Abstract
A liquid matrix-assisted laser desorption/ionization (liquid MALDI) method has been developed for high-throughput atmospheric pressure (AP) mass spectrometry (MS) analysis of the molecular content of crude bioliquids for disease diagnostics. The presented method is rapid and highly robust, enabling its application in environments where speed and low-cost high-throughput analyses are required. Importantly, because of the creation of multiply charged analyte ions, it provides additional functionalities that conventional solid MALDI MS profiling is lacking, including the use of high-performance mass analyzers with limited m/z range. The concomitant superior MS/MS performance that is achieved similar to ESI MS/MS adds greater analytical power and specificity to MALDI MS profiling while retaining the advantages of a fast laser-based analysis system and off-line large-scale sample preparation. The potential of this MALDI MS profiling method is demonstrated on the detection of dairy cow mastitis, which is a substantial economic burden on the dairy industry with losses of hundreds of dollars per diseased cow per year, equating to a total annual loss of billions of dollars, as well as leading to the use of large quantities of antibiotics, adding to the proliferation of antimicrobial resistance. Only small amounts of aliquots obtained from the daily farm milking process were prepared for liquid MALDI MS profiling using a simple one-pot/two-step analyte extraction. Automated analysis was performed using a custom-built AP-MALDI ion source, enabling the simultaneous detection of lipids, peptides, and proteins. Diagnostic, multiply charged, proteinaceous ions were easily sequenced and identified by MS/MS experiments. Samples were classified according to mastitis status using multivariate analysis, achieving 98.5% accuracy (100% specificity) determined by “leave 20% out” cross-validation. The methodology is generally applicable to AP-MALDI MS profiling on most commercial high-resolution mass spectrometers, with the potential for expansion into hospitals for rapid assessment of human and other biofluids.
Highlights
Conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) profiling has been applied in many areas of disease analysis with varying success
Raw milk samples were collected from individual cows at the Centre for Dairy Research at the University of Reading, UK, and prepared for liquid MALDI MS analysis by a one-pot/twostep extraction, taking less than 5 min
Samples were taken from individual udder quarters of 109 cows for a total of 135 samples, with some cows being sampled on multiple dates
Summary
Conventional (solid) matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) profiling has been applied in many areas of disease analysis with varying success. MALDI a prime candidate for the development of a powerful MS profiling method that can add the advances made in modern ESI MS to the speed, robustness, and easiness of MALDI MS analysis. Validated milk molecular biomarkers for diagnosing mastitis have yet to be described,[9] but several candidates have been proposed.[18−23] Once validated molecular biomarkers are available, targeted assays for their specific detection and quantitation, such as immunoassays, are typically developed These classical diagnostic tests often reach their limits with respect to marker specificity and multiplexing, to name but a few. The potential of “on-the-fly” MS/MS analysis of multiply charged analyte ions for greater analytical specificity, as well as the use of modern bioanalytics developed for clinical applications, significantly adds to the power of this MS method and is demonstrated in this study
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