Development and qualification of an LC-MS/MS method for quantification of MUC5AC and MUC5B mucins in spontaneous sputum

Abstract

Aim: Airway mucins in sputum are promising respiratory disease biomarkers, despite posing substantial analytical challenges due to their physicochemical properties and rare and heterogenous nature of the matrix. We aimed to identify a suitable sputum collection and processing method, and qualify a bioanalytical method for MUC5AC and MUC5B quantification in clinical samples.

Method: Mucins were quantified in induced and spontaneous sputum collected from the same COPD patients, following various sample processing procedures. LC-MS/MS method used truncated recombinant mucins as surrogate analytes in surrogate matrix.

Results: Frozen spontaneous sputum was found to be a suitable and convenient matrix for mucin quantification and fit-for-purpose method qualification was performed.

Conclusion: Our methodology provides accurate and reliable MUC5AC and MUC5B quantification and facilitates multi-site clinical sputum collection.

Keywords: LC-MS/MS; MUC5AC; MUC5B; bioanalytical method; method qualification; sputum collection; sputum processing.

Figure 1.

Summary of the LC-MS/MS method development, sputum collection and processing assessment, and method qualification. IS: internal standard; BSA: bovine serum albumin; PBS: phosphate buffered saline; DTT: dithiothreitol. Created with BioRender.com.

Figure 2.

MUC5AC and MUC5B quantification across various sample collection and processing methods and total peptide normalization assessment. (a) Univariate dot plots comparing mucin quantification in fresh induced sputum, frozen induced sputum and frozen spontaneous sputum in PBS and DTT fractions. Each data point represents the mean of measurements from duplicate samples from five patients diagnosed with COPD. Concentration values (μg/mL) are plotted against the y-axis. Horizontal black lines = mean concentration; horizontal red lines = LLOQ, lower limit of quantification. PBS: phosphate buffered saline; DTT: dithiothreitol. (b) Scatter plots comparing analyte concentrations in DTT fractions from fresh induced, frozen induced and frozen spontaneous sputum before (colored axes) and after total peptide normalization (black axes) for MUC5AC (blue) and MUC5B (purple). Each data point represents the mean of measurements from duplicate samples. Unweighted linear regression lines, R² and p-values are shown for each data set.

Figure 3.

Accuracy and precision for MUC5AC and MUC5B. (a) replicate QC data for all accuracy and precision runs (n = 2 runs, n = 6 per run). Values are depicted in raincloud plots consisting of jittered univariate dot plots, tukey-style box-and-whisker plots, and density (half-eye) plots. (b) Mean intra- and mean inter-run accuracy values appear as colored dots and as horizontal black lines, respectively. (c) Intra- and inter-run precision values appear as colored circles and vertical bars, respectively. Where applicable, acceptance limits are plotted as horizontal, dashed, red lines. LLOQ: lower limit of quantification; QC: quality control; DT: difference from theoretical; CV: coefficient of variation; low-: low-qc; mid-: mid-qc; high-: high-qc; QCs with shade are primary matrix QCs.

Figure 4.

Stability and dilutional study results. Shaded gray bands indicate the acceptance ranges for these studies (±20% from theoretical). At least six replicates were run in each experiment. MUC5B overspike-QCs are represented as black diamonds. The * symbol denotes tests which did not meet acceptance criteria. QC: quality control; OTC: over-the-curve.

Figure 5.

Matrix parallelism evaluation. Dilution-factor corrected concentration values for each replicate are depicted as circular data points (n = 3 replicates per dilution, n = 6 lots of human sputum; left axis). Mean intra-lot dilutional concentrations appear as horizontal, black lines. Mean intra-lot precision values (%CV) appear as vertical bars (right axis). Acceptance criteria appear as red dashed lines (right axis). The * symbol denotes matrix lots that did not meet acceptance criteria.