Enhancing precision medicine through clinical mass spectrometry platform

Abstract There is an extraordinary flood of new technologies in medicine nowadays. Sophisticated diagnostics based on genome assays, mass spectrometry and cell sorting platforms are driving the technological transfer and promote the entrance of individualized patient management in clinical practice. Mass spectrometry (MS) could be viewed as one of the major tools that promote the development of precision medicine (PM), which employs a patient’s genotype and phenotype investigation to establish individually tailored drug treatment. While genetic testing allows the physician to choose appropriate medicine, MS assays provide the patient’s actual phenotype, with all of the environmental, pharmacological and pathological variables. Therefore, MS is an essential technology for personalized patient management, and high-resolution MS systems are employed to resolve challenging analytical demands. The great technological advance of MS resulted in the introduction of methods with unprecedented identification power, extreme sensitivity, specificity and extended linearity range, which are simpler to use in the medical laboratories, and are based on the current reference analytical principles. Further, the ability to perform panel profiling with simultaneous measurement of bioactive compounds, their precursors and metabolites in a single sample, enormously amplifies the informative value of results, with ultimate improvement of patient care. Typical examples include newborn screening, therapeutic drug management, toxicology, endocrinology, microbiology, clinical omics assays and others. It should be specially emphasized that clinical MS integrates chemical and anatomical pathology: MS imaging and iKnife-MS guidance in surgery, although still in the research phase, open new horizons for personalized treatment and individualized patient care.


Introduction
Precision medicine (also known as personalized medicine, PM), as a strategy for customization of medical practices, is becoming integral part of health care. It utilizes in-depth molecular understanding of diseases and employs patient's genotype and phenotype investigation to establish individually tailored diagnosis, drug selection, and drug treatment [1,2]. PM is an active component of clinical management today, and evolves further research and translational efforts to broaden its application in achieving the final goal -to provide the best possible patient care in the form of monitoring (and improving) of health and disease, individualized prevention, early and proactive diagnosis, and personalized treatment [3]. It is clear that PM will become essential in the way healthcare is provided, and therefore, it is not surprising that Precision Medicine Initiative (PMI) announced by the White House in 2016 (https://obamawhitehouse.archives.gov) has gained global attention. However, translating data generated at the molecular level into clinically relevant information requires understanding and clinical validation of complex new technologies, such as third-generation sequencing, mass spectrometry (MS), cell sorting platforms, nuclear magnetic resonance spectroscopy and others. Advanced genetic and non-genetic testing combined with tools, such as bioinformatics, big data analysis, sophisticated machine learning and artificial intelligence (AI), drive and pave the entrance of PM in healthcare [4].
MS is among the leading technologies that drive the translation of research into clinical practice. In addition, MS has already achieved prominence in clinical medicine and therefore, it could be viewed as one of the major tools that promote the development of PM [5]. While genetic testing allows the physician to choose appropriate medicine, the performance of MS assays provides the patient's actual phenotype, with all of the environmental, pharmacological and pathological variables. Therefore, MS is essential technology for personalized patient management [1]. Triple quadrupole tandem MS (LC-MS/MS, QQQ) is the most utilized analytical platform today, but high-resolution MS systems are also employed to resolve challenging analytical demands. The great technological advance of MS resulted in the introduction of methods with unprecedented identification power, extreme sensitivity, specificity and extended linearity range, which are simpler to use in the medical laboratories, and are based on the current reference analytical principles [6]. Further, the ability to perform panel profiling with simultaneous measurement of bioactive compounds, their precursors and metabolites in a single sample, enormously amplifies the informative value of results, with ultimate improvement of patient care [1]. Typical examples include newborn screening, TDM, toxicology, endocrinology, microbiology, clinical omics assays and others. It should be specially emphasized that clinical MS integrates chemical and anatomical pathology: MS imaging and iKnife-MS guidance in surgery, although still in research phase, open new horizons for personalized treatment and individualized patient care.

Analytical principles of clinical mass spectrometry
MS is a physical chemistry branch analytical technique with history of more than 100 years, but its wide application in the medical laboratory was achieved within the last two or three decades, due to the introduction of excellent technical innovations, appropriate hardware and software tools, and clinically validated methods. Understanding the analytical principles of MS requires sufficiently deep knowledge of physical chemistry. This overview will only present a brief summary of selected MS variants with approved clinical application. MS is a powerful analytical technique used to identify and quantify analytes in biological fluids and tissues, most often coupled with a gas-chromatographic (GC) or liquid chromatographic (LC) separation step, which has three basic characteristics: (1) Analysis is performed in a vacuum. (2) Atoms and molecules are ionized before analysis. (3) Identification and quantitation of generated ions are based on their mass-tocharge (m/z) ratios. The MS analytical process consists of several consecutive steps. The first one is ionization; ions are thereafter separated according to their m/z ratios in a mass analyzer, and filtered ions are finally passed to a detector for counting [2].

Ion sources
The ion source is the first principal part of a mass spectrometer. During the ionization step ions are produced from the neutral atoms or molecules (M) in the ion source of the instrument by several mechanisms. Ions must be introduced in a gas phase in order to undergo separation by the mass analyzer within the vacuum system, no matter what the initial state of the sample is -gas, liquid or solid. Samples from a GC separation are already in a gas phase and are directly introduced in the mass spectrometer high-vacuum system.
The techniques most often used in the GC-MS systems for clinical use are electron impact (EI) ionization and chemical ionization (CI). EI is considered a hard-ionization technique in which high-voltage electrons bombard the molecules of interest inducing simultaneous production of ions and fragmentation of the molecules. Thus, a set or spectrum of fragment ions with respective abundance are produced of each analyte. The process is uniform and reproducible; the spectrum of ion fragments is unique for the specific compound, and this makes EI ionization very appropriate for clinical toxicology assays. Reliable identification of unknown component/s of a sample is achieved by comparing the experimental spectra of the unknown component/s with commercially available spectral libraries. CI methods involve EI of a reagent gas rather than direct bombardment of the molecules of interest. The resultant reagent gas ions transfer protons to the neutral molecule, thus producing molecular ions without significant fragmentation. Therefore, CI is considered a 'soft-ionization' method, suitable for quantitation and molecular mass determination.
Samples from a LC separation are in a liquid phase and cannot be directly introduced to the mass spectrometer. The most commonly used ionization techniques in LC-MS are electrospray ionization (ESI), atmospheric pressure CI (APCI) and atmospheric pressure photo ionization (APPI). They do not require a vacuum chamber and are performed at a near-atmospheric pressure, before a sample is introduced to the mass analyzer. Those are 'soft-ionization' methods in which molecules of interest are not destroyed, and ionization products are usually referred to as molecular ions, most often with added or distracted proton (M + H + or M − H + ). In ESI liquid phase samples coming out of the LC column are subjected to an electric field that expels a spray of large droplets. Application of gas flow along with heat induce continuous solvent evaporation with formation of smaller and smaller droplets with progressively increasing similar surface charges repulsing each other, eventual expulsion of ions from the droplet, until final evaporation of the liquid and formation of individual molecular ions in a gas phase, suitable to enter in the mass spectrometer. APCI is similar to ESI, which along with APPI is used to complement ESI to encompass the widest possible range of sample molecules. APCI is a spray technique evolving a two-step process starting with application of heat to turn the liquid phase into a gas phase. In the second step a corona discharge needle releases an electron cloud that bombards the gas phase molecules, producing single-charged molecular ions and little fragmentation. APPI is similar to APCI with one principal difference -it uses ultraviolet light instead of a corona discharge needle to ionize the gas phase molecules.
Matrix assisted laser desorption ionization (MALDI) usually coupled to time of flight (TOF) mass spectrometers is suitable for MS analysis of solid samples. Biological samples are applied to a support surface onto which analyte and matrix molecules crystallize in a solid form. Matrix molecules absorb UV energy near to the wavelength of the laser beam. The laser beam bombards onto a discrete small surface area, causes excitation, vaporization and ionization of analyte molecules, which are then transferred to the mass analyzer. The molecules of interest must be able to form crystals with the matrix molecules but must not interact with them. Because of matrix complexity and heterogeneity, MALDI techniques are not sufficiently reproducible to be used for quantitation and their primary clinical application is for identification, especially in microbiology [2,7].
Mass analyzers separate ions based on their m/z ratios and send them to a detector. They are two principal types: beam-type (magnetic sectors, quadrupole, TOF) and trapping-type (ion trap, orbitrap). Magnetic sector mass analyzers, the first introduced, separate ions through a magnetic field and a detector located at a 90° angle to the ion beam. Ions deflect at a different degree depending on their m/z ratios -heavier ones are deflected less than lighter ones. These instruments are very sensitive and provide high m/z resolution and accuracy, but are quite expensive and complex to use and therefore are not commonly found in medical laboratories. Quadrupole mass analyzers (Q), also referred to as 'mass filters' consist of four rods arranged in square fashion, forming a channel through which the ion beam is passed. The opposite rod pairs are charged by direct current (DC) and radio frequency (RF) voltages, which could be set differently to manipulate the trajectories of the individual ions entering the Q. Thus, in one case, under a particular set of DC and RF voltages, only a selected individual ion with a specific m/z ratio could have a stable trajectory, is able to pass through the Q channel and reaches the detector. Ions with different m/z ratios (non-resonant ions) have unstable trajectories, and are eliminated from the Q. Alternatively, DC and RF could be set to produce larger voltage range to detect all possible ions in the passing beam. In contrast to magnetic sectors, Q analyzers are not high resolution and exact mass instruments, but they are less expensive, easy to use, are conveniently interfaced with various ion sources, all of which makes them suitable for many clinical applications and the most common mass spectrometers used in medical laboratories nowadays.
Trapping-type mass analyzers are primarily divided into Q ion traps (QIT), ion cyclotron resonance mass spectrometers (ICR-MS) and orbitraps. QIT operate similarly to Q, but they collect and retain ions for a specific time, and are able to induce excitation and fragmentation before sending selected molecular and fragment ions to the detector. QIT are not commonly used in the clinical setting because of their limited dynamic range and size of the trap itself. They are not well suited for quantitation, and are used for identification or structural analyses. ICP-MS and orbitraps along with TOF-MS belong to the family of high-resolution or exact mass instruments. ICR-MS also known as Fourier transform ICR (FT-ICR) separates ions with extremely high resolution, accuracy and wider dynamic range, based on their trap and movement in a magnetic field, but its usage for clinical laboratory applications is very limited due to the complexity and the high costs of the instrumentation. The orbitrap MS is the newest edition of clinical MS with significant advantages and benefits compared to QIT and FT-ICR: it is less expensive, much easier to use, has a wide dynamic range comparable to Q-MS, along with highest mass accuracy and resolution. Although still not widely used in the medical setting, this instrument has the potential to identify and quantitate compounds of interest from a large set of analytes in complex matrices, and could be an extremely powerful tool in analytical toxicology and clinical proteomics.
TOF-MS utilizes an exceptionally straightforward separation principle: lighter is faster. These instruments measure the time required for each ion to pass through a tube from the ion source to the detector. An equal kinetic force is applied at the tube entrance, and thus ion velocity depends on its mass, and the flight time of an ion is directly proportional to its individual m/z ratio. Under these conditions small and lighter ions move faster than the large and heavier ions, and reach the detector earlier. TOF-MS is a rapid speed, high-resolution, accurate mass and sensitive instrument, often combined with MALDI (MALDI-TOF) for variety of applications in research and medical laboratories, mainly for proteomic studies, biomarker discovery and clinical microbiology analyses.
The detectors of the MS instruments are most often electron multipliers which register ions as they strike the detector. An initial low number of electrons is amplified by serial dynodes up to 10 8 electrons for each initial one, and this large signal is counted, digitalized, processed and recorded in nanoseconds for each individual ion [2,7].

Mass spectrometry changes the scene of chemical pathology
MS is currently widely used in a clinical setting for TDM and toxicology, newborn screening and genetic disorder programs, endocrinology and metabolism, paediatric clinical chemistry, cancer diagnostics, clinical immunology, microbiology, and even -point-of-care testing. Clinical MS techniques exponentially enhance our knowledge and abilities to improve patient care through deeper understanding of chemical pathology on an individual level, thus being one of the major tools paving the entrance of PM in medical practice.
Newborn screening was the first clinical area for MS/ MS applications, which shifted the paradigm towards omics-based strategies and PM in inborn errors of metabolism (IEM) investigations. High-throughput MS technologies in the post-genomic era transverse the entrance of systems biology in diagnosing IEM [9][10][11]. Classic and MS/MS methods for newborn screening are compared in Table 1.
Therapeutic drug management (earlier known as therapeutic drug monitoring, TDM) is a multidisciplinary endeavour encompassing skills from clinical pharmacology, laboratory analysis and clinical medicine. Its final goal is to provide the best possible patient care through individualization of drug therapy. The major approach of TDM is the development, validation and clinical application of objective and quantitative pharmacokinetic, pharmacodynamics and pharmacogenetic criteria for personally tailored pharmacotherapy [12]. TDM should be viewed as the beginning of PM in clinical practice. In 2009, Charles Pippenger, one of the founding fathers of TDM and the founder of the journal Therapeutic Drug Monitoring, wrote: 'In 1979, the world of TDM recognized the importance of personalized drug therapy; only we called it individualized drug therapy. But, regardless of what it is called, we know TDM is one of the core components of successful therapies' [13]. Currently, triple quadrupole LC-MS/MS is the most utilized analytical platform for routine TDM, providing fast, simultaneous and sensitive analysis of multiple drugs in micro-volume samples. The advantages, future perspectives and application of MS in the TDM services as a tool of PM have been reviewed [14]. Drugs of abuse and clinical toxicology is another field in which MS methods fully dominate and provide personalized diagnostics and treatment. GC-MS, with its ability for production of universal, reproducible and specific mass spectra, is traditionally a 'working horse' in analytical toxicology, but nowadays LC-MS/MS and LC-orbitrap techniques sustain essential part of the analytical arsenal in toxicology assays and significantly enhance the abilities for further in-depth understanding of the molecular mechanisms in that clinical field [15,16].
Endocrinology and metabolism benefitted enormously from clinical MS development. LC-MS/MS methods for steroid hormones, vitamin D metabolites quantitation, thyroid hormones, free metanephrines, angiotensins, oxytocin, ADH and many others, over-perform immunoassays and become routine and preferred techniques in many clinical laboratories [17][18][19]. In particular, steroid profiling allows for the simultaneous determination of the active compounds, their precursors and excretory products in a single sample, thus presenting the physiologic and pathologic relationships of the synthetic and metabolic chain on an individual level.
MALDI-TOF MS revolutionized diagnostic microbiology. While traditional microbiological methods require 48-72 h and are restricted regarding the number of microorganisms identified, MALDI-TOF MS differentiates thousands of cultured individual pathogens in a matter of minutes by detecting highly conserved microbial proteins and peptides (mainly ribosomal) and by matching the proteomic fingerprint from the sample to a known database. It is expected that in a very near future identification of microbes will be performed directly from patient samples [20,21].
Future trends happen today. Recently, Ashrafian et al. [3] described metabolomics as the stethoscope for the 21st century, reviewed technologies, clinical applications, translational challenges and stressed on the pivotal role of MS assays in PM. Beebe and Kennedy [22] defined metabolomics in the form of the individual's biochemical profile, as a key element to sharpen PM. Indeed, MS omics investigations are on the front line in many new and exciting research and clinical areas. In the human proteome project characterization of 16,092 out of 17,470 proteins is based on MS results [23]. MS methods are critical for in-depth understanding of post-translational protein modifications, protein signalling and protein-protein interactions [24,25]. MS empowers proteomic, peptidomic and metabonomic investigations in neurology, dermatology, intracrinology, gastroenterology, haematology, immunology, oncology and nephrology [26][27][28][29][30][31][32][33][34]. MS lipidomics studies and apolipoprotein profiling are used as a personalized approach for the diagnosis and treatment of dyslipidemia [35,36]. MS methods allow for the elucidation of post-translationally modified cardiovascular proteome and epigenetic landscape and their implication in coronary artery disease PM [37,38]. Metabolome MS analysis characterizes host-gut microbiota interactions and contributes to elucidating the role of the gut microbiome for drug disposition, drug pharmacokinetics and pharmacodynamics variability [39,40]. New MS technologies contribute to comprehensive and high throughput scale omics measurements of single cells [41]. LC-and GC-based high-resolution MS methods are applied for defining the role of exposome (the influence of environment on health and disease) in PM [42]. In the arena of personalized cancer medicine, very often mutation patterns do not predict tumour phenotype, and MS-based proteomic studies pave the way towards clinical implementation [43]. Mapping the tumour HLA ligandome (the entirety of human leucocyte antigens) with orbitrap MS enabled immence advance of cancer immunopeptidomics both answering basic scientific questions and contributing to the development of peptide specific cancer immunotherapies [44,45]. Oncoproteogenomic and glycoproteomic investigations utilizing MS are considered a novel platform for next-generation cancer neoantigen discovery for cancer vaccines [46,47].

From imagining to imaging through mass spectrometry
Mass spectrometry imaging (MSI) is a remarkably sophisticated molecular imaging technique providing high-end sensitivity for localization and specificity for identification of molecules submitting a label-free visualization that no other analytical approach is capable of. Mapping the spatial distribution and abundance of thousands of small biomolecules and metabolites in tissue samples brings scientific discoveries to a new level of knowledge on the interaction mechanisms in health and disease. Initially developed for studying proteins and peptides, the current potentialities of MSI extend far beyond proteomics combining biochemical and positional data with almost limitless application in omics profiling. It can also image lipids, sugars, metabolites, oligonucleotides and xenobiotics [48], elucidating the way to solving obscure clinical challenges. Thus, MSI provides a more comprehensive understanding of genotypic, phenotypic and metabolic responses of the tissue [49], which cannot be visualized by conventional modalities.
Cancer is a leading cause of death and disability worldwide, hence a principal driving force of precision medicine. Biomarker tracking and accurate detection of molecular signatures by MSI and MS profiling are nowadays increasing in-depth methodologies for investigation of tumour microenvironment and margins [50]. Tumours are heterogeneous by nature [51], as is their metabolism.
Proteins and peptides are preferred targets for molecular imaging. MALDI-MSI allows sparing investigation of the abundance and spatial distribution of multiple peptide ions [52] and precise mapping of tumour tissues and borderline regions. In the 'omics' era, MALDI-MSI gives an intense impulse to oncoproteomic studies overcoming some of the limitations of the classic histological imaging methods [53]. A number of researchers describe the superior performance characteristics of mass protein profiling in human breast cancer allowing for the identification of thousands of proteins from small tissue regions, thus suggesting an approach to improve the diagnostic effectiveness of widely used methods [54,55]. That brings the accuracy of breast cancer diagnostics to an unprecedented level. MALDI-MS protein profiling was used by Schwartz et al. [56] to determine the expression of characteristic peptide patterns in tumour tissue of patients with primary glioma. In addition, a small subset of proteins substantially involved in cell metabolism appeared to stand out as a survival discriminant. The technique was also used to evaluate the distinguishing profiles of different brain tumour types against non-tumour brain specimens [57]. MSI can be used for both characterization of normal pulmonary section patterns [58] and lung cancer tissue [59]. The capability of untargeted molecular investigation practically gives ground to MSI application for credible as well as cost-effective visualization of all tumour types. MALDI imaging and profiling was successfully used for tracing valuable markers for identification, grading and prognosis of ovarian [53], prostate [60], urothelial [61] and a variety of other types of cancer.
Although proteins are known to be key-point biomarkers in clinical research and applied analysis, tumour heterogeneity is not limited to the proteome. Promising studies of carnitine pathways seek to answer important questions about malignant tumours and their management. Harbouring a quaternary amine group which can attain a positive charge, most of the studied biomolecules of the carnitine system wield good ionization properties [62]. First described by Caprioli et al. [63], MALDI-MSI was proposed by Sun et al. [64] for visualization of altered carnitine metabolism in heterogeneous breast cancer, where the study demonstrated significantly higher levels of L-carnitine and acylcarnitines in cancer tissue in comparison to normal tissues. A recent MS lipidomic study provided information on the potential of 11 lipid classes for rapid intraoperative determination of breast cancer margins [65]. Reprogramming and accretion is associated with increased activity and coordinate expression of several lipogenic enzymes in tumour cells [66] and superimposed impaired lipid-related biochemical processes [67], such as inhibition of β-oxidation in hypoxic tumour regions. Untargeted lipidomic analysis of brain tumours reached even more profound realization of the intermediates in a dysregulated carnitine shuttle system [68], offering an encouraging perspective of MSI in the field of neurobiological science. The molecular imaging application in brain tumours is still limited, nonetheless there are a few studies demonstrating mass spectra-based distinctive lipid profiles of different types of malignancies [69]. Ovarian carcinoma lipidomic profiling showed drastic changes in the lipid composition of the entire cell proving the occurrence of a shift from lipid uptake to de novo lipogenesis [70].
Glycosylation is one of the most common post-translational modifications and serves many functions [71]. As a general feature of a multiplicity of disorders, aberrant glycosylation can also be used for identification and characterization of tumour tissue [72]. The large number of structures in the glycome requires effective separation methods [73], and owing to its eminent sensitivity and large peak capacity, MS reveals high potentiality for capturing and visualization of glycans. In addition, mapping of glycan distribution and identification of their glycoprotein carriers by MALDI-MSI provides a novel perspective to precision oncology. The reasoning of proteomic-glycomic characterization strategies would set a sustainable template for subsequent studies in this field [74].
Mass spectrometry application in cancer research is just a fragment of the global multi-omic painting. Molecular profiling of socially significant disturbances and diseases that gives a deeper insight into the complexity of intra-and inter-cellular interactions adduced as a helpful tool for management of a variety of clinical entities, including cardiovascular diseases [75,76], infections [71,77], male and female infertility [78,79], particularly endometriosis affecting approximately 10% of women in their reproductive years [80].
Molecular studies on drug bioaccumulation and biotransformation introduce limitless opportunities for imaging and quantification of pharmaceutical compounds in biological specimens, far beyond drug disposition studies. MALDI-MSI was applied for drug distribution quantification using a mimetic tissue model where both lapatinib and nevirapine were quantified in tissue homogenates spiked with different drug concentrations [81]. Spatial profiling is a promising approach for studying the distribution of metal-based anticancer drugs, such as platinum-and ruthenium-based drugs, with a view to their effectiveness, adverse effects on healthy tissues, and drug resistance [82]. Monitoring of circulating drug levels through classic pharmacokinetic analysis cannot strictly mirror intra-tumour drug concentrations, respectively the drug effectiveness, mainly because of the irregular tumour vascularization. A recent study showed the superior role of MSI for determination of tumour tissue distribution of two Poly (ADP-ribose) polymerase inhibitors (PARPi), niraparib and olaparib, unravelling the discrepancies between their clinical efficacy in vivo and inhibition potency in vitro [83]. Additionally, MSI was used for accurate quantification of antibiotics and their metabolites in tissue sections, pooled plasma and biofilm-inhibiting multilayers [84][85][86]. Imaging data obtained through MALDI-technique confirmed inadequate antiretroviral penetration in persistent HIV replication [87]. In conclusion, the rapidly increasing number of visualization/quantification studies of drugs and their metabolites in animal models distinctly outline the rising role of MS-based imaging as a paramount tool in precision pharmacology.

From imaging to execution through intrasurgical mass spectrometry
Surgical oncology has reached an uncontestable summit in recent years through a couple of discerning modalities for real-time assessment of tumour margins. Precise techniques, such as rapid evaporative ionization mass spectrometry (REIMS), also known as intelligent knife (iKnife), having undergone a series of optimizations, now cover to a large extent some of the unanswered demands of tissue-conservation strategy [88]. The iKnife allows for a direct intra-operative molecular profiling in surgical aerosol [89]. Thereafter, the data-rich vapour is analyzed by time-of-flight (ToF) MS to discriminate promptly and accurately between healthy, premalignant and invasive tissue in studies on cervical diseases [90], ovarian tumours [91], breast cancer [89], colorectal cancer [92], etc. The resolution assets of intelligent surgery through mass spectrometry give a new insight towards tumour biology and therapeutic response that could reduce the need of revision interventions and prevent applying adjuvant chemotherapy. A recent study proposed the first-time perioperative use of iKnife along with a deep learning framework for detection of basal cell carcinoma signatures from tissue burns [93].
Desorption electrospray ionization (DESI) is the most extensively used soft ionization non-destructive technique that transfers the analytes intact into the gas phase, thus preserving the integrity of the area of analysis. The performance at ambient conditions is a huge advantage over MALDI, since no sample preparation is required. Combining DESI with field asymmetric waveform ion mobility (FAIMS) demonstrated a significant increase of protein ions signal-to-noise, thus enhancing the diagnostic effectiveness through improved imaging contrast and quality [94,95]. A sequential step ahead in precision surgical oncology, the MasSpec Pen is a handheld biocompatible device using ambient ionization MS capable of providing the highest degree of sparing online in vivo molecular diagnosis with revolutionary accuracy [96]. The drop-in version allows integration of the MasSpec Pen into minimally invasive robotic-assisted surgical systems [97], where the tool is used for immediate direct molecular profiling contributing to refinement of surgical resections and facilitated decision-making.

Final remarks
In conclusion, PM has been intensively promoted in recent years, and is becoming integral part of patient care. Many innovative technologies drive the research and clinical development of PM as a strategy to improve health and disease management at an individual level. Among these, MS, coupled to adaptive bioinformatic pattern-recognition tools, is of particular importance and has potential to enhance the personalization of healthcare [5]. Together with the approved clinical use of various MS techniques, advanced novel MS platforms enable the investigation and understanding of omics profiles which go beyond the general patient and open the era of personalized signatures and disease biomarkers [1,98,99]. In addition, MS is unique with its ability to link together and integrate chemical and anatomical pathology. Of course, the mass amount of data produced by novel MS techniques require the aid of big data analytics, AI, and enhanced translation of precision mathematics to PM [100]. Beyond any doubt, enhancing PM with MS platform results in exponential advancement in our understanding of health, disease prevention and individualization of therapy.

Disclosure statement
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Data availability statement
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Funding
The author(s) reported there is no funding associated with the work featured in this article.