbiological techniques, MALDI-ToF MS offers significantly shorter turnaround time in terms of species identification. A limitation of MALDI-ToF MS, however, is its relative lack of specificity in differentiating closely related strains of bacterial species. In molecular diagnostics, genotyping and mutational analysis in liquid biopsy have been in development using MALDI-ToF MS.

MS for microorganism identification. Currently, mass spectrometry-based approaches to identify microorganisms are being adopted in microbiology laboratories across the world owing to the shorter identification time, compared to conventional techniques, which is one of the most prominent clinical MS application to date. It is a game changer in microbiology labs. A patient-derived biofluid is cultured and a colony is analyzed by the MS instrument, producing a spectrum one can match to established databases. Although only FDA-cleared for a subset of microorganisms, applications for mycobacteria, filamentous fungi, and rarer microbes in research-use-only (RUO) databases are forthcoming. Improvements in the technique may also shorten analysis time further, such as testing antimicrobial susceptibility or direct evaluation from blood culture or biofilms.

MS for laboratory tests. As a highly sensitive and specific technique, MS is ideal for quantifying small molecules from biofluids, such as steroids, illicit drugs, or abused and therapeutic prescription drugs. For instance, regulating the concentration of immunosuppressants (such as tacrolimus) is essential; if levels are too low, this increases the risk of tissue rejection, whereas if the levels are too high, it could lead to toxicity and damage to the transplanted organ. MS is also amenable for detecting and quantifying larger biomolecules, such as proteins. For example, it may be employed to monitor thyroid cancer patients post treatment by assaying for thyroglobulin, a glycoprotein produced by thyroid follicular cells and a biomarker of recurrent thyroid cancer.

Mass spectrometry-based methods are also being used to identify cases of monoclonal gammopathy, which is the over production of gamma immunoglobulins that can occur during the course of hematological malignancies. Current laboratory tests employ protein gel electrophoresis, immunofixation, and free light-chain assays; however, they suffer from low sensitivity and high cost. On the other hand, MS offers a sensitive and low per-sample-cost alternative. MS can also be used to quantify serum levels of immunotherapeutic monoclonal antibodies, which it can differentiate from endogenous monoclonal antibodies.

Omics. Novel applications of MS methods are driving omics research (lipidomic, metabolomic, and proteomic) and biomarker discovery in a variety of specimen types including blood, urine, and biopsy tissues. Proteomic analysis using MS can be approached using two methods–top-down proteomics and bottom-up proteomics. Top-down proteomics involves the intact analysis of the entire protein. However, many proteins involved in human diseases share similar masses, making the top-down method ineffective for distinct clinical subtyping. The top-down proteomics method is also unable to accurately determine the prevalence of different protein variants, if they possess similar masses. In contrast, bottom-up proteomics involves enzymatic cleavage of the whole protein through peptidase digestion to generate a mixture of peptides.

In anatomical pathology, whole-proteome profiling from tissue slides is beginning to be established to examine global protein expression patterns and elucidate the pathophysiology of diseases. For instance, routine pathological diagnosis of renal diseases requires interpretation of morphological alterations observed by light microscopy (LM), immunofluorescence (IF), and electron microscopy (EM), with correlation to clinical parameters. Immunohistochemical (IHC) staining of proteins has additionally been used to identify the protein of interest. In difficult or equivocal cases, more sensitive techniques such as immunoelectron microscopy (IEM) and laser microdissection mass spectrometry (LMD-MS) analyses may assist in the confirmation of identity and localization of renal protein deposits. In particular, progress has been demonstrated in the clinical utility of amyloid and kidney proteome analysis, which holds importance for diagnosing amyloidosis and characterization of renal diseases.

Amyloidosis. Amyloidosis is a group of diseases that result from build-up of insoluble protein aggregates in various tissues and organs. The diagnosis of amyloidosis presents a unique challenge as the clinical presentation is often subtle, and may overlap with several different conditions. Clinical management depends on the disease phenotype, which depends on the protein composition of the amyloid fibrils.

Accurate and comprehensive subtyping of amyloid diseases is critical for appropriate clinical management, since there are distinct treatment options for different subtypes. To date, there are over 36 different subtypes of localized and systemic amyloidosis syndromes.

The inherent limitations of antibody-based techniques led to the development of novel subtyping methods such as LMD-MS. In LMD-MS, a laser is coupled to a light microscope, and used to selectively dissect a subpopulation of protein aggregates of interest from the tissue on the slide. The protein mixture is denatured, digested with trypsin, and analyzed via LC-MS/MS.

LMD-MS has a major advantage over conventional amyloid subtyping, where it is a single test that can simultaneously identify and semi-quantify the amyloid protein composition, thereby improving the cost-effectiveness and efficiency in amyloid diagnosis. LMD-MS can identify the subtype of amyloidosis in more than 92–95 percent of cases. Overall, LMD-MS has higher sensitivity and specificity, compared with IF and IHC, and is currently the gold standard for amyloid subtyping. LMD-MS is indicated for the confirmation of amyloid subtype, when there are equivocal Congo red or heavy- and/or light-chain staining, and when there are inadequate tissue samples for IF studies, in difficult cases like differentiating Congophilic fibrillar glomerulonephritis (FGN) and amyloidosis, and in rare cases such as familial and hereditary amyloidosis.

Fibrillar glomerulonephritis. FGN is a rare glomerular disease that is associated with autoimmune diseases, viral infections, and malignant neoplasm. The pathogenesis and therapy for FGN remain largely unknown. More recently, serum levels of DNAJB9 were measured by immunoprecipitation-based MRM and demonstrated to be elevated in patients with FGN, when compared with non-FGN glomerular diseases, such as immunoglobulin light-chain amyloidosis or multiple myeloma, with adjustment for estimated glomerular filtration rate (eGFR) differences. Serum DNAJB9 predicted FGN with a moderate sensitivity (67%) and high specificity (98%) in a discovery cohort.

Further studies are needed to elucidate new variants of FGN, clinical utility of serum DNAJB9 as a non-invasive FGN biomarker, pathogenetic role of DNAJB9, and possibility as a targeted therapy. Overall, LMD-MS is indicated to help differentiate difficult cases, such as when there are equivocal Congo red staining, in the presence of heavy-chain component to distinguish monoclonal heavy-chain FGN from AH amyloidosis and heavy-chain deposition disease (HCDD), and differentiating Congophilic FGN from amyloidosis, FGN with concurrent renal diseases (IgA, membranous and diabetic nephropathy), and FGN from immunotactoid glomerulopathy (ITG), associated with chronic lymphocytic leukemia (CLL).

Immunotactoid glomerulopathy. ITG is another rare glomerular disease that is frequently associated with monoclonal gammopathies and hypocomplementemia, but not cryoglobulinemia and autoimmune diseases. The pathogenesis of ITG is largely unknown, and treatment depends on the underlying hematological process. In pathology, ITG is characterized by proliferative glomerulonephritis pattern by LM, monotypic IgG (>90% cases) with κ or λ light-chain restriction and C3 staining by IF, and protein deposits defined by microtubular structure with distinct hollow centers measuring 10–90 nm in diameter, deposited in organized, parallel arrays in the mesangium, subendothelial, and subepithelial space of the glomeruli by EM. LMD-MS confirmed the presence of monotypic immunoglobulins, and the activation of classical and terminal pathways of complement as C3 and C4 was identified in ITG cases. Comparing the kidney proteome profiles of amyloidosis, FGN, ITG and cryoglobulinemic glomerulonephritis by LMD-MS in a discovery cohort, apolipoprotein E (apo E) was hypothesized to be required for fibrillogenesis, where the size and organization of the protein deposits are associated with the ratio of apo E to immunoglobulin/amyloidogenic protein. Overall, LMD-MS for ITG is indicated to confirm the identity of protein deposits, and to distinguish difficult cases. For instance, ITG associated with CLL typically has small microtubules that may not show the distinct hollow centers and may resemble FGN fibrils even by EM, and LMD-MS proteomic analysis can support the differential diagnosis.

MS imaging. Another emerging technology that has potential clinical applications in pathology is MS imaging (MSI), which employs MS to map the spatial distribution of molecules in a thin tissue sample section. The gold standard of diagnosis for tissue samples has traditionally been a combination of chemical staining and light microscopy, with results being interpreted by a skilled anatomical pathologist. MSI, however, is remarkable for its ability to image thousands of molecules, such as lipids, proteins, peptides, drugs, and metabolites in tissues. MSI also possesses practical advantages compared with current methodology, including analyzing tissue directly (in situ), identifying endogenous biomolecules and exogenous compounds in a label-free and multiplex manner, and at the same time correlating molecular spatial distribution to traditional histology. The technique allows for an understanding of the molecular basis, and mechanism of disease with relation to tissue morphology. Clinical applications for MSI have been proposed to include tumor typing and disease staging, tumor margin assessment, intraoperative tumor excision, drug localization, and biomarker discovery.

One of the most common and well-studied mass spectrometers for MSI is the MALDI-ToF. Under vacuum, an ultraviolet or infrared laser irradiates the matrix co-crystallized tissue sample to desorb and ionize the analytes of interest in a raster-stepped sampling of the entire tissue surface. One of the limitations of MALDI-ToF MSI is that additional sample preparation is required, where the prepared tissue is coated with a MALDI matrix–an organic acid chemical compound that co-crystallizes and extracts the analyte during the pulsed laser ionization process. The quality of the matrix deposition and its application technique have significant effects on the analytical sensitivity (i.e., signal-to-noise ratio) and spatial resolution of the MS image.

Another vacuum-based MSI technique is the secondary ionization MS (SIMS), which uses a focused ion beam to ionize molecules in the tissue sample. Secondary ions are then generated and analyzed by the mass spectrometer. MALDI-ToF MSI typically can achieve 10–20 µm spatial resolution; meanwhile, SIMS MSI can achieve <1 µm spatial resolution. However, the drawbacks of SIMS are that it is a destructive technique that obliterates all molecules that it ionizes, has limitations in analyzing large intact biomolecules (>2 kDa), and requires complex instrumentation.

Catch-22 situation
MS has many applications in the clinical laboratory, including clinical chemistry, microbiology, and more recently anatomical pathology, as there are significant advantages for MS-based assays in their multiplexing capacity, high analytical specificity and sensitivity, and potential for real time in vivo analysis. Prior to clinical implementation of these MS-based methods, a specific gap in clinical need or question needs to be addressed and the advantages and limitations of MS-based methods should be compared with traditional methods in pathology. Additionally, there are desirable and practical features to consider, including high capital costs, requirement of skilled personnel, lack of automation, lack of direct bidirectional interface between MS instruments and laboratory or hospital information system, lack of standardization, and regulatory requirements. Significant progress is being actively pursued by the manufacturers and clinical MS community in regard to regulatory requirements, standardization of methods, automation in instrumentation and data analysis, and flat file interface to laboratory information systems to facilitate seamless order-to-result workflows. Operational factors like standardized workflow, turnaround time, and comprehensive biocomputational data analysis and storage should also be considered.

While future innovations in technology and instrumentation will drive novel clinical applications of MS to the forefront, the perpetual need of skilled individuals to drive the segment will always need to be addressed.