Complexity, cost, and content are three important factors that can impede translation of clinical protein mass spectrometry (MS) tests at a larger scale. Complexity stems from the many components/steps involved in bottom-up protein MS workflows, making them significantly more complicated than enzymatic immunoassays (EIA) that currently dominate clinical testing. This complexity inevitably leads to increased costs, which is detrimental in the price-competitive clinical marketplace. To successfully compete, new clinical protein MS tests need to offer something new and unique that EIAs cannot – a new content of proteoform detection. The preferred method for proteoform profiling is intact protein MS analysis, in which all proteins are measured as intact species thus allowing discovery of new proteoforms. To illustrate the importance of intact proteoform testing with MS and its potential clinical implications, we discuss here recent findings from multiple studies on the distribution of apolipoprotein C-III proteoforms and their correlations with key clinical measures of dyslipidemia. Such studies are only made possible with assays that are low in cost, avoid unnecessary complexity, and are unique in providing the content of proteoforms.

November 22, 2019

Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation

Kendall W. Cradic, Paula M. Ladwig, Ann L. Rivard, Waddah Katrangi, Karl Florian Wintgens, Maria A.V. Willrich

Page range: 864-872

Abstract

Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

October 22, 2019

Development and evaluation of an element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection: can we apply the new assay in the clinical laboratory?

Wencan Jiang, Gongwei Sun, Xinyu Wen, Shasha Men, Wenbin Cui, Miao Jing, Xingwang Jia, Zhian Hu, Danna Pu, Sichun Zhang, Xiaozhou Yuan, Xiaoting Liu, Xinrong Zhang, Chengbin Wang

Page range: 873-882

Abstract

Introduction Element-tagged immunoassay coupled with inductively coupled plasma-mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis in clinical detection; however, a systematic evaluation with the standard guidelines of the assay is needed to ensure its performance meets the requirements of the clinical laboratory. Methods Carcinoembryonic antigen (CEA) was chosen for analysis using the proposed method. A systematic evaluation of the proposed assay was carried out according to the Clinical and Laboratory Standards Institute (CLSI). The 469 clinical samples were analyzed using the new method and compared with the electrochemiluminescent immunoassay (ECLIA) method. Results The measurement range of the assay was 1–900 ng/mL, with a detection limit of 0.83 ng/mL. The inter-assay and intra-assay imprecision were 4.67% and 5.38% with high concentration samples, and 9.27% and 17.64% with low concentration samples, respectively. The cross-reactivity (%) for different antigens was less than 0.05%, and the recovery was between 94% and 108%. Percentage deviation of all the dilutions was less than 12.5% during linearity estimation. The interference bias caused by different substances was less than 10%. The reference interval of the assay was 0–4.442 ng/mL. Comparison with the commercial ECLIA method for clinical sample detection, the proposed method showed a correlation of 0.9878 and no significant differences between the methods were observed (p = 0.6666). Conclusions The ICP-MS based immunoassay was successfully developed, and the analytical performance of the assay met the requirements of the CLSI, which fully proved the clinical transferability and application of the new method.

MALDI-MS for the clinic

March 25, 2020

Matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS): basics and clinical applications

Muhammad Zubair Israr, Dennis Bernieh, Andrea Salzano, Shabana Cassambai, Yoshiyuki Yazaki, Toru Suzuki

Page range: 883-896

Abstract

Background Matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS) has been used for more than 30 years. Compared with other analytical techniques, it offers ease of use, high throughput, robustness, cost-effectiveness, rapid analysis and sensitivity. As advantages, current clinical techniques (e.g. immunoassays) are unable to directly measure the biomarker; rather, they measure secondary signals. MALDI-MS has been extensively researched for clinical applications, and it is set for a breakthrough as a routine tool for clinical diagnostics. Content This review reports on the principles of MALDI-MS and discusses current clinical applications and the future clinical prospects for MALDI-MS. Furthermore, the review assesses the limitations currently experienced in clinical assays, the advantages and the impact of MALDI-MS to transform clinical laboratories. Summary MALDI-MS is widely used in clinical microbiology for the screening of microbial isolates; however, there is scope to apply MALDI-MS in the diagnosis, prognosis, therapeutic drug monitoring and biopsy imaging in many diseases. Outlook There is considerable potential for MALDI-MS in clinic as a tool for screening, profiling and imaging because of its high sensitivity and specificity over alternative techniques.

Publicly Available February 12, 2020

Clinical use of mass spectrometry (imaging) for hard tissue analysis in abnormal fracture healing

Sylvia P. Nauta, Martijn Poeze, Ron M.A. Heeren, Tiffany Porta Siegel

Page range: 897-913

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Abstract

Common traumas to the skeletal system are bone fractures and injury-related articular cartilage damage. The healing process can be impaired resulting in non-unions in 5–10% of the bone fractures and in post-traumatic osteoarthritis (PTOA) in up to 75% of the cases of cartilage damage. Despite the amount of research performed in the areas of fracture healing and cartilage repair as well as non-unions and PTOA, still, the outcome of a bone fracture or articular cartilage damage cannot be predicted. Here, we discuss known risk factors and key molecules involved in the repair process, together with the main challenges associated with the prediction of outcome of these injuries. Furthermore, we review and discuss the opportunities for mass spectrometry (MS) – an analytical tool capable of detecting a wide variety of molecules in tissues – to contribute to extending molecular understanding of impaired healing and the discovery of predictive biomarkers. Therefore, the current knowledge and challenges concerning MS imaging of bone and cartilage tissue as well as in vivo MS are discussed. Finally, we explore the possibilities of in situ , real-time MS for the prediction of outcome during surgery of bone fractures and injury-related articular cartilage damage.

Publicly Available October 30, 2019

Cellular resolution in clinical MALDI mass spectrometry imaging: the latest advancements and current challenges

Klára Ščupáková, Benjamin Balluff, Caitlin Tressler, Tobi Adelaja, Ron M.A. Heeren, Kristine Glunde, Gökhan Ertaylan

Page range: 914-929

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Abstract

Mass spectrometry (MS) is the workhorse of metabolomics, proteomics and lipidomics. Mass spectrometry imaging (MSI), its extension to spatially resolved analysis of tissues, is a powerful tool for visualizing molecular information within the histological context of tissue. This review summarizes recent developments in MSI and highlights current challenges that remain to achieve molecular imaging at the cellular level of clinical specimens. We focus on matrix-assisted laser desorption/ionization (MALDI)-MSI. We discuss the current status of each of the analysis steps and remaining challenges to reach the desired level of cellular imaging. Currently, analyte delocalization and degradation, matrix crystal size, laser focus restrictions and detector sensitivity are factors that are limiting spatial resolution. New sample preparation devices and laser optic systems are being developed to push the boundaries of these limitations. Furthermore, we review the processing of cellular MSI data and images, and the systematic integration of these data in the light of available algorithms and databases. We discuss roadblocks in the data analysis pipeline and show how technology from other fields can be used to overcome these. Finally, we conclude with curative and community efforts that are needed to enable contextualization of the information obtained.

December 19, 2019

Bacterial identification by lipid profiling using liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

Sophie E. Lellman, Rainer Cramer

Page range: 930-938

Abstract

Background In recent years, mass spectrometry (MS) has been applied to clinical microbial biotyping, exploiting the speed of matrix-assisted laser desorption/ionization (MALDI) in recording microbe-specific MS profiles. More recently, liquid atmospheric pressure (AP) MALDI has been shown to produce extremely stable ion flux from homogenous samples and ‘electrospray ionization (ESI)-like’ multiply charged ions for larger biomolecules, whilst maintaining the benefits of traditional MALDI including high tolerance to contaminants, low analyte consumption and rapid analysis. These and other advantages of liquid AP-MALDI MS have been explored in this study to investigate its potential in microbial biotyping. Methods Genetically diverse bacterial strains were analyzed using liquid AP-MALDI MS, including clinically relevant species such as Escherichia coli , Staphylococcus aureus and Klebsiella pneumoniae . Bacterial cultures were subjected to a simple and fast extraction protocol using ethanol and formic acid. Extracts were spotted with a liquid support matrix (LSM) and analyzed using a Synapt G2-Si mass spectrometer with an in-house built AP-MALDI source. Results Each species produces a unique lipid profile in the m / z range of 400–1100, allowing species discrimination. Traditional (solid) MALDI MS produced spectra containing a high abundance of matrix-related clusters and an absence of lipid peaks. The MS profiles of the bacterial species tested form distinct clusters using principle component analysis (PCA) with a classification accuracy of 98.63% using a PCA-based prediction model. Conclusions Liquid AP-MALDI MS profiles can be sufficient to distinguish clinically relevant bacterial pathogens and other bacteria, based on their unique lipid profiles. The analysis of the lipid MS profiles is typically excluded from commercial instruments approved for clinical diagnostics.

Clinical application of ’omics technologies

April 5, 2019

Individualized metabolomics: opportunities and challenges

Biswapriya Misra

Page range: 939-947

Abstract

The goal of advancing science in health care is to provide high quality treatment and therapeutic opportunities to patients in need. This is especially true in precision medicine, wherein the ultimate goal is to link disease phenotypes to targeted treatments and novel therapeutics at the scale of an individual. With the advent of -omics technologies, such as genomics, proteomics, microbiome, among others, the metabolome is of wider and immediate interest for its important role in metabolic regulation. The metabolome, of course, comes with its own questions regarding technological challenges. In this opinion article, I attempt to interrogate some of the main challenges associated with individualized metabolomics, and available opportunities in the context of its clinical application. Some questions this article addresses and attempts to find answers for are: Can a personal metabolome (n = 1) be inexpensive, affordable and informative enough (i.e. provide predictive yet validated biomarkers) to represent the entirety of a population? How can a personal metabolome complement advances in other -omics areas and the use of monitoring devices, which occupy our personal space?

February 18, 2020

Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre

Diana Canetti, Nigel B. Rendell, Janet A. Gilbertson, Nicola Botcher, Paola Nocerino, Angel Blanco, Lucia Di Vagno, Dorota Rowczenio, Guglielmo Verona, P. Patrizia Mangione, Vittorio Bellotti, Philip N. Hawkins, Julian D. Gillmore, Graham W. Taylor

Page range: 948-957

Abstract

Systemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined.

Publicly Available January 28, 2020

The “olfactory fingerprint”: can diagnostics be improved by combining canine and digital noses?

Giuseppe Lippi, Liam M. Heaney

Page range: 958-967

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Abstract

A sniffer (detecting) dog is conventionally defined as an animal trained to use its olfactory perceptions for detecting a vast array of substances, mostly volatile organic compounds (VOCs), including those exceptionally or exclusively generated in humans bearing specific pathologies. Such an extraordinary sniffing performance translates into the capability of detecting compounds close to the femtomolar level, with performance comparable to that of current mass spectrometry-based laboratory applications. Not only can dogs accurately detect “abnormal volatilomes” reflecting something wrong happening to their owners, but they can also perceive visual, vocal and behavioral signals, which altogether would contribute to raise their alertness. Although it seems reasonable to conclude that sniffer dogs could never be considered absolutely “diagnostic” for a given disorder, several lines of evidence attest that they may serve as efficient screening aids for many pathological conditions affecting their human companions. Favorable results have been obtained in trials on cancers, diabetes, seizures, narcolepsy and migraine, whilst interesting evidence is also emerging on the capability of early and accurately identifying patients with infectious diseases. This would lead the way to proposing an “olfactory fingerprint” loop, where evidence that dogs can identify the presence of human pathologies provides implicit proof of the existence of disease-specific volatilomes, which can be studied for developing laboratory techniques. Contextually, the evidence that specific pathologies are associated with abnormal VOC generation may serve as reliable basis for training dogs to detect these compounds, even (or especially) in patients at an asymptomatic phase.

Open Access March 26, 2020

Peptidomic and proteomic analysis of stool for diagnosing IBD and deciphering disease pathogenesis

Daniela Basso, Andrea Padoan, Renata D’Incà, Giorgio Arrigoni, Maria Luisa Scapellato, Nicole Contran, Cinzia Franchin, Greta Lorenzon, Claudia Mescoli, Stefania Moz, Dania Bozzato, Massimo Rugge, Mario Plebani

Page range: 968-979

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Abstract

Background The sensitivities and specificities of C-reactive protein (CRP) and faecal calprotectin (fCal), as recommended for inflammatory bowel diseases (IBD) diagnosis and monitoring, are low. Our aim was to discover new stool protein/peptide biomarkers for diagnosing IBD. Methods For peptides, MALDI-TOF/MS ( m / z 1000–4000) was performed using stools from an exploratory (34 controls; 72 Crohn’s disease [CD], 56 ulcerative colitis [UC]) and a validation (28 controls, 27 CD, 15 UC) cohort. For proteins, LTQ-Orbitrap XL MS analysis (6 controls, 5 CD, 5 UC) was performed. Results MALDI-TOF/MS spectra of IBD patients had numerous features, unlike controls. Overall, 426 features (67 control-associated, 359 IBD-associated) were identified. Spectra were classified as control or IBD (absence or presence of IBD-associated features). In the exploratory cohort, the sensitivity and specificity of this classification algorithm were 81% and 97%, respectively. Blind analysis of the validation cohort confirmed 97% specificity, with a lower sensitivity (55%) paralleling active disease frequency. Following binary logistic regression analysis, IBD was independently correlated with MALDI-TOF/MS spectra (p < 0.0001), outperforming fCal measurements (p = 0.029). The IBD-correlated m / z 1810.8 feature was a fragment of APC2, homologous with APC, over-expressed by infiltrating cells lining the surface in UC or the muscularis-mucosae in CD (assessed by immunohistochemistry). IBD-associated over-expressed proteins included immunoglobulins and neutrophil proteins, while those under-expressed comprised proteins of the nucleic acid assembly or those (OLFM4, ENPP7) related to cancer risk. Conclusions Our study provides evidence for the clinical utility of a novel proteomic method for diagnosing IBD and insight on the pathogenic role of APC. Moreover, the newly described IBD-associated proteins might become tools for cancer risk assessment in IBD patients.

January 15, 2020

The influence of hypoxia on the prostate cancer proteome

James A. Ross, Johannes P.C. Vissers, Jyoti Nanda, Grant D. Stewart, Holger Husi, Fouad K. Habib, Dean E. Hammond, Lee A. Gethings

Page range: 980-993

Abstract

Prostate cancer accounts for around 15% of male deaths in Western Europe and is the second leading cause of cancer death in men after lung cancer. Mounting evidence suggests that prostate cancer deposits exist within a hypoxic environment and this contributes to radio-resistance thus hampering one of the major therapies for this cancer. Recent reports have shown that nitric oxide (NO) donating non-steroidal anti-inflammatory drugs (NSAIDs) reduced tumour hypoxia as well as maintaining a radio-sensitising/therapeutic effect on prostate cancer cells. The aim of this study was to evaluate the impact of hypoxia on the proteome of the prostate and to establish whether NO-NSAID treatment reverted the protein profiles back to their normoxic status. To this end an established hormone insensitive prostate cancer cell line, PC-3, was cultured under hypoxic and normoxic conditions before and following exposure to NO-NSAID in combination with selected other common prostate cancer treatment types. The extracted proteins were analysed by ion mobility-assisted data independent acquisition mass spectrometry (MS), combined with multivariate statistical analyses, to measure hypoxia-induced alterations in the proteome of these cells. The analyses demonstrated that under hypoxic conditions there were well-defined, significantly regulated/differentially expressed proteins primarily involved with structural and binding processes including, for example, TUBB4A, CIRP and PLOD1. Additionally, the exposure of hypoxic cells to NSAID and NO-NSAID agents, resulted in some of these proteins being differentially expressed; for example, both PCNA and HNRNPA1L were down-regulated, corresponding with disruption in the nucleocytoplasmic shuttling process.

Laboratory automation and kit-based approaches

March 19, 2020

Mass spectrometry and total laboratory automation: opportunities and drawbacks

Gian Luca Salvagno, Elisa Danese, Giuseppe Lippi

Page range: 994-1001

Abstract

The diffusion of laboratory automation, initiated nearly 50 years ago with consolidation of preanalytical, clinical chemistry and immunochemistry workstations, is now also gradually embracing mass spectrometry (MS). As for other diagnostic disciplines, the automation of MS carries many advantages, such as efficient personnel management (i.e. improving working atmosphere by decreasing manual activities, lowering health risks, simplifying staff training), better organization (i.e. reducing workloads, improving inventory handling, increasing analytical process standardization) and the possibility to reduce the number of platforms. The development and integration of different technologies into automated MS analyzers will also generate technical and practical advantages, such as prepackaged and ready-to-use reagents, automated dispensing, incubation and measurement, automated sample processing (e.g. system fit for many models of laboratory automation, bar code readers), multiplex testing, automatic data processing, also including quality control assessment, and automated validation/interpretation (e.g. autoverification). A new generation of preanalytical workstations, which can be directly connected to MS systems, will allow the automation of manual extraction and elimination of time-consuming activities, such as tube labeling and capping/decapping. The use of automated liquid-handling platform for pipetting samples, along with addition of internal standards, may then enable the optimization of some steps of extraction and protein precipitation, thus decreasing turnaround time and increasing throughput in MS testing. Therefore, this focused review is aimed at providing a brief update on the importance of consolidation and integration of MS platforms in laboratory automation.

February 18, 2020

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Caroline Le Goff, Jordi Farre-Segura, Violeta Stojkovic, Patrice Dufour, Stéphanie Peeters, Justine Courtois, Adrien Nizet, Nathalie De Vos, Etienne Cavalier

Page range: 1002-1009

Abstract

Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an “ready-to-use” (RTU) kit for steroids analysis.

December 19, 2019

Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory

Sally C. Benton, Godwin K. Tetteh, Sarah-Jayne Needham, Jakob Mücke, Leanne Sheppard, Steven Alderson, Corinne Ruppen, Maurus Curti, Maurice Redondo, Anna M. Milan

Page range: 1010-1017

Abstract

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantages over immunoassay due to its increased specificity and ability to multiplex metabolites within a single run. Wide scale adoption of LC-MS/MS in routine clinical laboratories is restricted in part due to the high level of technical expertise required. The Thermo Scientific™ Cascadion™ SM Clinical Analyzer is the first fully automated, random access clinical analyser that utilises LC-MS/MS technology. We report an analytical validation of the 25-hydroxy vitamin D2 and D3 assays on the Cascadion Analyzer and an assessment of its performance within a routine clinical laboratory. Methods Analyser usability was assessed by staff with no previous experience of LC-MS/MS. An analytical validation included analysis of 154 patient samples on two different Cascadion Analyzers and a four-way method comparison of 146 patient samples on Roche and Siemens immunoassays and an in-house LC-MS/MS method. Accuracy was assessed using external quality assurance and reference materials. Seven third party IQC materials were tested on Cascadion. Results Cascadion proved easy to use by scientific and non-scientific staff. The assay passed all validation criteria. Excellent agreement was demonstrated between two different Cascadions (y = 0.97x + 3.9 nmol/L, r 2 > 0.99). A method comparison demonstrated no significant difference (p > 0.05) between the Cascadion and the Roche immunoassay. A significant difference (p < 0.0001) was observed between the Cascadion and an LC-MS/MS and Siemens methods. Results obtained from EQA and reference material showed a mean bias of +3.09% and all samples were within ±10% of assigned concentrations. All third party IQC samples tested were compatible for use with Cascadion. Conclusions The Cascadion Analyzer is a fully automated LC-MS/MS system that requires no prior LC-MS/MS expertise. The vitamin D assays demonstrated excellent performance with high levels of accuracy.

About this journal

Objective
Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. It is focused on basic and applied research and cutting-edge clinical laboratory medicine. CCLM is one of the leading journals in the field, with an impact factor over 8. All contributions submitted for publication in CCLM are single-blind peer reviewed by at least two experts in the field. CCLM is led by a multi-institutional editorial board. It is issued monthly, both in print and electronically. Letters to the Editor and Congress Abstracts are published online only.

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Topics

clinical biochemistry
clinical genomics and molecular biology
clinical haematology and coagulation
clinical immunology and autoimmunity
clinical microbiology
drug monitoring and analysis
evaluation of diagnostic biomarkers
disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
new reagents, instrumentation and technologies
new methodologies
reference materials and methods
reference values and decision limits
quality and safety in laboratory medicine
translational laboratory medicine
clinical metrology

Article formats
Research Articles, Reviews, Mini Reviews and Opinion Papers on all aspects of clinical chemistry and laboratory medicine, Guidelines and Recommendations, Point/Counterpoint Articles, Letters to the Editor, Editorials

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