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Abstract Introduction:
Matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has been widely used in clinical fields for the analysis of large biomolecules. However, MALDI-ToF MS on small molecules are limited due to the fragmented matrix molecules during ionization process. Furthermore, the analyte crystals usually form inhomogeneous shape, so the mass signals vary by the sample spot morphology even in the constant laser irradiations. Therefore, the quantitative analysis has been limited for conventional MALDI-ToF MS. To overcome this limitation, various solid matrices were suggested to replace the organic matrices by using their structural stability in laser radiation. Moreover, the concept of combi-matrix also reported by mixing organic matrices and other type of materials to enhance the mass spectrometry results.
Objectives:
In this study, a combi-matrix system was prepared by mixing organic matrix with inorganic silica aerogel. The nanoporous structure and the unique thermal insulating properties of silica aerogel was demonstrated through the material characteristic analysis. Furthermore, Laser desorption/ionization mass spectrometry (LDI-MS) based on combi-matrix was applied to the quantitative analysis of dodecanoyl-L-carnitine (DC) using patient samples for clinical diagnosis of colon cancer.
Methods:
Silica aerogels for combi-matrix component was synthesized from sodium silicate precursor by two-step sol-gel process with ambient pressure drying. The combi-matrix was prepared by mixing silica aerogel and organic matrix (α-4-hydroxycinnamic acid, CHCA) in ethanol condition. The optimized ratio was confirmed by calculating S/N ratio from LDI-MS results of model analytes. The feasibility of combi-matrix for quantitative analysis was confirmed by fluorescence image test, shot-to-shot, and spot-to-spot reproducibility test. The difference in material perspective between organic matrix and combi-matrix was confirmed by thermal analysis using DSC. The quantitative analysis on small molecules with LDI-MS using combi-matrix conducted in various sample condition including patient real samples.
Results:
Synthesized silica aerogel was prepared into powder form. Average hydrodynamic radius of silica aerogel was measured to be 100-200 nm by dynamic light scattering (DLS) analysis. A typical pore structure of aerogel was confirmed by the scanning electron microscope (SEM) and the Brunauer, Emmett, and Teller (BET) method. The morphological data of aerogel was estimated with an average pore size of 9.3 nm and a porosity greater than 82.1%.
The applicability of silica aerogel for LDI-MS combi-matrix was demonstrated step-by-step by mixing it with a CHCA organic matrix. Silica aerogel itself resulted no mass peaks from model amino acid (histidine). However, the silica aerogel could be used for the elimination of mass peaks of the organic matrix by the mixed zone of matrix and analyte within the nanopores of aerogel. The component ratio between organic matrix and silica aerogel was controlled in the range from 1:0.2 to 1:2.6×10^-6 (weight per weight, w/w). The optimal ratio was estimated to be 1:1.6×10^-3 with the highest signal-to-ratio of 154.2 (a.u.).
The homogeneous and dense distribution of analytes for quantitative analysis was demonstrated by the fluorescence imaging of sample spot. The morphology of crystals in sample spot was compared at different concentrations of silica aerogel. The coffee-ring effect on fluorescence intensity was minimized as the aerogel component was increased to the former optimal ratio of combi-matrix. Additionally, the shot-to-shot reproducibility (96.0%) and spot-to-spot reproducibility (90.4%) were confirmed through the combi-matrix.
Investigation on the thermal properties of silica aerogel in combi-matrix was conducted using differential scanning calorimetry (DSC). The DSC analysis showed that the low thermal conductivity of silica aerogel reduces the thermal dissipation of laser energy through the sample plate. In addition, using the Gibbs-Thomson equation for organic matrix (CHCA), the average crystal radius of CHCA in the combi-matrix system was calculated to be smaller than that of the bulk state. This phenomenon represents the silica aerogel induces a nanoscale crystal of the organic matrix.
LDI-MS analysis using combi-matrix was conducted for the quantitative analysis of a biomarker for colon cancer, dodecanoyl-L-carnitine (DC). A standard curve obtained a linear correlation in the wide concentration range with a linearity factor over 0.94 for buffer and spiked serum conditions, respectively. A clinical diagnosis of colon cancer was demonstrated using a combi-matrix with real samples from healthy volunteers and cancer patients. The results from real samples were compared with the conventionally used LC-MS. The correlation of two experimental results was statistically analyzed using Bland-Altman plot and Passing-Bablok regression. Two methods distributed within the 95% confidence level, and the Spearman correlation coefficient was 0.947 (p<0.0001). Therefore, the LDI-MS with combi-matrix and conventional LC-MS results in statistically coincidence.
Conclusion:
In this work, a combi-matrix was presented based on the mixture of organic matrix and nanoporous silica aerogel as an inorganic component for quantitative analysis using LDI-MS instrument. The optimal ratio for combi-matrix was determined and small molecule analysis was conducted. The feasibility of quantitative analysis was confirmed by various ways, including fluorescence distribution, reproducibility test, and thermal property analysis. The combi-matrix was applied for the quantitative LDI-MS analysis of a biomarker for colon cancer, dodecanoyl-L-carnitine. Furthermore, by using serum samples from healthy volunteers and patients, clinical diagnosis of colon cancer was demonstrated using combi-matrix system.
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= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
